DEPARTMENT OF CELL BIOLOGY 2011-2012 - Albert Einstein College

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DEPARTMENT OF CELL BIOLOGY 2011-2012 3’ 5’ L V DJ C μ C δ C γ3 C γ1 C γ2b C γ2a C ε C α 3’ Enh Enh intronic **** 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. ARC Insulin Hoechst PU.1 ChIP-Seq Peaks light chain heavy chain Variable

Transcript of DEPARTMENT OF CELL BIOLOGY 2011-2012 - Albert Einstein College

 

DEPARTMENT OF CELL BIOLOGY2011-2012

3’5’L V DJ Cμ Cδ Cγ3 Cγ1 Cγ2b Cγ2aCε Cα

3’ EnhEnh intronic

****

1.2.

3. 4.

5.

6. 7. 8.

9. 10. 11.

ARC Insulin Hoechst

PU.1 ChIP-Seq Peaks

light chain

heavy chain

Variable

 

Picture Legends 

1.Query  Lab:  Scheme  for  progression  of  pre‐mRNA  splicing,  highlighting  the  different conformations  required  for  the  first  and  second  catalytic  steps;  modulation  of  transition between the two catalytic steps by spliceosomal mutations results  in altered splicing  fidelity and splice site choice. 

2.  Schildkraut  Lab:    Fluorescent  antibodies  showing  replication  fork direction  in  single DNA molecules labeled with halogenated nucleotides. 

3. Stanley Lab: The Notch ligand Dll3 is upregulated in mid‐hind brain of E8.5 mouse  embryos lacking  O‐fucose glycans on  Notch receptors. 

4.  Skoultchi  Lab:    ChIP‐Seq  –  Chromatin  immunoprecipitation  followed  by massive  parallel sequencing  reveals  differences  in  the  DNA  binding  patterns  of  transcription  factor  PU.1  in normal red blood cells (ES‐EP) and malignant erythroleukemia cells (MEL).  

5.  Birshtein/Scharff  Labs:    Mouse  Immunoglobulin  Heavy  Chain  Locus  with  genes  and regulators, intronic enhancer (Enh and 3’ regulatory region (3’ Enh). 

6. Fyodorov  & Skoultchi Lab: Drosophila melanogaster, a model system to study biochemistry and genetics of chromatin. 

7. Kitsis Lab: Micrograph of pancreatic islet from normal mouse immunostained for ARC (panel A) and insulin (panel B) and counterstained with Hoechst 33342 to demarcate nuclei (panel C). These data  show  the unexpected presence of ARC  in pancreatic beta‐cells, which make and secrete  insulin. ARC plays  a  critical  survival  function  in  these  cells,  as demonstrated by  the acceleration of beta‐cell death and islet destruction in mice lacking ARC (not shown). 

8. Warner Lab: The structure of a yeast ribosome 

9.  Ye  Lab:  Immunofluorescence  staining  reveals  that  transcription  factors  BCL6  (red)  and STAT3 (green) are expressed  in separate populations of B cells within the germinal center,   a dynamic microenvironment critical for T‐cell dependent antibody response.  

10. Scharff/Birshtein Labs: Cartoon of an Antibody Molecule.  

11. Kielian lab: Assembly and budding of alphaviruses from the plasma  membrane of infected host cells.  

 

Department of Cell Biology Welcome to the Albert Einstein College of Medicine and the Department of Cell Biology. Our department is focused on molecular mechanisms in many important areas of cell biology, ranging from stem cells to viruses, DNA replication to RNA processing, gene expression to immunology, glycobiology to cancer. We share many common interests and enjoy an interactive and scientifically stimulating atmosphere that makes the Cell Biology Department a great place to work. Graduate students in Cell Biology participate in a variety of departmental activities. The department meets every Friday for a “work-in-progress” seminar in which post-doctoral fellows and graduate students describe the progress of their current research and discuss future directions. The department hosts a bi-weekly seminar program of invited outside speakers, with many opportunities for students and postdocs to meet the speaker for discussion and lunch. There is a departmental journal club series in which students present original articles and discuss over dinner. A Friday afternoon get-together encourages scientific interactions as well as social connections. Lastly, our departmental retreat takes us all to the seashore or mountains for a chance to talk about the big picture of our research, to enjoy poster presentations from students and postdocs, and to try to solve the zany puzzles organized by the skit committee. On the following pages you will find information about the research programs of the individual faculty members, as well as listings of the current students and postdocs in the department. You can also find out more about the department on our web page at http://www.einstein.yu.edu/cellbiology . Feel free to contact any of us for further discussions. Enjoy your first year!

Cell Biology Faculty

Barbara Eric Winfried Paul Birshtein Bouhassira

Winfried Edelmann

Paul Frenette

Dmitry Fyodorov

Margaret Kielian

Michael Keogh

Wenjun Guo

Charles Query

Richard Kitsis

Matthew Scharff

Stanley Nathenson

Arthur Skoultchi

Carl Schildkraut

David Shafritz

Robert Singer

Hilda Ye

Jonathan Warner

UlrichSteidl

Pamela Stanley

 

CELL BIOLOGY FACULTY 2011- 2012

Room Building Phone Barbara Birshtein, Professor 417A Chanin 2291

Eric Bouhassira, Professor 903A Ullman 2188 (joint appointment, Medicine/Hematology)

Winfried Edelmann, Professor 279 Price 1086

Paul Frenette, Professor 101 Price 1255 (Director, Gottesman Stem Cell Institute)

Dmitry Fyodorov, Associate Professor 414A Chanin 4021

Wenjun Guo, Assistant Professor 122 Price TBD

Michael Keogh, Assistant Professor 415 Chanin 8796

Margaret Kielian, Professor 515 Chanin 3638

Richard Kitsis, Professor G46 Forchheimer 2609 (joint appointment, Medicine/Cardiology

Stanley Nathenson, Distinguished Professor 407 Chanin 2226 (joint appointment, Microbiology and Immunology)

Charles Query, Professor 440 Chanin 4174

Matthew Scharff, Distinguished Professor 403 Chanin 3527

Carl Schildkraut, Professor 416 Chanin 2097

David Shafritz, Professor 603 Ullman 2098 (joint appoinment, Medicine/Liver)

Robert Singer, Professor 601 Golding 8646 (joint appointment, Anatomy and Structural Biology)

Arthur Skoultchi, Chair & Professor 402 Chanin 2168

Pamela Stanley, Professor 516 Chanin 3346

Ulrich Steidl, Assistant Professor 606 Chanin 3437

Jonathan Warner, Professor 413A Chanin 3022

B. Hilda Ye, Associate Professor 302C Chanin 3339

ASSOCIATES AND INSTRUCTORS

Name (Mentor) Room Building Phone Email Address

ASSOCIATES

Elena Avdievich (Edelmann) 269 Price 1087 [email protected] Emelyanov (Fyodorov) 414 Chanin 4022 [email protected] Ghosh (Skoultchi) 402 Chanin 2168 [email protected] Lajugie (Bouhassira) 911 Ullman 2188 [email protected] Lailler (Bouhassira) 911 Ullman 2188 [email protected] Muller (Stanley) 516 Chanin 3470 [email protected] Prophete (Frenette) 107 Price 1204 [email protected] Simkin (Steidl) 606 Chanin 3551 [email protected] Werling (Edelmann) 269 Price 1087 [email protected]

Yong Wei Zhang (Edelmann) 269 Price 1087 [email protected] Yan (Skoultchi) 402 Chanin 2168 [email protected]

INSTRUCTORS

William Drosopoulos (Schildkraut) 416 Chanin 3193 [email protected] Lucas (Frenette) 107 Price 1204 [email protected] Magnon (Frenette) 107 Price 1204 [email protected] Sanchez San Martin (Kielian) 515 Chanin 3639 [email protected] Sandler (Bouhassira) 911 Ullman 2188 [email protected] Scheiermann (Frenette) 107 Price 1204 [email protected]

RESEARCH FELLOWS

Name (Mentor) Room Building Phone Email Address Boris Bartholdy (Steidl) 515 Chanin 3786 [email protected] Batista (Stanley) 516 Chanin 3470 [email protected] Bi (Ye) 302 Chanin 3341 [email protected] Bruns (Frenette) 101 Price 1204 [email protected] Cartier (Steidl) 515 Chanin 3786 [email protected] Chahwan (Scharff) 404 Chanin 2170 [email protected] Chen (Frenette) 101 Price 1204 [email protected] Christopeit 515 Chanin 3786 [email protected] Desprat (Bouhassira) 911 Ullman 3119 [email protected] Gerhardt (Schildkraut) 416 Chanin 3193 [email protected] Guo (Nathenson) 407 Chanin 2259 [email protected] Hanoun (Frenette) 101 Price 1204 [email protected] James (Kitsis) G01 Golding 2613 [email protected] Kavi (Skoultchi) 402 Chanin 2168 [email protected] Konstantinidis (Kitsis) G01 Golding 2613 [email protected] Kokavec (Skoultchi) 402 Chanin 2168 [email protected] Lacombe (Frenette) 101 Price 1204 [email protected] Ju Kim (Skoultchi) 402 Chanin 2168 [email protected] Kunasaki (Frenette) 101 Price 1204 [email protected] Lee (Edelmann) 269 Price 1087 [email protected] Xingwu Lu (Skoultchi) 402 Chanin 2168 [email protected] (Maria) Martinez (Kielian) 515 Chanin 3639 [email protected] Medina (Kitsis) G01 Golding 2613 [email protected] Miwa (Stanley) 516 Chanin 3470 [email protected] Moldon (Query) 415B Chanin 4175 [email protected] Pinho (Frenette) 101 Price 1204 [email protected] Samanta (Nathenson) 407 Chanin 2259 [email protected] Schaetzlein (Edelmann) 269 Price 1087 [email protected] Soo Kim (Keogh) 415 Chanin 4787 [email protected] Stiles (Kielian) 515 Chanin 3639 [email protected] Surena (Bouhassira) 911 Ullman 3119 [email protected] Tashima (Stanley) 516 Chanin 3470 [email protected] Van Oers (Edelmann) 269 Price 1087 [email protected] Vigdorovich (Nathenson) 407 Chanin 2259 [email protected] Volpi (Birshtein) 417 Chanin 3184 [email protected] Wei (Stanley) 516 Chanin 3470 [email protected] Wang (Scharff) 403 Chanin 3504 [email protected] Wei (Scharff) 403 Chanin 3504 [email protected] Will (Steidl) 515 Chanin 3786 [email protected] Yan (Stanley) 416 Chanin 3193 [email protected] Ying Yang (Kitsis) G01 Golding 2613 [email protected] Zheng (Kielian) 515 Chanin 3639 [email protected]

PREDOCTORAL FELLOWS

Name (Mentor) Room Building Phone Email Address

Laura Barreyro de Pujat (Steidl) 606 Chanin 3786 [email protected] Basak (Query) 415B Chanin 4175 [email protected] Chang (Bouhassira) 911 Ullman 3119 [email protected] Fields (Kielian) 515 Chanin 3639 [email protected] Ford (Fyodorov)* 414 Chanin 4021 [email protected] Grieco (Bouhassira) 911 Ullman 3119 [email protected] Ketchum (Frenette) 107 Price 1204 [email protected] Kung (Kitsis) G01 Golding 2613 [email protected] Limi (Skoultchi) 402 Chanin 2168 [email protected] Liu (Nathenson) 407 Chanin 2259 [email protected] McKimpson (Kitsis) G01 Golding 2613 [email protected] Cynthia Okoye (Steidl)* 606 Chanin 3786 [email protected] C. Onyinyechukwu (Kielian)* 515 Chanin 3639 [email protected] Shin Ooi (Kielian) 515 Chanin 3639 [email protected] Pandolfi (Steidl)* 606 Chanin 3786 [email protected] Rybin (Kitsis) G01 Golding 2613 [email protected] Am. S-Armogan (Bouhassira) 911 Ullman 3119 [email protected] C. Silva (Keogh) 415 Chanin 4787 [email protected] Stanley (Steidl)* 606 Chanin 3786 [email protected] Tosti (Edelmann) 269 Price 1087 [email protected] Whelan* (Kitsis) G01 Golding 2613 [email protected] Zhang (Frenette) 107 Price 1204 [email protected] Zheng (Kielian) 515 Chanin 3639 [email protected]

*MD/PhD Student

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Regulation of Antibody Heavy Chain Gene Rearrangements and Expression

The immune system is our spacesuit for life in an environment containing enormous

numbers of infectious agents. An essential part of the immune system is the B cell, which is the only cell type that produces antibodies. Antibody (Ig) genes are constructed via a series of DNA rearrangements. Occasionally, mistakes occur during antibody construction, which activate oncogenes and lead to cancers. My long term goal is to understand the mechanisms that initiate and control antibody gene rearrangements within the 3 megabase heavy chain gene (IgH) locus. Our experiments focus on a complex 3' IgH regulatory “ignition” region that lies immediately downstream of the antibody heavy chain gene cluster in mouse and humans. The ~50 kb 3’ IgH regulatory region (3’ RR), containing multiple regulatory elements, and a 1 kb intronic enhancer, are the only two currently known regulatory regions for the Igh locus. We have identified an extension of the 3’ RR (hs5, 6, 7), which like the other 3’ enhancers, has binding sites for Pax5, a transcription factor essential for B cell development. In addition, the 3’ RR extension contains insulator activity, i.e. prevention of communication between an enhancer and its target promoter. This is associated with binding sites for CTCF, a protein identified in all mammalian insulators. Chromatin analysis shows that the 3’ RR extension is likely to be active throughout B cell development while other 3’ RR segments become progressively and stage-specific active.

