Cell Reports Article - CAU
Transcript of Cell Reports Article - CAU
Cell Reports
Article
Cell-Cycle-Regulated Interaction between Mcm10and Double Hexameric Mcm2-7 Is Required forHelicase Splitting and Activation during S PhaseYun Quan,1,4 Yisui Xia,1,4 Lu Liu,1,4 Jiamin Cui,1 Zhen Li,1 Qinhong Cao,1 Xiaojiang S. Chen,3 Judith L. Campbell,2
and Huiqiang Lou1,*1State Key Laboratory of Agro-Biotechnology, College of Biological Sciences, China Agricultural University, 2 Yuan-Ming-Yuan West Road,
Beijing 100193, China2Braun Laboratories, California Institute of Technology, Pasadena, CA 91125, USA3Molecular and Computational Biology, USC Norris Cancer Center, and Chemistry Department, University of Southern California,Los Angeles, CA 90089, USA4Co-first author
*Correspondence: [email protected]://dx.doi.org/10.1016/j.celrep.2015.11.018
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
SUMMARY
Mcm2-7 helicase is loaded onto double-strandedorigin DNA as an inactive double hexamer (DH) inG1 phase. The mechanisms of Mcm2-7 remodelingthat trigger helicase activation in S phase remain un-known. Here, we develop an approach to detect andpurify the endogenous DHs directly. Through cellularfractionation, we provide in vivo evidence that DHsare assembled on chromatin in G1 phase and sepa-rated during S phase. Interestingly, Mcm10, a robustMCM interactor, co-purifies exclusively with the DHsin the context of chromatin. Deletion of the maininteraction domain, Mcm10 C terminus, causesgrowth and S phase defects, which can be sup-pressed through Mcm10-MCM fusions. By moni-toring the dynamics of MCM DHs, we show a signif-icant delay in DH dissolution during S phase in theMcm10-MCM interaction-deficient mutants. There-fore, we propose an essential role for Mcm10 inMcm2-7 remodeling through formation of a cell-cy-cle-regulated supercomplex with DHs.
INTRODUCTION
The assembly of the DNA replication machinery and initiation of
synthesis are controlled in a tightly orchestratedmanner accord-
ing to different stages of the cell cycle (Costa et al., 2013; Heller
et al., 2011; Labib, 2010; Siddiqui et al., 2013). The origin
licensing step involving recruitment and assembly of the replica-
tive helicase mini-chromosome maintenance (MCM) into the
pre-replication complexes (pre-RCs) has been reconstituted
in vitro through purified yeast proteins (Evrin et al., 2009; Remus
et al., 2009). Notably, two Mcm2-7 hexameric rings are sequen-
tially loaded onto double-stranded DNA (dsDNA) as an inactive
head-to-head double hexamer (DH) (Evrin et al., 2009; Gambus
et al., 2011; Remus et al., 2009; Ticau et al., 2015). These findings
raise an intriguing question: how is the double hexameric MCM
activated to initiate bidirectional DNA replication in eukaryotes
(Boos et al., 2012; Li and Araki, 2013; Tognetti et al., 2015)?
Mcm2-7 in solution exhibits primarily a single hexameric struc-
ture stabilized upon ATP binding (Bochman and Schwacha,
2009; Coster et al., 2014). The pre-RC intermediates are very
sensitive to salt wash, while the MCM DHs remain very stable
on chromatin in the presence of high salt (Gambus et al., 2011;
Remus et al., 2009). It is of particular importance to ensure that
MCM hexamers be poised on chromatin before S phase ready
for activation, given the fact that helicase reloading is blocked
during S phase (Bell and Dutta, 2002; Masai et al., 2010). The
DH state may be maintained in the initial holo-helicase Cdc45-
Mcm2-7-GINS (CMG) complex (Costa et al., 2014). However,
the two helicase rings need to be separated and remodeled to
encircle the leading strands to initiate bidirectional replication
(Fu et al., 2011; Yardimci et al., 2010). The two rings are dimer-
ized through an interface composed of the N termini of Mcm2-
7 subunits (Evrin et al., 2009; Fletcher et al., 2003; Remus
et al., 2009), which bear multiple critical target sites for protein
kinases, such as Dbf4-dependent kinase Cdc7 (DDK) and cy-
clin-dependent kinase (CDK) (Hoang et al., 2007; Sheu and Still-
man, 2010; Sheu et al., 2014). Phosphorylation is thought to be
required but not sufficient to activate the helicase (On et al.,
2014; Yeeles et al., 2015).