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.Murine germline Igh locus, with variable (VH), diversity (DH), joining (JH) and constant region genes (μ, δ, γ, ε, α). Regulatory elements, Eμ and the 3’ regulatory region (3’RR) are depicted

A major goal of our laboratory is to understand the mechanisms by which the 3’ RR

functions in both mouse and human, both by acting on the Igh locus and by insulating the locus from its non-Igh neighboring genes. We predict that the 3’ RR promotes Igh DNA rearrangement events throughout B cell development. In addition to its known essential contribution to the expression of different classes of antibodies (class switch recombination), the 3’ RR is likely to regulate high levels of expression of antibodies in fully differentiated plasma cells. The 3’ RR is also implicated in the formation (VDJ joining) and subsequent hypermutation (SHM) of the variable region. CTCF binding sites are anticipated to play a major role in these activities by promoting interactions between distal DNA sequences through loop formation.

We are interested in identifying the extent of B cell-specific regulation of the IgH locus,

by analyzing DNA demethylation, histone modifications in and the binding of CTCF and Pax5 , and other factors to 3' RR sequences during B cell development and class switching.. Furthermore, we are examining the interaction of the 3’ RR with target sequences within the IgH locus, as assessed by the chromosome capture conformation technique (3C). Together, this approach is designed to understand how molecular acrobatics can occur only within the

Barbara K. Birshtein, Ph.D. Professor

Chanin Building, Room 417A (718) 430-2291

[email protected]

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IgH locus without intruding on downstream non-IgH genes, or leading to chromosomal translocations involved in malignancies.. Publications: Manis, J. P., Michaelson, J. S., Birshtein, B, K., and Alt, F, W. (2003) “Elucidation of a

downstream boundary of the 3’ IgH regulatory region”. Mol. Immunol. 39:753-760. Gordon, S. J., Saleque, S. and Birshtein, B. K. (2003) Yin Yang 1 (YY1) is an LPS-inducible

activator of the murine 3' Igh enhancer, hs3". J. Immunol., 170:5549-5557. Barlev, N. A.,* Emelyanov, A. V.,* Castagnino, P., Zegerman, P., Bannister, A., Sepulveda, M.

A., Kouzarides, T., Birshtein, B. K., and Berger, S. L. (2003) "A novel human Ada2 homologue functions with Gcn5 or Brg1 to coactivate transcription." *Co-first authors. Mol. Cell. Biol. 23:6944-6957.

Sepulveda, M.A,, Emelyanov, A. V., and Birshtein, B. K. (2004) “NF-κB and Oct-2 synergize to activate the human 3’ Igh hs4 enhancer in B cells.” J. Immunol. 172:1054-1064.

Cogne’, M. and Birshtein, B.K. (2004). “Regulation of Class Switch Recombination” in Molecular Biology of B Cells, ed. T. Honjo, F.W.Alt and M. Neuberger. Elsevier Science USA. 289-305.

Sepulveda, M. A., Garrett, F., Price-Whelan, A., Birshtein, B.K., (2005) “Comparative analysis of human and mouse 3’ Igh regulatory regions identifies distinctive structural features.” Mol. Immunol.. 42:605-615.

Garrett, F.E. Emelyanov, A.V., Sepulveda, M.A., Flanagan, P., Volpi, S., Li, F., Loukinov, D., Eckhardt, L.A., Lobanenkov, V.V. and Birshtein, B.K. (2005). “Chromatin architecture near a potential 3’ end of the Igh locus Involves modular regulation of histone modifications during B-cell development and in vivo occupancy at CTCF sites” Mol. Cell Biol. 25 (4):1511-1525.

Zhou, J., Saleque, S., Ermakova, O., Sepulveda, M. A., Yang, Q., Eckhardt, L. A., Schildkraut, C. L. and Birshtein, B. K. (2005). Changes in replication, nuclear location and expression of the Igh locus after fusion of a pre-B cell line with a T cell line. J. Immunol. 175: 2317-2320i

Schweitzer, B. L., Huang, K. J., Kamath, M. B., Emelyanov, A. V., Birshtein, B. K. and DeKoter R. P. (2006). “Spi-C has opposing effects to PU.1 on gene expression in progenitor B cells”, J. Immunol. 177:2195-2207.

Ju, Z., Volpi, S. A., Hassan, R., Martinez, N., Giannini, S. L., Gold, T., and Birshtein, B. K. (2007). "Evidence for physical interaction between the immunoglobulin heavy chain variable region and the 3' regulatory region." J. Biol. Chem. 282:35169-35178. Epub 2007 Oct 5, cited by Faculty of 1000.

Giambra, V., Volpi, S., Emelyanov, A. V., Pflugh, D., Bothwell, A. L. M., Norio, P., Fan, Y., Ju, Z., Skoultchi, A. I., Hardy, R. R., Frezza, D., and Birshtein, B. K. (2008). "Pax5 and Linker Histone H1 Coordinate DNA Methylation and Histone Modifications in the 3' Regulatory Region of the Immunoglobulin Heavy Chain Locus". Mol Cell. Biol. 28:6123-6133. Epub 2008 Jul 21

Frezza, D., Giambra, V., Mattioli, C., Piccoli, K., Massoud, R., Siracusano, A., Di Giannantonio, M., Birshtein, B. K. and Rubino, I. A. (2009) "Allelic Frequencies of 3' Ig Heavy Chain Locus Enhancer HS1,2-A Associated with Ig Levels in Patients with Schizophrenia and Healthy Control Subjects". Intl. J. Immunopath. Pharm. 22: 115-23.

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Degner, S. C., Verma-Gaur, J., Wong, T. P., Bossen, C., Iverson, G. M., Torkamani, A., Vettermann, C., Lin, Y. C., Ju, Z., Schulz, D., Murre, C. S., Birshtein, B. K., Schork, N. J., Schlissel, M. S., Riblet, R., Murre, C., and Feeney, A. J. (2011). “CCCTC-Binding Factor (CTCF) and Cohesin Influence the Genomic Architecture of the Igh Locus and Antisense Transcription in Pro-B Cells”. Proc. Natl. Acad. Sci., 108:.9566-9571.

Yan, Y., Pieretti, J., Ju, Z., Christin, J. R., Bah, F., Birshtein, B. K., and Eckhardt, L. A. (2011). “Homologous Elements hs3a and hs3b in the 3’ Regulatory Region of the Immunoglobulin Heavy Chain (Igh) Locus Are Both Dispensable for Class-switch Recombination,” J. Biol. Chem. 286:27123-27131.

Chatterjee, S., Ju, Z., Hassan, R., Volpi, S., Emelyanov, A., and Birshtein, B. K., (2011). “Dynamic Changes in Binding of Immunoglobulin Heavy Chain 3’ Regulatory Region to Protein Factors during Class Switching”. J. Biol. Chem. In press.

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Key Words: Epigenetic, gene regulation, stem cells, reprogramming HUMAN EMBRYONIC STEM CELLS, EPIGENETICS AND REPROGRAMMING: Epigenetic is the study of mitotically or meiotically heritable changes in gene function not associated with changes in DNA sequence. Epigenetic regulations are mediated by changes in chromatin structure that alter access of transcription factors to their cognate binding sites, and therefore, expression levels of genes and transgenes. Understanding these regulations is critical for gene therapy, cancer therapy and generally to gain a greater ability to modify mammalian genomes. The main focus of my lab is to understand the molecular bases of some of these epigenetic regulations. We are particularly interested in using massively parallel sequencing technologies to understand the mechanisms of establishment, maintenance, and inheritance of chromatin structures. We have recently developed a method termed TimEX to determine the timing of DNA replication genome-wide. We are using TimEX in conjunction with Chip-Seq and RNA-seq to study epigenome organization. The second major focus of the lab is to use the epigenetic information obtained above to improve the reprogramming of somatic cells into induced pluripotent stem cells and to develop methods to reprogram somatic cells into other stem or progenitor cells by over-expression of transcription factors without reverting to an embryonic state Finally, we are currently developing a system to produce normal human red blood cells with embryonic, fetal or adult characteristics by forced differentiation of human ES cells using a genetic approach. The goal is to generate large amount of genetically homogenous, genetically modifiable normal erythroid cells at different stages of differentiation. This system could allow us to produce red cells for transfusion medicine, to study the silencing of the gamma-globin gene, to develop silencing resistant gene therapy cassettes that will be useful to cure the hemoglobinopathies, and to generally study the establishment and maintenance of epigenetic signals directly in untransformed human cells. We also have active research projects on the role of DNA methylation, DNA replication, histone modification, linker histones, and transcriptional interferences in the determination of expression levels. FOR MORE DETAILS: http://cellbio.aecom.yu.edu/Lab/Bouhassira/ Selected References: 1. Desprat R, Thierry-Mieg D, Lailler N, Lajugie J, Schildkraut C, Thierry-Mieg J, Bouhassira EE. Predictable dynamic program of timing of DNA replication in human cells. Genome Res. 2009 Nov 5. 2. Guan Z, Hughes CM, Kosiyatrakul S, Norio P, Sen R, Fiering S, Allis CD, Bouhassira EE, Schildkraut CL. Decreased replication origin activity in temporal transition regions. J Cell Biol. 2009 Nov 30;187(5):623-35.

ERIC BOUHASSIRA, Ph.D. Professor Medicine, Hematology & Cell Biology ULLMAN BLDG. – ROOM 903

718 430-2188 [email protected]

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3. Li H, Rybicki AC, Suzuka SM, von Bonsdorff L, Breuer W, Hall CB, Cabantchik ZI, Bouhassira EE, Fabry ME, Ginzburg YZ. Transferrin therapy ameliorates disease in beta-thalassemic mice. Nat Med. Nat Med. 2010 Feb;16(2):177-8 4. Qiu C, Olivier EN, Velho M, Bouhassira EE. Globin switches in yolk sac-like primitive and fetal-like definitive red blood cells produced from human embryonic stem cells. Blood. 2008 Feb 15;111(4):2400-8.

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Genomic Instability and Cancer in Murine Models

The maintenance of genomic integrity in all organisms requires multiple DNA repair pathways that are involved in the processes of DNA replication, repair and recombination. Perturbations in these pathways can lead to increased mutation rates or chromosomal rearrangements that ultimately result in cancer. MMR is one of the repair systems that mammalian cells employ to maintain the integrity of its genetic information by correcting mutations that occur during erroneous replication. Mutations in MMR genes are linked to one of the most prevalent human cancer syndromes, Lynch syndrome and a significant number of sporadic colorectal cancers. At the molecular level tumors that develop in these patients display increased genomic mutation rates as indicated by increased instability at microsatellite repeat sequences (termed microsatellite instability, MIN). MMR in eukaryotes is complex and involves several homologs of the bacterial MutS and MutL proteins. In mammals, the initiation of the repair process requires two complexes formed by three different MutS homologs (MSH): A complex between MSH2-MSH6 for the recognition of single base mismatches and a complex between MSH2-MSH3 for the recognition of insertion/deletions. The repair reaction also requires a complex between the two MutL homologs MLH1 and PMS2 that interacts with the MSH complexes to activate subsequent repair events which include the excision of the mismatch carrying DNA strand and its re-synthesis. These steps are carried out by exonculeases, polymerases and a number of replication associated proteins. In addition to correcting DNA mismatches, the MMR system mediates an apoptotic response to DNA damage and suppresses recombination between non-identical sequences in mammalian genomes. All of these functions are thought to be important for genome maintenance and tumor suppression. We have generated knockout mouse lines with inactivating mutations in all the different MutS and MutL homologs, and also in genes that function in the later MMR steps to study their roles in genome maintenance and tumor suppression. In addition, we have generated knock-in mouse lines with missense mutations and conditional knockout mouse lines that inactivate specific MMR functions and/or model mutations found in humans. Our studies indicate that specific MMR functions play distinct roles in maintaining genome stability and that defects in these functions have important consequences for tumorigenesis and the response of tumors to chemotherapeutic treatment. They have also revealed that some of the MMR proteins play essential roles in the control of meiotic recombination in mammals. Selected References: Wang J.Y.J and Edelmann W. 2006. Mismatch Repair Proteins as Sensors of Alkylation DNA Damage. Cancer Cell 6:417-418. Avdievich E. et al. 2008. Distinct effects of the recurrent Mlh1G67R mutation on MMR functions, cancer and meiosis. Proc. Natl. Acad. Sci. USA 105:4247-4252. Takedo M.M. & Edelmann W. 2009. Mouse models of colorectal cancer. Gastroenterology 136(3):780-798. PMID: 19263594 Kucherlapati M.H., Lee K., Nguyen A.A., Clark A.B., Hou H. Jr., Rosulek A., Li H., Yang K., Fan K., Lipkin M., Bronson R.T., Jelicks L., Kunkel T.A., Kucherlapati R. and Edelmann W. 2010. An Msh2

WINFRIED EDELMANN, Ph.D. Professor

Price BLDG. – ROOM 269/277 718 678-1086

[email protected]

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Conditional Knockout Mouse for Studying Intestinal Cancer and Testing Anti-cancer Agents. Gastroenterology 138:993-1002. Wang Y., Zhang W., Edelmann L., Kolodner R.D., Kucherlapati R. And Edelmann W. 2010. Cis lethal genetic interactions attenuate and alter p53 tumorigenesis. Proc Natl Acad Sci U S A 107:5511-5515. PMID: 20212136 van Oers J.M., Roa S., Werling U., Liu Y., Genschel J., Hou H. Jr., Sellers R.S., Modrich P., Scharff M.D., Edelmann W. 2010. PMS2 endonuclease activity has distinct biological functions and is essential for genome maintenance. Proc Natl Acad Sci U S A. (in press)

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Our laboratory is interested in understanding how hematopoietic stem cells (HSCs) and

mature blood cells traffic in vivo. We have uncovered a key role for the nervous system in regulating HSC trafficking, and are evaluating its role in the inflammatory response in diseases such as sickle cell disease. In addition, we are also exploring whether the traffic paradigms uncovered for healthy stem cells applies to cancer cell migration and metastasis. Molecular and cellular constituents of the stem cell niche. HSCs continuously traffic from the bone marrow to the blood compartment (and vice-versa) under homeostasis. Ongoing studies have focused on the role of the nervous system in the regulation of the HSC niche in the bone marrow. This effort is based on our recent observations suggesting a critical function of adrenergic signals emerging from the sympathetic nervous system (SNS) in HSC egress. While investigating further the mechanisms by which HSCs were mobilized, we have found that exposure to constant light significantly reduced mobilization efficiency following the administration of the hematopoietic cytokine G-CSF. G-CSF is the most commonly used HSC mobilizer in the clinic to harvest stem cells for transplantation. This finding prompted us to assess how HSC are released from the bone marrow under steady-state conditions. We have described the phenomenon and its mechanisms. These studies revealed that stromal cells in the bone marrow are subjected to circadian adrenergic signals transmitted by the �3 adrenergic receptor that lead to the degradation of the transcription factor Sp1 and diurnal changes in the expression of the chemokine Cxcl12. Recent investigations are focused on the identification and regulation of the stromal target for the SNS. These studies have led to the identification of a nestin+ mesenchymal stem cell as a candidate niche cell required for HSC maintenance in the bone marrow. Other studies have evaluated the role of macrophages and found a role in the retention of HSC in the niche by regulating nestin+ cells. Mechanisms of sickle cell vaso-occlusion. This project emerged from our intravital microscopy observations suggesting that sickle cell vaso-occlusion was mediated by the direct interaction between sickle erythrocytes and adherent leukocytes in small venules. Further analyses using novel high-speed multichannel fluorescence microscopy techniques have revealed that E-selectin-mediated activating signals emanating from the inflamed endothelium led to the activation of specific microdomains on the leading edge of adherent neutrophils, which then induce intravascular heterotypic interactions between erythrocytes or platelets with adherent leukocytes. Ongoing studies dissect further the molecular basis of this phenomenon. Role of the nervous system in cancer. We are exploring the role of the autonomic nervous system in cancer formation and metastasis using xenogeneic and transgenic models of prostate cancer. Ultimately, the goal of these studies is to obtain new insight on the cellular and molecular cues that regulate the tumour microenvironment and allow cancer cells to spread.

PAUL S. FRENETTE, M.D. Professor

PRICE BLDG. - ROOM 101B 718-678-1255

[email protected]

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Selected Recent Publications: Chow A, Lucas D, Hidalgo A, Mendez-Ferrer S, Hashimoto D, Sheiermann C, Battista M, Leboeuf M, Prophete C, van Rooijen N, Merad M, and Frenette PS. Bone Marrow CD169+ Macrophages Promote Retention of Hematopoietic Stem and Progenitor Cells in the mesenchymal stem cell niche. J. Exp. Med. 2011. Feb 14;208(2):261-71.

Jang JE, Hod EA, Spitalnik SL and Frenette PS. CXCL1 and its receptor, CXCR2, mediate murine sickle cell vaso-occlusion during hemolytic transfusion reactions. J. Clin. Invest. 2011. Apr 1;121(4):1397-401.

Méndez-Ferrer S, Michurina T, Ferraro F, Mazloom AR, MacArthur BD, Lira SA, Scadden DT, Maayan A, Enikolopov GN, and Frenette PS. (2010) Mesenchymal and hematopoietic stem cells form a unique bone marrow niche. Nature. Aug 12;466(7308):829-34.

Chang J, Patton JT, Sarkar A, Ernst B, Magnani JL, and Frenette PS. GMI-1070, a novel pan-selectin antagonist, reverses acute vascular occlusions in sickle cell mice. Blood 2010, In press. Hidalgo A, Chang J, Jang JE, Peired AJ, Chiang EY and Frenette PS. Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury. Nat. Med. 2009 Apr;15(4):384-91. Lucas D, Battista M, Shi PA, Isola L and Frenette PS. Mobilized hematopoietic stem cell yield depends on species-specific circadian timing. Cell Stem Cell 2008 Oct 9;3(4):364-6. Méndez-Ferrer S, Lucas D, Battista M, and Frenette PS. Haematopoietic stem cell release is regulated by circadian oscillations. Nature 2008 Mar 27;452(7186):442-447. Chang J, Shi PA, Chiang EY, and Frenette PS. Intravenous immunoglobulins reverse acute vaso-occlusive crises in sickle cell mice through rapid inhibition of neutrophil adhesion. Blood 2008 Jan 15;111(2):915-23. Epub 2007 Oct 11. Hidalgo A, Peired AJ, Wild M, Vestweber D, and Frenette PS. Complete identification of E-selectin ligand activity on neutrophils reveals distinct functions of PSGL-1, ESL-1 and CD44. Immunity. 2007 Apr;26(4):477-89. Chiang EY, Hidalgo A, Chang J, and Frenette PS. Imaging receptor microdomains on leukocyte subsets in live mice. Nat. Methods. 2007 Mar;4(3):219-22. Katayama Y, Battista M, Kao WM, Hidalgo A, Thomas SA and Frenette PS. Signals from the sympathetic nervous system regulate hematopoietic stem cell egress from bone marrow. Cell. 2006 Jan 27;124(2):407-21.

1

BIOCHEMISTRY AND GENETICS OF CHROMATIN ASSEMBLY

In the eukaryotic nucleus, hundreds of millions of base pairs of DNA are packed into

chromosomes. Chromatin, the central nucleoprotein filament of a chromosome, has many different forms and organization levels. Chromatin is the natural state of DNA in the nucleus and the native substrate for nuclear reactions such as DNA replication, recombination, repair and transcription. The assembly of DNA into chromatin and dynamic conversion between its different forms are critical steps in the maintenance and regulation of the eukaryotic genome. The ultimate goal of our research is to understand how chromosomes are assembled and how chromatin assembly regulates the structure and activity of eukaryotic chromosomes. The crucial first step in this direction is a systematic study of factors that mediate this process. To this end we use biochemical approaches to analyze mechanisms of chromatin assembly by histone chaperones and ATP-dependent enzymes. We also dissect their function in vivo by methods of Drosophila genetics. Thus, we are trying to uncover the intricate network of assembly factors and their roles in the hierarchical organization of the chromosome.

1. Molecular mechanisms of nucleosome assembly ACF (ATP-utilizing chromatin assembly factor) was identified on the basis of its ability to

mediate ATP-dependent reconstitution of chromatin in vitro. ACF consists of two subunits, a SNF2-like ATPase ISWI and another evolutionary conserved polypeptide termed Acf1. In the presence of a core histone chaperone NAP-1, ACF/ISWI mediate deposition of histone octamers onto DNA and form arrays of regularly spaced nucleosomes. We study ACF and ISWI as prototype factors to elucidate elementary molecular events that take place during ATP-dependent formation of nucleosomes. Upon reaction initiation, ACF commits to the DNA template and assembles nucleosomes as a processive, ATP-driven, DNA-translocating motor. Multiple conserved domains of Acf1 and ISWI are required for this activity.

2. Biological function of chromatin assembly factors ACF is the major ATP-dependent chromatin assembly factor in Drosophila. To expose its

biological functions, we study fly mutants that do not express ACF. ACF-deficient animals have multiple defects of chromatin organization. However, ACF is not essential for fly viability due to the presence of additional, redundant ACF-like factors. We discovered a novel ISWI-containing complex ToRC (comprising Tou, ISWI and CtBP) that can functionally substitute ACF in vivo. SNF2-like protein CHD1 is another ATP-dependent nucleosome assembly factor. We disrupted Chd1 in flies and discovered that CHD1 is specifically required for replication-independent deposition of histones into chromatin in vivo.

3. Higher-order chromatin forms ACF can mediate deposition of both core and linker histones (H1) in vitro. Thus, it can

assemble the 30 nm chromatin fiber in a defined system. To reconstitute other higher-order chromatin structures, we incorporate modified core histones, histone variants and heterochromatin proteins. In vitro assembled chromatin vectors can turn into useful tools in research and therapy. Among other outcomes, these studies will eventually lead to the discovery of techniques to reconstitute functional metazoan chromosomes.

Sperm DNA is compacted with protamines to form enzymatically static sperm “chromatin”. We have begun to analyze protein factors that mediate protamine deposition during spermatogenesis and their removal from DNA after fertilization.

DMITRY FYODOROV, Ph.D. Associate Professor

CHANIN BLDG. – ROOM 414 718 430-4021

[email protected]

2

References Lu, X., Wontakal, S.N., Emelyanov, A.V., Morcillo, P., Konev, A.Y., Fyodorov, D.V., and

Skoultchi, A.I. (2009). Linker histone H1 is essential for Drosophila development, the establishment of pericentric heterochromatin, and a normal polytene chromosome structure. Genes Dev. 23, 452-465.

Konev, A.Y., Tribus, M., Park, S.Y., Podhraski, V., Lim, C.Y., Emelyanov, A.V., Vershilova, E., Pirrotta, V., Kadonaga, J.T., Lusser, A., and Fyodorov, D.V. (2007). CHD1 motor protein is required for deposition of histone H3.3 into chromatin in vivo. Science 317, 1087-1090.

Fyodorov, D.V., Blower, M.D., Karpen, G.H., and Kadonaga, J.T. (2004). Acf1 confers unique

activities to ACF/CHRAC and promotes the formation rather than disruption of chromatin in vivo. Genes Dev. 18, 170-183.

Fyodorov, D.V., and Kadonaga, J.T. (2003). Chromatin assembly in vitro with purified

recombinant ACF and NAP-1 [37]. Meth. Enzymol. 371, 499-515. Fyodorov, D.V., and Kadonaga, J.T. (2002). Dynamics of ATP-dependent chromatin

assembly by ACF. Nature 418, 896-900.

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Key Words: yeast, chromatin, transcription, DNA repair, genome stability, cancer HOW DOES CHROMATIN WORK? Eukaryotic DNA usually resides in a nucleoprotein complex known as chromatin. The basic repeating unit is a nucleosome: ~150bp of DNA wound around eight histone proteins. A simple repeating array would suffice if the only function of the biopolymer was packaging. However, cells need to gain region-specific access to the DNA for replication, transcription, repair or chromosome transmission. Families of enzymes distinguish specific nucleosomes by replacing a major histone with a related variant, or covalently modifying the histones with small chemical moieties. These modifications are then thought to play distinct roles. Current projects in the lab include:

(i)How chromatin controls the usage of inappropriate transcription start sites (ii)How the acetylation of histone H2A.Z regulates its function (iii)How cells establish and maintain mitotic chromosome structure (iv)How genetic interaction networks in yeast can be used to design novel therapies for human cancers

Selected References: 1:Chowdhury D, Keogh MC, Ishii H, Peterson CL, Buratowski S & Lieberman J. �H2AX dephosphorylation by protein phosphatase 2A facilitates DNA double-strand break repair. Mol Cell (2005) 20: 801-9. 2:Keogh M-C, Kurdistani S, Morris SA, Ahn SH, Podolny V, Collins SR, Shuldiner M, Chin K, Punna T, Thompson NJ, Boone C, Emili A, Weissman JS, Hughes TR, Strahl BD, Grunstein M, Greenblatt JF, Buratowski S & Krogan NJ. Co-transcriptional Set2 methylation of histone H3 lysine 36 recruits a repressive Rpd3 complex. Cell (2005) 123: 593-605. 3:Keogh MC, Kim JA, Downey M, Fillingham J, Chowdhury D, Harrison JC, Onishi M, Datta N, Galicia S, Emili A, Lieberman J, Shen X, Buratowski S, Haber JE, Durocher D, Greenblatt JF & Krogan NJ. A phosphatase complex that dephosphorylates �H2AX regulates DNA damage checkpoint recovery. Nature (2006) 439: 497-501. 4:Fillingham J, Keogh MC, Krogan NJ. �H2AX and its role in DNA double-strand break repair. Biochem Cell Biol (2006) 84: 568-77. 5:Keogh MC, Mennella TA, Sawa C, Berthelet S, Krogan NJ, Wolek A, Podolny V, Carpenter LR, Greenblatt JF, Baetz K & Buratowski S. The Saccharomyces cerevisiae histone H2A variant Htz1 is acetylated by NuA4. Genes Dev (2006) 20: 660-5.