Mcm10 is among the recently reported minimal set of the
essential firing factors for reconstituted DNA synthesis in vitro
(Yeeles et al., 2015), and it has been inferred to be important in
Mcm2-7 helicase activation post-CMG formation, as indicated
by Mcm10 depletion in yeast (Kanke et al., 2012; van Deursen
et al., 2012; Watase et al., 2012) and Xenopus (Pacek et al.,
2006). However, the mechanistic details of Mcm10 function
have yet to be defined (Thu and Bielinsky, 2013, 2014).
In this study, we developed an approach to purify the endog-
enous MCM complexes from yeast cells, which allowed us to
monitor the formation and separation of MCMDHs in vivo. Using
this assay, we were able to show that Mcm10 defines an
2576 Cell Reports 13, 2576–2586, December 22, 2015 ª2015 The Authors
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Cellular and molecular regulation of theactivation of mammalian primordialfollicles: somatic cells initiate follicleactivation in adulthoodHua Zhang1,* and Kui Liu2,*
1State Key Laboratory of Agrobiotechnology, College of Biological Science, China Agricultural University, Beijing 100193, China2Department of Chemistry and Molecular Biology, University of Gothenburg, SE-405 30 Gothenburg, Sweden
*Correspondence address. E-mail: [email protected] (H.Z.)/[email protected] (K.L.)
Submitted on February 9, 2015; resubmitted on May 29, 2015; accepted on July 13, 2015
table of contents
† Introduction: the mammalian ovary and primordial follicles† Methods† Focusing on a single primordial follicle: the intra-follicle signaling networks in regulating the activation of primordialfollicles
Earlier studies of signaling networks in dormant oocytes that regulate primordial follicle activationRecent studies of the cellular and molecular regulation of follicle activation with a focus on pfGCsCurrent progress on the intra-follicle signaling that regulates primordial follicle activation
† Regulatory roles of endocrine and nutrient factors in the activation of primordial folliclesThe suppressing effect of anti-Mullerian hormone in the activation of primordial folliclesThe relationship between nutrient status and activation of primordial follicles
† Summary and future perspectives
background: The first small follicles to appear in the mammalian ovaries are primordial follicles. The initial pool of primordial follicles servesas the source of developing follicles and oocytes for the entire reproductive lifespan of the animal. Although the selective activation of primordialfollicles is critical for female fertility, its underlying mechanisms have remained poorly understood.
methods: A search of PubMed was conducted to identify peer-reviewed literature pertinent to the study of mammalian primordial follicleactivation, especially recent reports of the role of primordial follicle granulosa cells (pfGCs) in regulating this process.
results: In recent years, molecular mechanisms that regulate the activation of primordial follicles have been elucidated, mostly through theuse of genetically modified mouse models. Several molecules and pathways operating in both the somatic pfGCs and oocytes, such as the phos-phatidylinositol 3 kinase (PI3K) and the mechanistic target of rapamycin complex 1 (mTORC1) pathways, have been shown to be important forprimordial follicle activation. More importantly, recent studies have provided an updated view of how exactly signaling pathways in pfGCs and inoocytes, such as the KIT ligand (KL) and KIT, coordinate in adult ovaries so that the activation of primordial follicles is achieved.
conclusions: In this review, we have provided an updated picture of how mammalian primordial follicles are activated. The functional rolesof pfGCs in governing the activation of primordial follicles in adulthood are highlighted. The in-depth understanding of the cellular and molecularmechanisms of primordial follicle activation will hopefully lead to more treatments of female infertility, and the current progress indicates that theuse of existing primordial follicles as a source for obtaining fertilizable oocytes as a new treatment for female infertility is just around the corner.