Michael-Christopher Keogh, Ph.D. Assistant Professor CHANIN BLDG – ROOM 415 718 430-8796

[email protected] sites.google.com/site/mckeogh2

2

6:Fillingham J, Recht J, Silva AC, Suter B, Emili A, Stagljar I, Krogan NJ, Allis CD, Keogh MC & Greenblatt JF. Chaperone control of the activity and specificity of the histone H3 acetyltransferase Rtt109. Mol Cell Biol (2008) 13: 4342-53. 7:Ahn SH, Keogh M-C & Buratowski S. Ctk1 promotes dissociation of basal transcription factors from elongating RNA polymerase II. EMBO J (2009) 28: 205-12. 8:Fiedler D, Braberg H, Mehta M, Chechik G, Cagney G, Mukherjee P, Silva AC, Shales M, Collins SR, van Wageningen S, Kemmeren P, Holstege, FCP, Weissman JS, Keogh M-C, Koller D, Shokat KM & Krogan NJ. Functional organization of the S. cerevisiae phosphorylation network. Cell (2009) 136: 952-63. 9:Kim H-S, Vanoosthuyse V, Fillingham J, Roguev A, Watt S, Kislinger T, Treyer A, Carpenter LR, Bennett CS, Emili A, Greenblatt JF, Hardwick KG, Krogan NH, Bähler J & Keogh M-C. An acetylated form of histone H2A.Z regulates chromosome architecture in Schizosaccharomyces pombe. Nat Struct Mol Biol (2009) 16: 1286-93. 10:Mehta M, Kim H-S & Keogh M-C. Sometimes one just isn't enough: do vertebrates contain an H2A.Z hyper variant? J Biol (2010) 9: 3.

1

Molecular Mechanisms of Virus Entry and Exit. For more information please see our lab homepage:

https://sites.google.com/site/kielianlab/

During infection all enveloped viruses use the essential steps of membrane fusion to enter a cell, and membrane budding to produce infectious progeny viruses. Molecular information on these processes is critical to understanding the infection pathways of enveloped viruses and as a very useful model for cellular membrane fusion and budding reactions.

Our research focuses on the molecular mechanisms of virus-membrane fusion and virus budding using alphaviruses such as Semliki Forest virus (SFV) and flaviviruses such as dengue virus (DV). The flaviviruses and alphaviruses include many important human pathogens such as dengue, West Nile, and chikungunya viruses. DV is currently of particular concern as it has dramatically reemerged to become endemic in >100 countries, with an estimated 100 million cases of dengue infection per year. There are currently no vaccines or antiviral therapies for DV, and new therapeutic strategies for the flaviviruses and alphaviruses are urgently needed. SFV is a highly developed system to study virus fusion and budding, and is an important experimental model for both alphaviruses and flaviviruses.

Both SFV and DV enter cells by endocytic uptake and then fuse their membrane with the endosome membrane in a reaction triggered by the low pH of the endocytic vesicle. The flavivirus and alphavirus membrane fusion proteins are structurally related proteins and refold during fusion to a homotrimer conformation that mediates virus fusion and infection. In collaboration with Dr. Félix Rey, we determined the structure of the homotrimer of the SFV fusion protein E1. This structure is strikingly similar to the DV homotrimer.

Using the structures as a guide, our lab has developed fragments of the SFV and DV fusion proteins that act as dominant-negative inhibitors of SFV and DV fusion and infection. We are currently using expressed fusion protein constructs to characterize the mechanism of trimer formation, to define the stages of the fusion reaction, and to reconstitute trimerization in vitro. We are also developing these in vitro trimers as screens for small molecule inhibitors of virus fusion reactions that will be lead compounds for new antiviral therapies.

E1-membrane insertion and alphavirus fusion are strikingly dependent on the presence of cholesterol in the cell membrane. The control of E1-membrane insertion and the mechanism of E1’s cholesterol and pH-dependence are not understood, and we are using biochemistry, fluorescence methods, virus genetics and in vitro mutagenesis of SFV and DV infectious clones to address these questions.

SFV exits by budding through the plasma membrane of the infected host cell. Little is known about budding of alphaviruses or flaviviruses, although it is clear that budding is highly specific and produces very organized virus particles. How does this happen and what are the roles of cellular and viral components? The available data strongly suggest that the exit pathway of the alphaviruses involves unique cellular proteins, and their de novo identification will require a broad-based, unbiased approach. We are testing the role of specific candidate

MARGARET KIELIAN, Ph.D. Professor

CHANIN BLDG. – ROOM 515 718 430-3638

[email protected]

2

cellular proteins in alphavirus budding. We are using an siRNA screening approach to identify novel host cell proteins involved in the alphavirus entry and exit pathways.

Our lab uses a wide variety of approaches including molecular biology, virus genetics, protein biochemistry, cell biology, fluorescence spectroscopy, small molecule and RNAi screens, and structural biology.

Potential research projects include: screens for virus fusion inhibitors, characterization of the role of viral and cellular factors in virus budding, mutagenesis of virus infectious clones to characterize specific steps in fusion.

Selected references: Gibbons, D.L., M.-C. Vaney, A. Roussel, A. Vigouroux, B. Reilly, J. Lepault, M. Kielian*, and F. A. Rey*.

(2004) “Conformational change and protein-protein interactions of the fusion protein of Semliki Forest virus.” Nature 427, 320–325. *Joint corresponding authors.

Liao, M., and M. Kielian. (2005) “Domain III from class II fusion proteins functions as a dominant-negative inhibitor of virus-membrane fusion.” J. Cell Biol. 171:111-120.

Taylor, G.M., P. I. Hanson, and M. Kielian. (2007). “Ubiquitin depletion and dominant-negative VPS4 inhibit rhabdovirus budding without affecting alphavirus budding.” J. Virol. 81:13631-13639.

Umashankar, M., C. Sánchez-San Martín, M. Liao, B. Reilly, A. Guo, G. Taylor, and M. Kielian. (2008) “Differential cholesterol binding by class II fusion proteins determines membrane fusion properties.” J. Virol. 82:9245-9253.

Sánchez-San Martín, C., H. Sosa and M. Kielian. (2008) “A stable prefusion intermediate of the alphavirus fusion protein reveals critical features of class II membrane fusion.” Cell Host and Microbe 4:600-608.

Qin, Z., Y. Zheng, and M. Kielian. (2009) “Role of conserved histidine residues in the low pH-dependence of the Semliki Forest virus fusion protein.” J. Virol., 83:4670-4677.

Liu, C.Y. and M. Kielian. (2009) “E1 mutants identify a critical region in the trimer interface of the Semliki Forest virus fusion protein.” J. Virol. 83:11298-11306.

Kielian, M., C. Chanel-Vos and M. Liao. (2010) “Alphavirus entry and membrane fusion.” Invited review, Viruses, 2: 796-825.

Liao, M., C. Sánchez-San Martín, A. Zheng, and M. Kielian. (2010) “In vitro reconstitution reveals key intermediate states of trimer formation by the dengue virus membrane fusion protein.” J. Virol. 84:5730-5740.

Zheng, A., M. Umashankar, and M. Kielian. (2010) “In vitro and in vivo studies identify important features of dengue virus pr-E protein interactions.” PLoS Pathog 6(10): e1001157.

Liu, C.Y., C. Besanceney, Y. Song, and M. Kielian (2010) “Pseudorevertants of a Semliki Forest virus fusion-block mutation reveal a critical interchain interaction in the core trimer” J. Virol. 84:11624-11633.

Zheng, Y., C. Sánchez-San Martín, Z. Qin, and M. Kielian. (2011) The Domain I-Domain III Linker Plays an Important Role in the Fusogenic Conformational Change of the Alphavirus Membrane Fusion Protein”. J. Virol. 85:6334-6342. [selected for spotlight by the editors]

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1

Studies on the Structural Basis for T Cell Activation and Regulation

Destruction of invading pathogenic organisms and transformed cells occurs through the specific recognition of these threats by the adaptive immune system. In order to recognize such a diverse and constantly changing set of targets, the immune system must exhibit broad reactivity, yet to avoid destruction of self tissues, activation must be tightly regulated. Broad reactivity arises from generation of a polyclonal T-cell population by recombination of the T-cell receptor (TCR) protein to allow the recognition of diverse antigenic peptide-Major Histocompatibility complex (MHC) protein complexes. No single receptor protein, however, is responsible for regulation of the immune response, (costimulation) and instead arises from action of both inhibitory and stimulatory signals delivered through both the TCR and diverse coreceptors present within the immunological synapse. The research program of the Nathenson lab has followed a simple philosophy, structure follows from function and conversely we can extend structural insights into a testable functional hypothesis. Historically we have used this guiding principle to study the role of MHC in T-cell activation. Biochemical studies using in vitro complex formation of MHC/peptide were used to evaluate the precise mechanism of peptide binding, and in conjunction with the x-ray structure, to determine the structural rules, which govern the selection and binding of such peptides to the MHC molecule. Further biochemical studies were carried out to start defining interactions that are important for recognizing the peptide and the MHC complex by the TCR. Our initial studies on T cell regulation in collaboration with the Almo laboratory focused on the stimulatory coreceptor CD28 and the inhibitory coreceptor CTLA-4, which, despite their difference in function, bind to the same ligands B7-1 and B7-2. Structural studies suggested that CTLA-4 is capable of forming an extended oligomeric structure, which led to the hypothesis that formation of an extended lattice is necessary for CTLA-4 function but not for CD28. Further biophysical studies using FRET on CTLA-4 and heterodimeric mutants are currently being employed to test this mechanism. Subsequently we have investigated additional members of the CD28 and B7 receptor families, most notably co inhibitory proteins PD-1 and PD-L1/2. Mutagenesis studies guided by the crystal structure of PD-1 allowed us to generate mutants which either have enhanced or disrupted binding to both the PD-L1 and PD-L2 ligands and more interestingly one mutant that differentially affects ligand binding. Studies with the Nosanchuk lab demonstrated that in the fungal disease Histoplasmosis, the PD-1 pathway is up regulated in order to evade the immune response. Anti PD-1 antibodies reverse the defect in immunity. The mutant PD-1 proteins provide not only reagents to elucidate the signaling pathway but also as a potential therapeutic. We have recently determined the structures of a number of non-CD28/B7 members of the immunoglobulin and TNF superfamilies with costimulatory activities that have been localized or been postulated to function within the Immunological Synapse. The (SLAM) family includes homophilic and heterophilic receptors that modulate both adaptive and innate immune

STANLEY G. NATHENSON, M.D. Distinguished Professor CHANIN BLDG. – ROOM 40 718 430-2226

[email protected]

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responses. These receptors share a common ectodomain organization: a membrane proximal IgC domain and a membrane distal IgV domain that is responsible for ligand recognition. We found that three members of the family, NTB-A. CD84, and LY-9 self-associate with a Kd in the nanomolar to sub-micromolar range. These data, in combination with previous reports, demonstrate that the SLAM family homophilic affinities span at least three orders of magnitude, and suggest that differences in the affinities may contribute to the distinct signaling behavior exhibited by the individual family members. These structural data also suggest that, like NTB-A, all SLAM family homophilic dimers adopt a highly kinked organization spanning an end-to-end distance of ~140 Å and are of particular interest in that this kinked geometry can be accommodated by either formation of the same cell (cis) or opposing cells (trans). We believe that the change from cis to trans binding can act as a biological switch to prevent signaling from occurring outside a fully formed immunological synapse. The Tim family of receptors regulates effector CD4+ T cell functions, and polymorphisms are implicated in autoimmune and allergic diseases. The ectodomains of Tim family members are composed of a membrane distal IgV domain, which represents the ligand recognition domain, and membrane proximal mucin domains. Examination of the sequence identified four cysteines, which are invariant within the Tim receptor family and structural studies demonstrate they form two non-canonical disulfide bonds that support formation of a surface (“cleft”) not present in other IgSF superfamily members. Binding and mutagenesis studies demonstrate that this unique structural feature mediates a previously unidentified galectin-9-independent binding site. Biochemical and biophysical studies indicate that the unique structural features observed in Tim-3 are conserved among the entire Tim family. GITRL (Glucocorticoid-induced TNF receptor) ligand, a recently identified member of the TNF superfamily, binds to its receptor GITR on both effector and regulatory T cells and generates positive costimulatory signals implicated in a wide range of T cell functions. Structural studies demonstrate that hGITRL ectodomain self-assembles into a homotrimer that differs from conventional TNF family members by adopting an atypical "open flower-like" assembly and, due too the decreases of interacting residues, hGITRL exhibits a relatively weak tendency to trimerize. Such a unique assembly behavior has direct implications for hGITRL:GITR signaling, as this dynamic equilibrium appears to reduce the strength of the signal transduced through the hGITRL:GITR pathway to a biologically optimal level, as opposed to the maximal achievable level. As more components of the immunological synapse are discovered we will expand our program to not only look at individual receptor proteins but how the network of coreceptors within the immunological synapse interact to ultimately control immunity. References: Cao, E., Ramagopal, U., Federov, A., Fedorov, L., Yan, Q., Lary, J., Cole, J., Nathenson,

S.G., and Almo, S.C. NTB-A crystal structure: homophilic interactions and signaling by the SLAM family of receptors, Immunity, 2006, 25:559-570.

Chattopadhyay, K., Bhatia, S., Fiser, A., Almo, S.C. and Nathenson, S.G. Structural basis of ICOSL costimulatory function: determination of the cell surface oligomeric state and

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functional mapping of the receptor binding site of the protein. 2006, J. Immunology, 2006, 177:3920-3929.

Cao, E., Zang, X., Ramagopal, Mukhopadhaya, Federov, E., Zencheck, W.D., Lary, J.W., Cole, J.L., Deng, H., Xiao, H., DiLorenzo, T.P., Allison, J.P., Nathenson, S.G. and Almo, S.C. (2007) T cell immunoglobulin mucin-3 crystal structure reveals a novel ligand binding surface, Immunity, 26(3):311-321.

Chattopadhyay, K., U.A. Ramagopal, A. Mukhopadhaya, V.N. Malashkevich, T.P. DiLorenzo, M. Brenowitz, S.G. Nathenson, and S.C. Almo Assembly and structural properties of glucocorticoid-induced TNF receptor ligand: Implications for function, 2007, Proc. Nat’l Acad. of Sci., 104 (49): 19452-19457.

Yan, Q., Malashkevich, V.N., Federov, A., Federov, E., Cao, E., Lary, J.W., Cole, J.L., Nathenson, S.G. and Almo, S.C. The structure of CD84 provides insight into SLAM family function. 2007, Proc. Nat’l Acad. Sci., 104(25): 10583-10588.

Chattopadhyay, K., Ramagopal, U.A., Brenowitz, M., Nathenson, S.G. and Almo, S.C. Evolution of GITRL immune function: Murine GITRL exhibits unique structural and biochemical properties within the TNF superfamily, 2008, Proc. Nat’l. Acad. Sci., 105:635-640.

Lazar-Molnar, E., A. Gacser, G.J. Freeman, S.C. Almo, S.G. Nathenson, and J.D. Nosanchuk, The PD-l/PD-L costimulatory pathway critically affects host resistance to the pathogenic fungus Histoplasma capsulatum, 2008, Proc. Nat’l Acad.of Sci., 105:2658-63.

Chattopadhyay K, Lazar-Molnar E, Yan Q, Rubinstein R, Zhan C, Bonanno J, Vigdorovich V, Ramagopal UA, Nathenson SG, Almo SC: Sequence, Structure, Function, Immunity: Structural Genomics of Costimulation. 2009, Immunological Reviews 229(1):356-86.

Lazar-Molnar, E., Chen, B., Sweeney, K.A., Wang, E.J., Liu, W., Porcelli, S.A., Almo, S.C., Nathenson, S.G., Jacobs, W.R. Jr., Programmed death-1 (PD-1) deficient mice are extraordinarily sensitive to tuberculosis, 2010, Proc. Nat’l Acad of Sci., PMID: 20624978, 107: 13402-13407.

Samanta, D., Mukherjee, G., Ramagopal, U.A., Chaparro, R.J., Nathenson, S.G., DiLorenzo, T.P., Almo, S.C., Structural and functional characterization of a single-chain peptide-MHC molecule that modulates both naive and activated CD8+ T cells, 2011, Proc. Nat’l. Acad. Sci., In press.

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Mechanisms of RNA Processing Our laboratory is interested in mechanisms by which the spliceosome assembles and catalyzes two related chemical reactions. Recently, we formulated a general model of spliceosome function based on concepts of thermodynamic equilibrium, which impacts our understanding of substrate selectivity and provides general insights into RNP machines whose functions rely on dynamic rearrangements. Background. Intron removal, an essential maturation step for eukaryotic pre-mRNAs and a control point for regulation, is catalyzed by the spliceosome, a 50-60S complex composed of five snRNAs and >100 proteins. The spliceosome is both compositionally and conformationally dynamic. Each transition along the splicing pathway presents an opportunity for progression, pausing or discard, allowing splice site choice to be regulated throughout both the assembly and catalytic phases of the reaction. Orthogonal systems for in vivo investigation of catalytic center interactions during pre-mRNA splicing. During pre-mRNA splicing, the branch site (BS) base pairs with a universally conserved sequence in U2 snRNA; this interaction is essential for both spliceosome assembly and splicing catalysis. Detailed investigation of the behavior/dynamics of the BS-U2 duplex has been limited by the highly deleterious nature of U2 mutations that disrupt BS-U2 pairing. Thus, we developed an orthogonal system in which a dedicated second copy of U2 with a grossly substituted BS-binding sequence mediates the splicing of a cognate reporter gene. This orthogonal BS-U2 pair produces a non-essential second spliceosome that allows in vivo characterization of the BS-U2 helix, positioning of the first-step nucleophile, and its interaction with the spliceosome core, with few constraints. The introduction of further complementary sets of mutations will allow our present system to form the basis of a truly orthogonal spliceosome and allow for further mechanistic investigation. How Can Catalytic Activity Be Modulated? Many splice/branch sites (MOST in mammals!) are not optimal sequences. How can they be used for splicing? We have proposed a two-state model of the catalytic spliceosome, in which conformations of the complex supporting the two catalytic steps are in kinetic competition: modulation of the stability of the first and second step conformations results in improvement of one catalytic step to the detriment of the other. This model explains how a large number of spliceosomal mutants can suppress defects due to a large number of intron mutations. This mechanism resembles tRNA miscoding caused by altered equilibrium between open/closed ribosomal conformations; such mechanistic commonality suggests that alteration of rearrangements represents an evolutionarily convenient way of modulating substrate selectivity. Similar modulation of conformational states

Charles Query, M.D., Ph.D. Professor CHANIN BLDG. – ROOMS 440 & 415B 718 430-4174 [email protected]

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may explain the ability of mammalian spliceosomes to act on the typically poor splice sites of alternatively spliced introns. In addition, this model poses a large number of testable predictions concerning crucial mechanistic aspects of splicing. Recent Publications Moldón, A., and Query, C.C. (2010). Crossing the exon. Molecular Cell 38, 159–161. Smith, D.J., Konarska, M.M., and Query, C.C. (2009). Insights into branch nucleophile

positioning and activation from an orthogonal pre-mRNA splicing system in yeast. Molecular Cell 34, 333–343.

Smith, D., Query, C.C., and Konarska, M.M. (2008). Nought may endure but mutability:

Spliceosome Dynamics and the Regulation of Splicing. Molecular Cell 30, 657–666. Xu, Y.-Z. and Query, C.C. (2007). Competition between the ATPase Prp5 and branch region–

U2 snRNA pairing modulates fidelity of spliceosome assembly. Molecular Cell 28, 838–849.

Liu, L., Query, C.C., and Konarska, M.M. (2007). Opposing classes of prp8 alleles modulate

the transition between the catalytic steps of pre-mRNA splicing. Nature Struct. Mol. Biol. 14, 519–526.

Konarska, M.M., Vilardell, J. and Query, C.C. (2006). Repositioning of the reaction

intermediate within the catalytic center of the spliceosome. Molecular Cell 21, 543–553.

Our laboratory is studying how antibody-forming cells respond to antigen by undergoing somatic hypermutation and class switch recombination so that they can produce higher affinity antibodies with more useful effector functions. The molecular and biochemical mechanisms of antibody variable region hypermutation and class switch recombination is being studied in mice that have mutations in various repair proteins in collaboration with Dr. Winfried Edelmann. In order to examine detailed molecular mechanisms, we are also studying how mutation is targeted to antibody genes and some oncogenes in human Burkitt’s lymphoma cell lines which are undergoing variable region mutation in culture. These cell lines and genetically defective mice are being used to study the role of activation induced deaminase (AID), mismatch repair and error prone polymerases in the variable region hypermutation and isotype switching. The analysis of these events also involves the examination of AID activity biochemically and, in collaboration with Drs, Aviv Bergman and Thomas MacCarthy, computationally to analyze and simulate the details of the mutational activity that leads to the generation of antibody diversity.

The highly mutagenic processes required to generate antibody diversity also leads to B cell lymphomas so we are trying to understand how AID causes mouse B cell lymphomas and human Chornic Lymphocytic Leukemia (in collaboration with Dr. Nicholas Choirazzi) and how mismatch repair protects B cells from undergoing malignant transformation while also contributing to the generation of antibody diversity.

We are also using somatic mutation and isotype switching to generate better monoclonal antibodies that will protect the host from lethal toxins and emerging infections. Monoclonal antibodies to such toxins and viruses are generated and the impact of affinity and isotype are determined in vitro and in vivo. Selected References: 1. The Biochemistry of Somatic Hypermutation. Peled, J.U., F. L. M. D. Kuang, Iglesias-Ussel, S. Roa, S.L. Kalis, M.F. Goodman and M.D. Scharff. Annual Reviews of Immunology. 26:481-511, 2008 2. Ubiquitylated PCNA plays a role in somatic hypermutation and class-switch recombination and is required for meiotic progression.Roa S, Avdievich E, Peled JU, Maccarthy T, Werling U, Kuang FL, Kan R, Zhao C, Bergman A, Cohen PE, Edelmann W, Scharff MD. Proc Natl Acad Sci U S A. 105:16248-53. 2008 3. H3 tri-methyl K9 and H3 acetyl K9 chromatin modifications are associated with Class Switch Recombination. Fei Li Kuang, Zhonghui Luo, Matthew D. Scharff, Proc Natl Acad Sci USA 106:5288-93, 2008 4. V-region mutation in vitro, in vivo and in silico reveal the importance of the enzymatic properties of AID and the sequence environment. Thomas MacCarthy, Susan Kalis, Sergio Roa, Phuong Pham, Myron F. Goodman, Matthew D. Scharff, and Aviv Bergman. Proc Natl Acad Sci USA 106:8629-34. 2009 5. MolecThe RNF8/RNF168 ubiquitin ligase cascade facilitates class switch recombination. Ramachandran S, Chahwan R, Nepal RM, Frieder D, Panier S, Roa S, Zaheen A, Durocher D, Scharff MD, Martin A.Proc Natl Acad Sci U S A. 107:809-14. 2010 (PMID: 20080757) 6. MSH2/MSH6 complex promotes error-free repair of AID-induced dU:G mispairs as well as error-prone hypermutation of A:T sites. Roa S, Li Z, Peled JU, Zhao C, Edelmann W, Scharff MD. PLoS One. 2010 Jun 17;5(6):e11182. PMID: 20567595 7. Msh6 protects mature B cells from lymphoma by preserving genomic stability. Peled JU, Sellers RS, Iglesias-Ussel MD, Shin DM, Montagna C, Zhao C, Li Z, Edelmann W, Morse HC 3rd, Scharff MD. Am J Pathol. 177(5):2597-608, 2010

MATTHEW D. SCHARFF, M.D. Professor

CHANIN BLDG. – ROOM 403 718 430-35277

[email protected]

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INDIVIDUAL FLUORESCENT DNA MOLECULES

AFTER REPLICATION IN THE RED TO GREEN DIRECTION

MOLECULAR GENETICS OF NUCLEAR REPROGRAMMING OF THE MAMMALIAN GENOME

Our laboratory is a part of the Einstein Center for Human Embryonic Stem Cell Research. Long term interests: ♦ Regulation and reprogramming of DNA replication during human embryogenesis and in human embryonic stem (ES) cells and induced pluripotent stem cells (iPS). ♦ Understanding how replication initiation sites affect early steps in human embryonic development. ♦ Replication of chromosome telomeres Current projects include a wide range of interests: ♦ Study of human ES cell DNA replication dynamics. Thorough understanding of replication programs to advance the availability of immunologically compatible hES cells for patients. ♦ Role of changes in the DNA replication program during embryonic development. The identification of factors that drive changes in the replication program essential for nuclear programming will provide a better understanding of contribution of this process to embryonic differentiation. ♦ Reprogramming and regulation of DNA replication in human and mouse ES and iPS cells. How the replication program changes during the conversion of somatic cells to iPS cells when ES cells differentiate along various developmental pathways.

♦ The relationship between telomere dynamics and the replication of these essential protective structures. Telomeres shorten during replication which results in cellular senescence. Do variations in telomere length and associated proteins affect telomere replication? How do ES cells manage to deal with these issues?

♦ The importance of the protein components of the telomere-protective shelterin complex during telomere replication.

SELECTED PUBLICATIONS: Schultz, S. S., Desbordes, S. C., Du, Z., Kosiyatrakul, S., Lipchina,, I., Studer, L. and Schildkraut, C. L. Single Molecule Analysis Reveals Changes in the DNA Replication Program for the POU5F1 Locus Upon hESC Differentiation. Mol. Cell. Biol., In Press. (2010). Sfeir, A., Kosiyatrakul, S. T., Hockemeyer, D., MacRae, S. L., Karlseder, J., Schildkraut, C.L., and De Lange, T., Mammalian telomeres resemble fragile sites and require TRF1 for efficient replication. Cell, 138: 90-103. (2009).

CARL SCHILDKRAUT, Ph.D. Professor

CHANIN BLDG. – Room 416 (718) 430-2097

[email protected]

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Desprat, R., Thierry-Mieg, D., Lailler, N., Schildkraut, C. L., Thierry-Mieg, J., and Bouhassira, E. E. Predictable Dynamic Program of Timing of DNA Replication in Human Cells. Genome Research. Genome Res. 19:2288-2299 (2009). Guan, Z., Hughes, C. M., Kosiyatrakul, S. T., Norio, P., Sen, R., Fiering, S., Allis, C. D Bouhassira, E. E., and Schildkraut, C. L. Decreased Replication Origin Activity in Temporal Transition Regions. Journal of Cell Biology. 187:623-35 (2009). Norio, P., Kosiyatrakul, S.,Yang, Q., Guan, Z.,Brown, N., Thomas, S., Riblet, R., and Schildkraut, C.L. Progressive activation of DNA replication initiation in large domains of the immunoglobulin heavy chain locus during B cell development. Mol. Cell 20: 575 - 587(2005). Norio P, Schildkraut C.L. Plasticity of DNA replication initiation in Epstein-Barr virus episomes. PLoS Biol 2: 816 - 833. (2004). Norio, P., Schildkraut, C.L. Visualization of DNA Replication on Individual Epstein-Barr Virus Episomes. Science 294: 2361-2364 (2001).