Key words: primordial follicle activation / primordial follicle granulosa cells / mechanistic target of rapamycin complex 1 / KIT ligand / KIT / PI3kinase signaling
& The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.For Permissions, please email: [email protected]
Human Reproduction Update, Vol.0, No.0 pp. 1–8, 2015
doi:10.1093/humupd/dmv037
Human Reproduction Update Advance Access published July 30, 2015 at U
niversity of Prince Edw
ard Island on August 13, 2015
http://humupd.oxfordjournals.org/
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GENERAL CONTROL NONREPRESSEDPROTEIN5-Mediated Histone Acetylation of FERRICREDUCTASE DEFECTIVE3 Contributes to IronHomeostasis in Arabidopsis1
Jiewen Xing, Tianya Wang, Zhenshan Liu, Jianqin Xu, Yingyin Yao, Zhaorong Hu, Huiru Peng,Mingming Xin, Futong Yu, Daoxiu Zhou, and Zhongfu Ni*
State Key Laboratory for Agrobiotechnology, Key Laboratory of Crop Heterosis and Utilization, Beijing KeyLaboratory of Crop Genetic Improvement (J.Xi., T.W., Z.L., Y.Y., Z.H., H.P., M.X., Z.N.), and College ofResources and Environmental Sciences (J.Xu, F.Y.), China Agricultural University, Beijing 100193, China;National Plant Gene Research Centre, Beijing 100193, China (J.Xi., T.W., Z.L., Y.Y., Z.H., H.P., M.X., Z.N.); andInstitut de Biologie des Plantes, Unité Mixte de Recherche 8618, Université Paris Sud, 91405 Orsay, France (D.Z.)
ORCID IDs: 0000-0003-3503-0739 (J.Xi); 0000-0002-1815-648X (Z.H.); 0000-0001-8672-3610 (F.Y.); 0000-0002-1540-0598 (D.Z.).
Iron homeostasis is essential for plant growth and development. Here, we report that a mutation in GENERAL CONTROLNONREPRESSED PROTEIN5 (GCN5) impaired iron translocation from the root to the shoot in Arabidopsis (Arabidopsis thaliana).Illumina high-throughput sequencing revealed 879 GCN5-regulated candidate genes potentially involved in iron homeostasis.Chromatin immunoprecipitation assays indicated that five genes (At3G08040, At2G01530, At2G39380, At2G47160, and At4G05200)are direct targets of GCN5 in iron homeostasis regulation. Notably, GCN5-mediated acetylation of histone 3 lysine 9 and histone 3lysine 14 of FERRIC REDUCTASE DEFECTIVE3 (FRD3) determined the dynamic expression of FRD3. Consistent with the function ofFRD3 as a citrate efflux protein, the iron retention defect in gcn5was rescued and fertility was partly restored by overexpressing FRD3.Moreover, iron retention in gcn5 roots was significantly reduced by the exogenous application of citrate. Collectively, these datasuggest that GCN5 plays a critical role in FRD3-mediated iron homeostasis. Our results provide novel insight into the chromatin-based regulation of iron homeostasis in Arabidopsis.
Iron is an essential trace nutrition element for plants;its availability affects root morphogenesis, photosynthe-sis, nitrogen fixation, respiration, and flower color andfertility (Guerinot and Yi, 1994; Walker and Connolly,2008). Iron deficiency impairs fundamental processesand causes a decrease in chlorophyll production, thusinfluencing crop productivity and quality (Schikora andSchmidt, 2001; Curie and Briat, 2003). Conversely, iron inexcess is toxic to plants. Therefore, to ensure the effectiveacquisition of iron from the soil and to avoid excess ironin cells, iron uptake and distribution are strictly controlled
in plants (Guerinot and Yi, 1994). Once taken up into theroot, iron must be moved to the central vascular cylinder,where it can be loaded into the xylem and translocated tothe aerial part of the plant (Rogers, 2006).