Liver Stem/Progenitor Cells for Transplantation and Gene Therapy

Our research has focused on regulation of liver gene expression, cell growth control, liver regeneration and liver reconstitution thru cell transplantation. Currently, there are four major projects: 1) Isolation and characterization of liver stem cells, 2) Repopulation of the liver by transplantation of fetal liver stem cells and identifying genes that regulate this process by a mechanism of cell competition, 3) Identifying genes that increase liver replacement in aging rats by augmenting cell competition and 4) Treatment of inherited metabolic disorders by ex vivo gene therapy, using stem cells and re-engineering hepatocytes with augmented proliferative activity to more effectively repopulate the liver. A number of years ago, we developed a cell transplantation system to follow the proliferation, lineage fate and repopulation capacity of liver stem/progenitor cells, using a marker gene, dipeptidyl peptidase IV (DPPIV). This cell transplantation system has also been used to identify stem cells in the fetal liver that are bipotent, proliferate extensively for up to one year after they have been transplanted, differentiate into both hepatocytes and bile duct cells, form completely new liver lobules and replace 25%-30% of hepatic mass in normal adult rats. Liver replacement occurs by cell competition, a mechanism originally described in Drosophila during embryonic wing development more than 30 yrs ago. We are currently conducting laser capture microdissection studies in conjunction with microarray analysis to identify the specific genes that regulate cell competition in liver repopulation, compared to those genes regulating cell competition in Drosophila. Liver regenerative capacity decreases with aging and we have recently discovered that tissue levels of activin A increase progressively as the liver ages. Activin A induces expression of the cellular senescence gene, p15INK4b, in adult hepatocytes, whereas fetal liver stem/progenitor cells are resistant to activin A signaling because they lack expression of activin A receptors. This creates a tissue microenvironment in which there is increased cell competition between fetal liver stem/progenitor cells and host hepatocytes, leading to augmented liver repopulation by fetal liver stem/progenitor cells in the aging liver. Finally, we have purified both rat and mouse fetal liver stem/progenitor cells and by microarray analysis are defining unique genes expressed by these cells that control their proliferative potential. In other studies, we have transplanted human cord blood stem cells into NOD/Scid mice with conversion of some of these cells into hepatocytes. We are also studying the ability of human and mouse embryonic stem (ES) cells to differentiate into hepatocytes and repopulate the liver and are planning to study liver repopulation by transplanted iPS cells. We will use laser capture microdissection to define the gene expression program of differentiating ES cells by Q-PCR and microarray analysis. Finally, we have transduced fetal liver stem/progenitor cells and mature hepatocytes with lentiviruses and are characterizing this transduction system in conjunction with cell transplantation as a method for ex vivo gene therapy in rodent models of Wilson's disease and α1-antitrypsin deficiency, as well as to develop genetically re-engineered hepatocytes with increased competitive potential for liver repopulation. References:

DAVID A. SHAFRITZ, M.D. Professor

ULLMANN BLDG. – ROOM 603 718 430-2098

[email protected]

Laconi, E., Oren, R., Mukhopadhyay, D., Hurston, E., Laconi, S., Pani, P., Dabeva, M.D., and Shafritz, D.A. (1998) Long term, near total liver replacement by transplantation of isolated hepatocytes. Am. J. Pathol. 153:319-329.

Kollet, O., Shivtiel, S., Chen, Y.-Q., Suriawinata, J., Thung, S.N., Dabeva, M.D., Kahn, J., Spiegel, A. , Dar, A., Samira, S., Goichberg, P., Kalinkovich,A., Arenzana-Seisdedos, F., Nagler, A., Hardan, I., Revel, M., Shafritz, D.A. and Lapidot, T. (2003) HGF, SDF-1 and MMP-9 are involved in stress-induced human CD34+ stem cell recruitment to the liver. J. Clin. Invest. 112:160-169.

Nierhoff, D., Ogawa, A., Oertel, M., Chen, Y.-Q., and Shafritz, D.A. (2005) Purification and characterization of mouse fetal liver epithelial cells with high in vivo repopulation capacity. Hepatology 42:130-139.

Oertel, M., Menthena, A., Dabeva, M.D., and Shafritz, D.A. (2006) Cell competition leads to a high level of normal liver reconstitution by transplanted fetal liver stem/progenitor cells. Gastroenterology 130:507-520.

Gouon-Evans, V., Boussemart, L., Gadue, P., Nierhoff, D., Koehler, C.I., Kubo, A., Shafritz, D.A., and Keller. G. (2006) BMP-4 is required for hepatic specification of mouse embryonic stem cell-derived definitive endoderm. Nature Biotechnology 24:1402-1411.

Nierhoff, D., Levoci, L., Schulte, S., Goeser, T., Rogler, L.E. and Shafritz,D.A. (2007) New cell surface markers for murine fetal hepatic stem cells identified through high density cDNA microarrays. Hepatology 46:535-547.

Shafritz, D.A. (2007) A human hepatocyte factory. Nature Biotechology, News & Views (Editorial) 25:871-872.

Oertel, M., Menthena, A., Chen, Y.Q., Teisner, B., Harken-Jensen, C., and Shafritz, D.A. (2008) Purification of fetal liver stem/progenitor cells containing all the repopulation potential for normal adult rat liver. Gastroenterology 134:823-832.

Shafritz, D.A. and Oertel, M. (2011) Model systems and experimental conditions that lead to effective repopulation of the liver by transplanted cells, Int J Biochem. & Cell Biol, 43:198-213

Menthena, A., Koehler, C.I., Sandhu, J.S., Yovchev, M.I., Hurston, E., Shafritz, D.A. and Oertel, M (2011) Activin A, p15INK4b Signaling, and Cell Competition Promote Stem/Progenitor Cell Repopulation of Livers in Aging Rats. Gastroenterology 140:1009-1020.

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Our work is focussed on the travels of RNA within the cell: from the site of its birth to its ultimate biological destiny in the cytoplasm where it makes proteins in specific locations. All we have learned results from the development of new technology, known as in situ hybridization, to visualize specific nucleic acid sequences within individual cells. Using our approach, synthetic nucleic acid probes are labeled with a variety of detectors such as fluorochromes or antigens. Subsequently these molecules are hybridized to the cell and detected using high resolution digital imaging microscopy. This enables the detection of specific nucleic acid molecules within the structural context of the cell. We have developed imaging algorithms capable of detecting a single RNA molecule within a cell. As a result of this approach, we have found that specific RNA sequences are located in particular cellular compartments. An example is the messenger RNA for beta-actin which is located in the periphery of the cell where actin protein is needed for cell motility. These transcripts are not free to diffuse. The transcripts may be associated with a cellular matrix or skeleton from the moment of their synthesis through translation. We are investigating how this spatial information is encoded within the gene and how the RNA transcript is processed within the nucleus and then transported to its correct compartment in the cytoplasm resulting in asymmetric protein distribution. We have recently discovered that RNA localization also occurs in yeast. During budding, a nuclear factor represses mating type switching asymmetrically, only in the daughter cell. This is because the factor is synthesized only in the bud because the mRNA was transported there by an actomyosin system. This discovery allows us to investigate the genetic mechanism responsible for this RNA's travels. In addition, we have constructed genetically altered yeast and vertebrate cells carrying chimeric genes modified by recombinant DNA techniques to elucidate the sequences responsible for mRNA localization. A reporter gene can be "delivered" to a variety of cellular compartments by using specific sequences, or "zipcodes", from the mRNAs found in those compartments. These "zipcodes" consist of short sequences in the 3' untranslated region of the mRNA that control translation and localization. By tagging the RNA with a ligand that binds GFP, we can see fluorescent RNA in living cells moving from its site of transcription to its site of localization. New technology has allowed us to interrogate many sites of transcription to determine the pattern of expression of many genes simultaneously and describe this mathematically. Selected References: Larson DR, Zenklusen D, Wu B, Chao JA, Singer RH, (2011) Real-Time Observation of

Transcription Initiation and Elongation on an Endogenous Yeast Gene. Science 332:475-478.

Grünwald D, Singer RH, (2010) In vivo imaging of labelled endogenous β-actin mRNA during nucleocytoplasmic transport. Nature 467:604-607.

Chao JA, Patskovsky Y, Patel V, Levy M, Almo SC, Singer RH, (2010) ZBP1 recognition of β-actin zipcode induces RNA looping. Genes Dev 24:148-158.

Huttelmaier S, Zenklusen D, Lederer M, Dictenberg J, Lorenz M, Meng X, Bassell GJ, Condeelis J, Singer RH, (2005) Spatial regulation of beta-actin translation by Src-dependent phosphorylation of ZBP1. Nature 438:512-515.

Shav-Tal Y, Darzacq X, Shenoy SM, Fusco D, Janicki SM, Spector DL, Singer RH (2004) Dynamics of single mRNPs in nuclei of living cells. Science 304:1797-1800.

Robert H. Singer, Ph.D. www.singerlab.org Professor and Co-Chair Golding Building, Room 601 Department of Anatomy & Structural Biology (718) 430-8646 [email protected]

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Janicki SM, Tsukamoto T, Salghetti SE, Tansey WP, Sachidanandam R, Prasanth KV, Ried T, Shav-Tal Y, Bertrand E, Singer RH, Spector DL. (2004) From silencing to gene expression: Real-time analysis in single cells. Cell 116:683-698.

Levsky JM, Shenoy SM, Pezo RC, Singer RH. (2002) Single-cell gene expression pro-filing. Science 297: 836-840.

Singer RH. (1998) Triplet-repeat transcripts: A role for RNA in disease. Science 280:696-697. Femino AM, Fay FS, Fogarty K, Singer RH. (1998) Visualization of single RNA transcripts in

situ. Science (Cover Article) 280:585-590. Chicurel ME, Singer RH, Meyer CJ, and Ingber DE. (1998) Integrin binding and mechanical

tension induce movement of mRNA and ribosomes to focal adhesions. Nature 392:730-733.

Bertrand E, Chartrand P, Schaefer M, Shenoy SM, Singer RH, Long RM. (1998) Localization of ASH1 mRNA particles in living yeast. Mol Cell 2:437.

Long RM, Singer RH, Meng X, Gonzalez I, Nasmyth K, Jansen, RP (1997) Mating type switching in yeast controlled by asymmetric localization of ASH1 mRNA. Science 277:383-38.

Key Words: Leukemia, proliferation, differentiation, chromatin, transcription

Our laboratory is interested in understanding the mechanisms controlling mammalian development and cell differentiation. Our approach is to investigate systems in which these processes are disturbed, either by malignant transformation (leukemia) or by directed gene inactivation in mice and Drosophila. Currently there are three major projects underway in the lab. Molecular Mechanisms of Leukemia: In this project we are investigating the molecular mechanisms for a block to differentiation present in blood cell tumors (leukemias). We have traced the cause of the differentiation block to a transcription factor called PU.1. We are trying to learn how dysregulation of PU.1 expression causes the leukemia cells to stop differentiating and start proliferating in an uncontrolled manner by studying the effect of PU.1 on other gene products, including, other transcription factors like GATA-1 and co-factors like RB that promote differentiation, and cyclins, cyclin-dependent kinases (cdks) and cdk inhibitors that promote proliferation. This project includes genome-wide approaches involving chromatin immunoprecipitation and high throughput sequencing (ChIP-Seq) and gene expression profiling with microarrays. Role of H1 Linker Histones and Chromatin Remodeling Factors in Chromatin Structure, DNA Methylation, Gene Expression and Development in Mice and Drosophila. Recent studies show that posttranslational modifications of core histones (H2A, H2B, H3, H4) (the Histone Code) play a very important role in control of gene expression. The H1 linker histones are more diverse than the core histones. Mice contain 8 H1 histone subtypes including differentiation-specific and tissue-specific subtypes, whereas Drosophila has only one type of H1. H1’s are thought to be responsible for the final level of packaging DNA into the compact chromatin structure but we know very little about their role in gene expression and development. We are studying the functional roles of H1 linker histones by inactivating (knocking-out) specific H1 genes in mice and the single H1 in Drosophila. We are also reintroducing mutant H1 linker histones into H1 depleted mouse cells and flies, to perform structure-function studies. We have also established a new connection between H1 histones, DNA methylation and genomic imprinting and we are learning how H1 regulates DNA methylation. We also have a new knock-out mouse for the SNF2H chromatin remolding ATPase, which assembles H1 histone into chromatin. Human ES Cell Proliferation and Totipotency. Continuous cell proliferation is required to maintain human embryonic stem cell totipotency. We have found that certain central regulators of the cell cycle also control differentiation decisions in the hematopoietic system. We are investigating which cell cycle regulators control human ES cell proliferation and whether these molecules also control their totipotency. Selected Recent Publications: Wontakal S.N., Guo X., Will B., Shi M., Raha D., Mahajan M.C., Weissman S., Snyder M., Steidl U., Zheng D., and A.I. Skoultchi. (2011). A Large Gene Network in Immature Erythroid Cells Is Controlled by the Myeloid and B Cell Transcriptional Regulator PU.1. PLoS Genet 7(6), e1001392. Maclean, J.A., et al. (2011). The rhox homeobox gene cluster is imprinted and selectively targeted for regulation by histone h1 and DNA methylation. Mol Cell Biol 31,1275-1287. Choe, K. S., O. Ujhelly, S. N. Wontakal, and A.I. Skoultchi (2010). PU.1 directly regulates CDK6 gene expression, linking the cell proliferation and differentiation programs in erythroid cells. J. Biol. Chem. 285(5):3044-3052 (Epub December 2, 2009).

DR. ARTHUR I. SKOULTCHI, Ph.D. Professor & Chairman CHANIN CANCER CENTER

ROOM 402 718-430-2169

[email protected]

Dr. Skoultchi - Selected Recent Publications: (Continued from page 1)

Papetti, M., S. N. Wontakal, T. Stopka, and A.I. Skoultchi (2010). GATA-1 directly regulates p21 gene expression during erythroid differentiation. Cell Cycle 9(10):1972-1980. (Epub May 18, 2010). Lu, X., S. N. Wontakal, A. V. Emelyanov, P. Morcillo, A. Y. Konev, D. V. Fyodorov, and A. I. Skoultchi. (2009). Linker histone H1 is essential for Drosophila development, the establishment of pericentric heterochromatin, and a normal polytene chromosome structure. Genes Dev 23:452-465. Nishiyama, M., et al. (2009). CHD8 suppresses p53-mediated apoptosis through histone H1 recruitment during early embryogenesis. Nat Cell Biol 11:172-182. Chong, S., et al. (2007). Modifiers of epigenetic reprogramming display paternal effects in the mouse. Nature Genetics 39(5):614-622. Murga, M., et al. (2007) Global chromatin compaction limits the strength of the DNA damage response. J Cell Biol 178:1101-1108. Papetti, M and A. I. Skoultchi (2007). Reprogramming Leukemia Cells to Terminal Differentiation and Growth Arrest by RNA Interference of PU.1. Mol. Cancer Res. 5:1053-1062. Fan, Y., Nikitina, T., Zhao, J., Fleury, T.J., Bhattacharyya, R., Bouhassira, E.E., Stein, A., Woodcock, C.L., and Skoultchi, A.I. (2005). Histone H1 depletion in mammals alters global chromatin structure but causes specific changes in gene regulation. Cell 123:1199-1212. Stopka, T., D. F. Amanatullah, M. Papetti, and A. I. Skoultchi. (2005). PU.1 inhibits the erythroid program by binding to GATA-1 on DNA and creating a repressive chromatin structure. Embo J 24:3712-3723. Lang, D., et al. (2005). Pax3 functions at a nodal point in melanocyte stem cell differentiation. Nature 433:884-887. Lin, Q., Inselman, A., Han, X., Xu, H., Zhang, W., Handel, M.A., and Skoultchi, A.I. (2004). Reductions in linker histone levels are tolerated in developing spermatocytes but cause changes in specific gene expression. J Biol Chem 279:23525-23535. Choe, K.S., Radparvar, F., Matushansky, I., Rekhtman, N., Han, X., and Skoultchi, A.I. (2003). Reversal of tumorigenicity and the block to differentiation in erythroleukemia cells by GATA-1. Cancer Res 63:6363-6369. Rekhtman, N., Choe, K.S., Matushansky, I., Murray, S., Stopka, T., and Skoultchi, A.I. (2003). PU.1 and pRB interact and cooperate to repress GATA-1 and block erythroid differentiation. Mol Cell Biol 23:7460-7474. Matushansky, I., Radparvar, F., and Skoultchi, A.I. (2003). CDK6 blocks differentiation: coupling cell proliferation to the block to differentiation in leukemic cells. Oncogene 22:4143-4149. Konishi A., et al. (2003). Involvement of histone H1.2 in apoptosis induced by DNA double-strand breaks. Cell 114(6):673-688 Fan, Y., Nikitina, T., Morin-Kensicki, E.M., Zhao, J., Magnuson, T.R., Woodcock, C.L., and Skoultchi, A.I. (2003). H1 linker histones are essential for mouse development and affect nucleosome spacing in vivo. Mol Cell Biol 23:4559-4572.

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Glycan Functions in Development, Cancer and Notch Signaling

Glycosylation is the most abundant and varied post-translational modification of proteins and is a critical factor in regulating their biological functions. The complement of glycans that may be produced by an organism is called the glycome. Changes in glycans expressed on the cell surface occur during development and transformation into a cancer cell. Cell surface changes that promote metastasis of cancer cells are also correlated with the appearance or disappearance of particular sugar residues on glycoproteins. Specific glycans on the Notch receptor modulate signal transduction by Notch ligands. This is a novel paradigm of signal transduction whereby the transfer of a single type of sugar residue alters the ability of Notch receptors to signal. We are using CHO cell glycosylation mutants, Notch signaling assays, glycosyltransferase gene knockout mice, and biochemical approaches including MALDI-TOF mass spectrometry, to identify biological functions of growth factor receptor and Notch glycans, and the underlying mechanisms by which glycans mediate biological events. Notch receptors span the cell membrane. When a Notch ligand like Delta or Jagged on an apposing cell binds to Notch receptor, it induces cleavage of Notch extracellular domain, followed by a second cleavage that releases Notch intracellular domain. The Notch intracellular domain goes to the nucleus and activates target genes that ultimately lead to a change in cell fate or cell growth control. Using a CHO glycosylation mutant that adds few O-fucose glycans to Notch extracellular domain, we showed that Notch signaling is markedly reduced when fucose is limiting. Using a panel of different CHO glycosylation mutants developed in this lab, we showed that inhibition of Notch signaling by the fringe glycosyltransferase requires the addition of a Gal residue to O-fucose glycans on Notch. We are continuing to use Notch signaling assays to define the mechanisms of action of Fringe and other glycosyltransferases that modulate Notch signaling. We are also targeting glycosyltransferase genes that encode enzymes that modify Notch in the mouse, and generating Notch mutants that cannot accept an O-fucose glycan at a specific site in Notch. Mice lacking O-fucose in the ligand binding domain have defective T cell development and are being investigated for other immunological defects. We have shown that mice lacking a single type of sugar, the bisecting GlcNAc, on complex N-glycans exhibit enhanced progression of mammary tumors. When the bisecting GlcNAc is overexpressed in developing mammary glands, tumor development is inhibited. We are currently investigating the mechanism by which the bisecting GlcNAc modulates growth factor signaling using isolated mammary tumor epithelial cells. We recently discovered a novel inhibitor of complex N-glycan synthesis that is expressed mainly in testicular germ cells in a highly regulated manner during spermatogenesis. Expression of this gene causes cells to bind strongly to Sertoli cells and we predict that it will be important for germ-Sertoli cell interactions necessary for spermatogenesis. We are testing this hypothesis by conditional deletion of the inhibitor in spermatogonia. We have also found that complex N-glycans are essential for male fertility and are testing the hypothesis that they play an important role in spermatid/Sertoli cell interactions.

Finally, Chinese hamster ovary (CHO) cell glycosylation mutants developed in this laboratory continue to be isolated and used as hosts to characterize orphan glycosyltransferases identified in

PAMELA STANLEY, Ph.D. Horace W. Goldsmith Professor CHANIN BLDG. – ROOM 516 (718) 430-3346 [email protected] http://stanxterm.aecom.yu.edu

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genome databases. The genome encodes ~300 glycosyltransferase genes, many conserved through evolution, and the reactions they catalyze are not known for a significant orphan subgroup. We also use the CHO mutants to develop new methods such as a novel approach to tracking glycan epitopes on the cell surface. Selected References Ge, C and Stanley, P. (2008) The O-fucose glycan in the Notch1 ligand binding domain regulates embryogenesis and T cell development, Proc. Natl. Acad. Sci. USA 105:1539-44. Ge, C., Liu, T., Hou, X. and Stanley, P. (2008) In vivo consequences of deleting EGF repeats 8-12 including the ligand bindng domain of mouse Notch1. BMC Dev. Biol., 8, 48. Stanley, P and Guidos, C. J. (2009) Regulation of Notch signaling during T- and B-cell development by O-fucose glycans. Immunol. Rev. 230: 201-215. Guilmeau S, Flandez M, Bancroft L, Sellers RS, Tear B, Stanley P, and Augenlicht LH.(2008) Intestinal deletion of Pofut1 in the mouse inactivates Notch signaling and causes enterocolitis. Gastroenterology, 135, 849-860. Aguilan, J. T., Sundaram, S., Nieves, E. and Stanley, P. (2009) Mutational and Functional Analysis of Large in a Novel CHO Glycosylation Mutant. Glycobiology, 19, 971-986. North, S. J., Huang, H-H., Sundaram, S., Jang-Lee, J., Etienne, A. T., Trollope, A., Al-Chalabi, S., Dell, A., Stanley P. and Haslam, S. M. (2009) Glycomics profiling of Chinese hamster ovary (CHO) cell glycosylation mutants reveals N-glycans of a novel size and complexity. J. Biol. Chem., 285, 5759-5775. Song Y, Aglipay JA, Bernstein RD, Goswami S and Stanley P. (2010) The Bisecting GlcNAc on N-Glycans Inhibits Growth Factor Signaling and Retards Mammary Tumor Progression. Cancer Research, 70, 3361-71. Ge, C. and Stanley, P. (2010) Effects of varying Notch1 signal strength on embryogenesis and vasculogenesis in compound mutant heterozygotes. BMC Dev Biol, 10, 36. Stanley, P. and Okajima, T. (2010) Roles of Glycosylation in Notch Signaling. Current Topics in Developmental Biology, 92,131-164. Huang, H-H. and Stanley, P. (2010) A testis-specific regulator of complex and hybrid N-glycan synthesis. J Cell Biol 190, 893-910 Zheng, T., Jiang, H., Gros, M., Soriano del Amo, D., Sundaram, S., Lauvau, G., Marlow, F., Liu, Y., Stanley, P. and Wu, P. (2011) Tracking N-acetyllactosamine on Cell Surface Glycans in vivo. Angewandte Chemie, 50: 4113-4118.

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Transcriptional and Epigenetic Regulation of Normal and Cancer Stem Cells in Hematopoiesis and Leukemia Hematopoiesis maintains a life-long supply of the entire spectrum of highly specialized hematopoietic cells dependent on varying systemic needs. It relies on a tightly regulated balance of self-renewal, differentiation, and migration of a small number of multipotent hematopoietic stem cells (HSC). Acute myeloid leukemias (AML) are malignant hematological diseases that originate from a single transformed cell which has progressively acquired critical genetic changes that disrupt these key growth-regulatory pathways. Despite the use and optimization of polychemotherapy and the development of new agents that are effective at reducing the tumor burden in patients with leukemia, relapse continues to be the most common cause of death in AML. Newer experimental evidence demonstrates that AML arises from a small population of cancer/leukemia stem cells (LSC). Similar to normal HSC, LSC are quiescent in terms of cell cycle and thus, conventional cytotoxic therapies are not effective against LSC in the majority of cases. However, therapeutic eradication of the LSC within the leukemia clone will be essential for a cure of disease. Therefore, an improved understanding of the molecular pathways governing the formation and maintenance of LSC is required for the development of therapies that target LSC rather than the bulk tumor cells (blasts). Recent findings from our own group and others demonstrate a critical role of transcriptional master regulators (e.g. PU.1) and chromatin-remodeling factors (e.g. SATB1) in the genesis and function of LSC in AML, and that transcription factors are already deregulated in the early stem cell compartment.

The goal of our research is to delineate critical mechanisms that drive leukemia stem cell (LSC) function, and to better understand the mechanisms of how transcriptional regulators (e.g. transcription factors and chromatin-remodeling factors) act in normal HSC and cause formation of LSC. To identify implicated pathways we are utilizing rigorously defined stem and progenitor cell subsets isolated by means of multi-parameter high-speed fluorescence-activated cell sorting (FACS). Identified target genes are biochemically and functionally tested. We utilize lentiviral gene transfer allowing for forced expression or shRNA-mediated knockdown, followed by in vitro as well as in vivo assays for stem and progenitor cell functions including murine transplantation models. This allows for assessing the function of candidate genes in normal and leukemia stem cells. We are studying murine genetic models as well as primary human samples of patients with leukemia. Our studies aim at providing the basis for development of targeted, LSC-directed therapies. Projects in the lab include: 1. Identification of LSC genes in AML/MDS by stem cell profiling and functional testing utilizing lentiviral gene restoration and xenotransplantation assays. 2. Study the mechanisms of how reduced levels of PU.1 contribute to genomic instability in stem and progenitor cells, which occurs in PU.1 hypomorphic leukemia in mice and humans. 3. Study the role of the chromatin-remodeling protein SATB1, that we have identified as a long-range transcriptional regulator of PU.1, by an inducible stem cell-specific double transgenic mouse model. 4. Study the role of transcriptional and epigenetic regulators in stem cells in the molecular pathogenesis of myeloid malignancies. 5. Testing of novel drugs/compounds in normal and leukemia stem cells in AML/MDS.

Ulrich Steidl, M.D., Ph.D. Assistant Professor Chanin Bldg – Room 606

(718) 430-3437 [email protected]

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Selected publications: MHC class II transactivator CIITA is a recurrent gene fusion partner in lymphoid cancers.