At the molecular level, several genes involved in ironuptake and mobilization have been characterized, in-cluding FERRIC REDUCTASE DEFECTIVE3 (FRD3),FERRIC REDUCTASE OXIDASE2 (FRO2), ZIP familyIRON REGULATED TRANSPORTER1 (IRT1), and othergenes (Curie and Briat, 2003; Walker and Connolly,2008). In Arabidopsis (Arabidopsis thaliana), FRO2 andIRT1 are two important genes responsible for iron up-take. IRT1 is the major transporter responsible for high-affinity iron uptake from the soil and is a key player inthe regulation of plant iron homeostasis, as attested toby the severe chlorosis and lethality of the irt1 mutant(Korshunova et al., 1999; Varotto et al., 2002; Vert et al.,2002). Encoding a low-iron-inducible ferric chelate re-ductase, FRO2 is responsible for the reduction of iron atthe root surface. When iron is loaded into the root xylemfrom the pericycle, iron distribution between the rootand shoot is essential for tolerating fluctuations in ironavailability (Long et al., 2010). Several studies revealedthat FRD3, a multidrug and toxin efflux protein, facili-tates iron chelation to citrate and the subsequent trans-port of iron citrate from the root to the shoot (Green andRogers, 2004; Durrett et al., 2007).
1 This work was supported by the National Basic Research Pro-gram of China 973 Program (grant no. 2012CB910900), the NationalNatural Science Foundation of China (grant no. 31471478), the 863Project (grant no. 2012AA10A309), and the Chinese Universities Sci-entific Fund (grant no. 2014XJ019).
* Address correspondence to [email protected] author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Zhongfu Ni ([email protected]).
J.Xi. and Z.N. conceived the project and designed all the research;J.Xi. performed the experiments and analyzed the data with assis-tance from Z.L., Z.H., T.W., J.Xu., H.P., Y.Y., M.X., F.Y. and D.Z.;J.Xi. and Z.N. wrote the article.
www.plantphysiol.org/cgi/doi/10.1104/pp.15.00397
Plant Physiology�, August 2015, Vol. 168, pp. 1309–1320, www.plantphysiol.org � 2015 American Society of Plant Biologists. All Rights Reserved. 1309 www.plant.org on April 4, 2016 - Published by www.plantphysiol.orgDownloaded from
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
ARTICLE
Received 13 Oct 2014 | Accepted 27 May 2015 | Published 2 Jul 2015
Mechanistic insights into metal ion activation andoperator recognition by the ferric uptake regulatorZengqin Deng1,*, Qing Wang1,*, Zhao Liu2,*, Manfeng Zhang1,*, Ana Carolina Dantas Machado3, Tsu-Pei Chiu3,
Chong Feng1, Qi Zhang1, Lin Yu1, Lei Qi1, Jiangge Zheng1, Xu Wang1, XinMei Huo4, Xiaoxuan Qi1, Xiaorong Li1,
Wei Wu1, Remo Rohs3, Ying Li1 & Zhongzhou Chen1
Ferric uptake regulator (Fur) plays a key role in the iron homeostasis of prokaryotes,
such as bacterial pathogens, but the molecular mechanisms and structural basis of Fur–DNA
binding remain incompletely understood. Here, we report high-resolution structures
of Magnetospirillum gryphiswaldense MSR-1 Fur in four different states: apo-Fur, holo-Fur, the
Fur–feoAB1 operator complex and the Fur–Pseudomonas aeruginosa Fur box complex. Apo-Fur
is a transition metal ion-independent dimer whose binding induces profound conformational
changes and confers DNA-binding ability. Structural characterization, mutagenesis, bio-
chemistry and in vivo data reveal that Fur recognizes DNA by using a combination of base
readout through direct contacts in the major groove and shape readout through recognition of
the minor-groove electrostatic potential by lysine. The resulting conformational plasticity
enables Fur binding to diverse substrates. Our results provide insights into metal ion
activation and substrate recognition by Fur that suggest pathways to engineer magnetotactic
bacteria and antipathogenic drugs.