Steidl C, Shah SP, Woolcock BW, Rui L, Kawahara M, Farinha P, Johnson NA, Zhao Y, Telenius A, Neriah SB, McPherson A, Meissner B, Okoye UC, Diepstra A, van den Berg A, Sun M, Leung G, Jones SJ, Connors JM, Huntsman DG, Savage KJ, Rimsza LM, Horsman DE, Staudt LM, Steidl U, Marra MA, Gascoyne RD. Nature. 2011; 471:377-381

Will B, Steidl U. Multi-Parameter Fluorescence-Activated Cell Sorting and Analysis of Stem and

Progenitor Cells in Myeloid Malignancies. Best Pract Res Clin Haematol. 2010; 23:391-401 Will B, Kawahara M, Luciano JP, Bruns I, Parekh S, Erickson-Miller CL, Aivado MA, Verma A, Steidl

U. Effect of the non-peptide thrombopoietin receptor agonist Eltrombopag on bone marrow cells from patients with acute myeloid leukemia and myelodysplastic syndrome. Blood. 2009; 114:3899-908

Ebralidze A, Guibal F, Steidl U et al., PU.1 expression is modulated by the balance of functional

sense and antisense RNAs regulated by a shared cis-regulatory element. Genes Dev. 2008; 22:2085-92

Steidl U et al., A single nucleotide polymorphism alters long-range regulation of the PU.1 gene in

AML. J Clin Invest. 2007; 117:2611-2620 Steidl U et al., Essential role of Jun family transcription factors in PU.1-induced leukemic stem cells. Nat Genet. 2006; 38:1269-1277 Rosenbauer F, Owens B, Yu L, Tumang R, Steidl U et al., Lymphoid cell growth and transformation

are suppressed by a key regulatory element of the gene encoding PU.1. Nat Genet. 2006; 38:27-373

Rosenbauer F, Koschmieder S, Steidl U, Tenen DG. Effect of transcription factor concentrations on

leukemic stem cells. Blood. 2005; 106:1519-1524 Steidl U et al., Primary human CD34+ hematopoietic stem and progenitor cells express functionally

active receptors of neuromediators. Blood. 2004; 104:81-88

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The ribosome is a molecular machine composed of 4 RNA molecules and 80 different

proteins. The construction of a ribosome involves the integration of fundamental cellular processes: the transcription and processing of ribosomal RNA, the transcription, processing and translation of the mRNAs for ribosomal proteins, the assembly of the ribosomal proteins with the ribosomal RNAs, etc. In the yeast Saccharomyces cerevisiae, the synthesis of ribosomes consumes an extraordinary proportion of the cell's resources, accounting for >70% of all transcription, about 50% of all Pol II transcription initiation events, and >90% of all pre-mRNA splicing.

In mammalian cells the quantitative aspects of ribosome synthesis may be less substantial, though still important. However, mammalian cells monitor ribosome synthesis closely, and any evidence of problems with ribosome synthesis can bring on a process known as ‘nucleolar stress, that leads to accumulation of p53, with subsequent cell cycle stalling, or ultimately apoptosis. Naturally, such a response provides strong selection for suppression of the p53 response. Such selection is inherently carcinogenic, and aberrations in ribosomal genes, such as haploinsufficiency for a ribosomal protein gene, often leads to cancer. Ribosome Synthesis and Disease: On the basis of these considerations it is my contention that the massive amount of transcriptome data acquired from microarrays and modern sequencing has the potential to reveal far more about the role of aberrant ribosome synthesis in human disease. Therefore, we are learning and developing tools to mine such data, looking for unexpected relationships between the products of the 80 ribosomal protein genes, the 2000 ribosomal protein pseudogenes, the 75 mitochondrial ribosomal protein genes, and the ~200 genes encoding ribosome assembly factors in healthy and diseased cells. We will also be examining regulated alternative splicing of mRNAs that encode ribosomal proteins, SNPs that alter the amino acid sequence of ribosomal proteins, and both qualitative and quantitative variations in ribosomal protein genes as cells evolve into malignant cancer. Recent Publications: Vilardell, J., S.J. Yu, & J.R. Warner, Multiple Functions of an Evolutionarily Conserved RNA Binding Domain. Mol. Cell 5, 761-766, 2000 Vilardell, J., P. Chartrand, R. H. Singer, & J. R. Warner. The Odyssey of a Regulated Transcript. RNA 6, 773-1780, 2000 Yu, X., & J. R. Warner. Expression of a Micro-Protein, J. Biol. Chem. 276, 33821-33825, 2001 Warner, J.R., J. Vilardell, & J.H. Sohn, The Economics of Ribosome Biosynthesis Cold Spring Harbor Symposium on Quantitative Biology, LXVI, 567-574, 2001 Warner, J. R., Nascent Ribosomes Cell 107, 133-136, 2001 Warner, J. R. and P. M. Knopf, The Discovery of Polyribosomes TiBS 27, 376-380, 2002

JONATHAN R. WARNER, Ph.D. Professor

CHANIN BLDG. – ROOM 413A 718 430-3022

[email protected]

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Zhao, Y, J-H. Sohn, & J. R. Warner, Autoregulation in the Biosynthesis of Ribosomes Molec. Cell. Biol. 23, 699-707, 2003 PMID: 12509467 Rudra, D. & J.R.Warner What Better Measure than Ribosome Synthesis? Genes & Dev., 18, 2431-2436, 2004. PMID: 15489289 Rudra, D., Y. Zhao, & J.R. Warner Central Role of Ifh1p-Fhl1p Interaction in the Synthesis of Yeast Ribosomal Proteins, The EMBO Journal, 24, 533-542, 2005 PMID:15692568 Zhao, Y., K.B. McIntosh, D. Rudra, S. Schawalder, D. Shore, & J.R. Warner Fine-Structure Analysis of Ribosomal Protein Gene Transcription, Molec. Cell. Biol. 26, 4853-4862, 2006 PMID: 16782874 Rudra, D., J. Mallick, Y. Zhao, & J.R. Warner Potential Interface Between Ribosomal Protein Production and pre-rRNA Processing Molec. Cell. Biol. 27, 4815-4824, 2007 PMID:17452446 Bhattacharya, A, & J.R. Warner Tbf1 or not Tbf1? Mol. Cell, 29, 537-538, 2008 PMID:18342600 Warner, J.R. and K.B. McIntosh How Common are Extra-ribosomal Functions of Ribosomal Proteins? Mol. Cell, 34, 3-11, 2009 PMID: 19362532 Warner, J.R. and H.-S. Kim The Fast-Track is Co-Transcriptional Mol. Cell 38, 745-6 2010 PMID: 20347416 Bhattacharya, A, K.B. McIntosh, I. M. Willis, & J.R.Warner Molec. Cell. Biol. Why Dom34 Stimulates Growth of Cells with Defects of 40S Ribosomal Subunit Biosynthesis Molec. Cell. Biol. 30, 5562-5571, 2010 PMID: 20876302 Warner, J.R. 18S rRNA: A Tale of the Tail, J. Mol. Biol. 405, 1-2, 2011 PMID:20969874 McIntosh K.B., A Bhattacharya, I. M. Willis, & J.R.Warner Eukaryotic ells producing ribosomes deficient in Rpl1 are hypersensitive to defects in the ubiquitin-proteasome system, PLoS One, in press

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Transcription Regulation and Cell Signaling Control in Normal and Transformed Germinal Center B cells

Molecular pathogenesis of lymphomas situates at the crossroad of B cell differentiation,

cancer genetics, transcription regulation, and cell signaling. Thus, to address the questions of how and why various lymphomas initiate and develop in vivo, we constantly draw upon the most recent advances in these perspective filelds, testing and integrating new paradims in our investigations. As each lymphoma entity often corresponds to a specific lymphocyte activation/differentiation state that is phenotypically “frozen” by the malignant transformtion process, our work also provides valuable insights to the regulatory mechanisms that goven the normal immune system. There are three general goals of our research: to better understand mature B cell development in molecular terms, to decipher how this process is perturbed during lymphomagenesis, and to help develop better lymphoma therapy.

BCL6 and the germinal center reaction:

After their first antigen encounter, mature naïve B cells follow one of the two distinct developmental paths: to rapidly differentiate into short-lived plasma cells without T cell help, or to participate in a T-cell dependent process called the germinal center (GC) response which allows some of their selected progenies to become either memory B cells or plasma cells (both short and long term) that produce high affintiy antibodies. GCs are dynamic and specialized structures in the secondary lymphoid organs composed of mostly B cells undergoing rapid clonal expansion. Within GC, the B cell genome is subject to two types of genetic alterations, e.g. Ig class switch recombination (CSR; IgH only) and somatic hypermutation (SHM; both IgH and IgL). Both of these processes are aimed to increase Ig diversity and are dependent upon the mutator enzyme AID (activation induced cytidine deaminase). Prior to their GC exit, B cells bearing mutated surface Ig molecules undergo positive and negative selections through interaction with two other types of cells in the GC, e.g. follicular dendritic cells and follicular T helper cells. As a result, only those B cells with the proper Ig specificity and affinity are allowed to escape the fate of apoptosis or anergy, gaining license to terminally differentiate into memory or plasma cells. At the moment, the sequence and nature of events that coordinate the initiation and termination of GC response is not well understood. Among the few well established facts is the observation that the onset and maintenance of GC reaction critically require the transcription repressor, BCL6. Widely considered to be the master regulator of the GC response, BCL6 maintains the GC-specific gene expression program by silencing genes involved in B cell activation (CD69, CD80, NF-�B1), response to DNA damage (p53, ATR), cell-cycle regulation (cyclin D2, p21WAF, p27KIP) and plasma cell differentiation (STAT3, IRF4, and Blimp-1). Thus, neither the memory nor the plasma cell differentiation program can be initiated until BCL6 expression is extinguished by GC exit signals. One of our main interests lies in the functional interplay between BCL6 and STAT3 in late GC reaction and lymphoma B cells.

B. HILDA YE, Ph.D. Associate Professor

CHANIN BLDG. – ROOM 302C (718) 430-3339

[email protected]

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B cell Non-Hodgkin’s lymphoma:

Non-Hodgkin’s lymphoma (NHL) is the 5th most common type of cancer in the U.S. Most of these tumors have a B cell phenotype and are derived from GC B cells. From the genetic point of view, NHL is distinct from non-hematophoietic cancers in that most lymphomas carry recurrent chromosomal translocations while microsatellite instability or genome-wide chromosomal instability is rare. To some extent, this phenomenon is explained by the unique mutagenic cellular environment of GC B cells featuring AID activity. Evidence is accumulating that not only is AID responsible for Ig CSR and SHM, but its mutagenic action can also be targeted to other loci in the genome leading to somatic mutations and chromosomal translocations that underlie many mature B cell lymphomas. BCL6, in fact, was initially cloned through its involvement in lymphoma-associated chromosomal translocations and is the most frequently targeted proto-oncogene in NHL. Another important characteristic of mature B cell lymphomas is its heterogeneity. There are 3 most common forms of NHL: follicular lymphomas (FL) which carry the hallmark BCL2 translocations, Burkitt’s lymphomas (BL) which are invariably associated with c-Myc translocations, and diffuse large B cell lymphomas (DLBCL) which in nearly half of the case carry translocations or activating mutations deregulating BCL6 expression.

DLBCL accounts for 30-40% of newly NHL cases in the United States and yet up to

80% of NHL mortality due to transformation of FL to DLBCL. Based upon their gene expression similarities to either normal GC B cells or in vitro activated peripheral blood B cells, DLBCLs are subdivided into 3 groups: the GCB-DLBCL, ABC-DLBCL and an unclassified type III. In general, the GCB group expresses high levels of BCL6 and tends to respond better to conventional chemotherapy, while the ABC group has lower levels of BCL6, constitutively activated NF-kappaB and STAT3, and tends to be refractory to chemotherapeutic treatment. In normal B cells, NF-kappaB activation can be triggered by CD40-CD40L interaction or BCR signaling while STAT3 works downstream of a number of cytokines promoting plasma cell differentiation. In addition, both NF-kappaB and STAT3 are well-known oncogenes with a variety of cancer promoting properties (enhance proliferation, survival, angiogenesis and metastasis). Therefore, the distinct gene expression and cell signaling properties between the two DLBCL subtypes have very important implications in understanding their transformation pathways as well as facilitating development of biology-based, targeted lymphoma therapies.

Ongoing studies are designed to address the following questions: 1. How is the expression status of BCL6 coupled to B cell differentiation control? 2. During late stage GC response, how does cell signaling coordinate the transistion from the

BCL6 governed GC program to a Blimp-1-directed plasma cell program? 3. What are the cause and consequences of a constitutively activated STAT3 pathway in ABC-

DLBCL?

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Selected References: Yu, Y.-L.R., Wang, X., Pixley, F.J., Yu, J.J., Dent, A.L., Broxmeyer, H.E., Stanley, E.R., and Ye, B.H. BCL-6 negatively regulates macrophage proliferation by suppressing autocrine IL-6 production. Blood. 105(4):1111-1784, 2005. Li, Z.P., Wang, X., Yu, Y.-L. R., Ding, B.B., Yu, J.J., Dai, X.-M., Naganuma, A., Stanley, E.R., and Ye, B.H. BCL-6 negatively regulates expression of the NF-�B1 p105/p50 subunit. J. Immunol. 174: 205-214, 2005. Pixley, F.J., Xiong, Y., Yu, Y.-L.R., Sahai, E., Stanley, E.R., and Ye, B.H. BCL-6 suppresses RhoA activity to alter macrophage morphology and motility. J. Cell Science. 118:1873-1883, 2005 Ding, B.B., Yu, J.J., Yu, Y.-L.R., Mendez, L.M., Shaknovich, R., Zhang, Y, Cattoretti, G., and Ye, B.H. Constitutively activated STAT3 promotes cell proliferation and survival in the activated B cell subtype of diffuse large B-cell lymphoma. Blood 111:1515-1523, 2008 Mendez, L.M., Polo, J., Yu, J.J., Krupski, M., Ding, B.B., Melnick, A., Ye, B.H. CtBP1 is an essential corepressor for BCL6 autoregulation. Mol. Cell. Biol., 28:2175-2186, 2008

Cell Biology Picnic 2011