DOI: 10.1038/ncomms8642 OPEN
1 State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100193, China. 2 Experimental Research Center, China Academy of ChineseMedical Sciences, Beijing 100700, China. 3 Molecular and Computational Biology Program, Departments of Biological Sciences, Chemistry, Physics, andComputer Science, University of Southern California, Los Angeles, California 90089, USA. 4 Institute of Apicultural Research, Key Laboratory of Pollinating InsectBiology, Chinese Academy of Agricultural Science, Beijing 100093, China. * These authors contributed equally to this work. Correspondence and requests formaterials should be addressed to R.R. (email: [email protected]) or to Y.L. (email: [email protected]) or to Z.C. (email: [email protected]).
NATURE COMMUNICATIONS | 6:7642 | DOI: 10.1038/ncomms8642 | www.nature.com/naturecommunications 1
& 2015 Macmillan Publishers Limited. All rights reserved.
RESEARCH ARTICLE
Mutations in RECQL Gene Are Associatedwith Predisposition to Breast CancerJie Sun1☯, Yuxia Wang1☯, Yisui Xia2☯, Ye Xu1, Tao Ouyang1, Jinfeng Li1, TianfengWang1,Zhaoqing Fan1, Tie Fan1, Benyao Lin1, Huiqiang Lou2*, Yuntao Xie1*
1 Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Breast Center,Peking University Cancer Hospital & Institute, Beijing, the People’s Republic of China, 2 State KeyLaboratory of Agro-Biotechnology, College of Biological Sciences, China Agricultural University, Beijing, thePeople’s Republic of China
☯ These authors contributed equally to this work.* [email protected] (HL); [email protected] (YX)
AbstractThe genetic cause for approximately 80% of familial breast cancer patients is unknown.
Here, by sequencing the entire exomes of nine early-onset familial breast cancer patients
without BRCA1/2mutations (diagnosed with breast cancer at or before the age of 35) we
found that two index cases carried a potentially deleterious mutation in the RECQL gene
(RecQ helicase-like; chr12p12). Recent studies suggested that RECQL is involved in DNA
double-strand break repair and it plays an important role in the maintenance of genomic sta-
bility. Therefore, we further screened the RECQL gene in an additional 439 unrelated famil-
ial breast cancer patients. In total, we found three nonsense mutations leading to a
truncated protein of RECQL (p.L128X, p.W172X, and p.Q266X), one mutation affecting
mRNA splicing (c.395-2A>G), and five missense mutations disrupting the helicase activity
of RECQL (p.A195S, p.R215Q, p.R455C, p.M458K, and p.T562I), as evaluated through an
in vitro helicase assay. Taken together, 9 out of 448 BRCA-negative familial breast cancer
patients carried a pathogenic mutation of the RECQL gene compared with one of the 1,588
controls (P = 9.14×10-6). Our findings suggest that RECQL is a potential breast cancer sus-
ceptibility gene and that mutations in this gene contribute to familial breast cancer
development.
Author Summary
In this study, we aimed to find novel breast cancer susceptibility genes by whole-exome se-quencing in nine early-onset familial breast cancer patients without BRCA1/2mutations.We found that two index cases carried a potentially deleterious mutation in the RECQLgene (RecQ helicase-like). We further screened the RECQL gene in an additional 439 unre-lated familial breast cancer patients. In total, we found nine index cases carried a patho-genic mutation in the RECQL gene among the 448 BRCA-negative familial breast cancerpatients. The RECQL is one of five RecQ helicase proteins (named RECQL, BLM, WRN,RECQL4 and RECQL5). RECQL is considered to be genome caretaker, and mutations in
PLOSGenetics | DOI:10.1371/journal.pgen.1005228 May 6, 2015 1 / 12
a11111
OPEN ACCESS
Citation: Sun J, Wang Y, Xia Y, Xu Y, Ouyang T, Li J,et al. (2015) Mutations in RECQL Gene AreAssociated with Predisposition to Breast Cancer.PLoS Genet 11(5): e1005228. doi:10.1371/journal.pgen.1005228
Editor: Steven A Narod, Women's College ResearchInstitute, CANADA
Received: December 23, 2014
Accepted: April 16, 2015
Published: May 6, 2015
Copyright: © 2015 Sun et al. This is an open accessarticle distributed under the terms of the CreativeCommons Attribution License, which permitsunrestricted use, distribution, and reproduction in anymedium, provided the original author and source arecredited.
Data Availability Statement: All relevant data arewithin the paper and its Supporting Information files.
Funding: This study was supported by the NationalKey Technology Research and DevelopmentProgram of the Ministry of Science and Technology ofChina (No. 2014BAI09B08, YXie); the 973 project2013CB911004 (YXie); and grant from the NationalNatural Science Foundation of China (No.81372832,YXie). This study was also supported by Program forNew Century Excellent Talents in University of China(NCET-09-0733, HL); and the National NaturalScience Foundation of China (No.31271331, HL).The funders had no role in study design, data
Patterns of genomic changes with crop domesticationand breedingJunpeng Shi and Jinsheng Lai
Crop domestication and further breeding improvement have
long been important areas of genetics and genomics studies.
With the rapid advancing of next-generation sequencing (NGS)
technologies, the amount of population genomics data has
surged rapidly. Analyses of the mega genomics data have
started to uncover a previously unknown pattern of genome-
wide changes with crop domestication and breeding. Selection
during domestication and breeding drastically reshaped crop
genomes, which have ended up with regions of greatly reduced
genetic diversity and apparent enrichment of potentially
beneficial alleles located in both genic and non-genic regions.
Increasing evidences suggest that epigenetic modifications
also played an important role during domestication and
breeding.
Addresses
State Key Laboratory of Agrobiotechnology and National Maize
Improvement Center, Department of Plant Genetics and Breeding, China
Agricultural University, Beijing 100193, China
Corresponding author: Lai, Jinsheng ([email protected])
Current Opinion in Plant Biology 2015, 24:47–53
This review comes from a themed issue on Genome studies and
molecular genetics
Edited by Insuk Lee and Todd C Mockler
http://dx.doi.org/10.1016/j.pbi.2015.01.008
1369-5266/# 2015 Published by Elsevier Ltd.
IntroductionModern crop varieties contain a number of superior
agronomic traits to meet human needs and to adapt to
local agronomic environments. These varieties are the
products of extensive scientific breeding from landraces,
which are domesticated for more than ten thousand years.
Both breeding and domestication processes have been
the subject of extensive genetic and genomic research.
Recently, a number of insightful reviews have summa-
rized the studies on molecular genetics changes during
crop domestication and breeding [1–4].
Rapid advancements in next-generation sequencing
(NGS) technology have provided a unique opportunity
for population genomics of crop domestication and breed-
ing, because genome-wide sequencing information of
large numbers of wild relatives and modern cultivars of
many crops are relatively easily available. Here, we sum-
marize the latest advances in the availability of genomic
information for crop domestication and breeding im-
provement, the emerging methods to analyze the popu-
lation genomics data and the patterns in genomics and
epigenomics changes that occur with crop domestication
and breeding.
Rapidly increasing population genomics dataprovide unprecedented opportunity for studyof crop domestication and breedingTraditionally, studies on crop domestication and breeding
have been addressed using relatively small numbers of
specific traits or by analyzing sequencing data of targeted
regions [5]. However, the advent of NGS technologies has
dramatically reduced the cost of sequencing, and so the
number of crops with their entire genomes nearly
completely sequenced and re-sequencing data of large
numbers of individuals has increased very rapidly over the
last several years. As a summary, the list of crops that have
a completely sequenced genome and at the same time
with reasonable data of population re-sequencing of ei-
ther domesticated lines or wild relatives are shown in
Supplementary Table 1. Except for the major staple crops
such as rice, maize and sorghum that had their reference
genomes sequenced using traditional Sanger sequencing,
the others were primarily sequenced using NGS. Addi-
tionally, there are larger number of crops that had their
first genome reported only very recently. It is highly likely
that many of these crops will have population re-sequenc-
ing efforts underway.
Coupled with the availability of large amounts of geno-
mics data for many crops, the methods of analyzing these
data have also been rapidly developed. One of the most
general trends during crop domestication is a dramatically
reduced genetic diversity, known as a genetic bottleneck
[6,7]. Because the reduction is uneven along chromo-
somes, with putative selected genes experiencing more
severe bottlenecks than unselected ones, such distinct
genetic characteristics can be used to identify so-called
selective sweeps. For a given breeding population with-
out ancestor information, the extremely low genetic di-
versity (p) or Tajima’s D can be used to scan the selective
regions [8,9]. The composite likelihood ratio (CLR) ap-
proach has been shown to be very useful in identifying
selective sweeps, which accurately predict the location
and selection coefficient of each selective sweep by
taking into account complex demographics and varying
Available online at www.sciencedirect.com
ScienceDirect
www.sciencedirect.com Current Opinion in Plant Biology 2015, 24:47–53
RESEARCH ARTICLE
Post-transcriptional Regulation ofKeratinocyte Progenitor Cell Expansion,Differentiation and Hair Follicle RegressionbymiR-22Shukai Yuan1,2,3☯, Feifei Li1☯, Qingyong Meng1, Yiqiang Zhao1, Lei Chen4,Hongquan Zhang5, Lixiang Xue5, Xiuqing Zhang6, Christopher Lengner7,8,Zhengquan Yu1*
1 State Key Laboratories for Agrobiotechnology, College of Biological Sciences, China AgriculturalUniversity, Beijing, China, 2 Department of Biochemistry and Molecular Biology, Basic Medical College,Tianjin Medical University, Tianjin, China, 3 Department of Animal Science, Southwest University,Rongchang, Chongqing, China, 4 Chongqing Academy of Animal Science, Rongchang, Chongqing, China,5 Laboratory of Molecular Cell Biology and Tumor Biology, Department of Anatomy, Histology andEmbryology, Beijing, China, 6 College of Food Science and Nutritional Engineering, China AgriculturalUniversity, Beijing, China, 7 Department of Animal Biology, School of Veterinary Medicine, University ofPennsylvania, Philadelphia, Pennsylvania, United States of America, 8 Institute for Regenerative Medicine,University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
☯ These authors contributed equally to this work.* [email protected]
AbstractHair follicles (HF) undergo precisely regulated recurrent cycles of growth, cessation, and
rest. The transitions from anagen (growth), to catagen (regression), to telogen (rest) involve
a physiological involution of the HF. This process is likely coordinated by a variety of mecha-
nisms including apoptosis and loss of growth factor signaling. However, the precise molecu-
lar mechanisms underlying follicle involution after hair keratinocyte differentiation and hair
shaft assembly remain poorly understood. Here we demonstrate that a highly conserved
microRNA,miR-22 is markedly upregulated during catagen and peaks in telogen. Using
gain- and loss-of-function approaches in vivo, we find thatmiR-22 overexpression leads to
hair loss by promoting anagen-to-catagen transition of the HF, and that deletion ofmiR-22delays entry to catagen and accelerates the transition from telogen to anagen. Ectopic acti-
vation ofmiR-22 results in hair loss due to the repression a hair keratinocyte differentiation
program and keratinocyte progenitor expansion, as well as promotion of apoptosis. At the
molecular level, we demonstrate thatmiR-22 directly represses numerous transcription fac-
tors upstream of phenotypic keratin genes, including Dlx3, Foxn1, and Hoxc13. We con-
clude thatmiR-22 is a critical post-transcriptional regulator of the hair cycle and may
represent a novel target for therapeutic modulation of hair growth.
PLOS Genetics | DOI:10.1371/journal.pgen.1005253 May 28, 2015 1 / 23
OPEN ACCESS
Citation: Yuan S, Li F, Meng Q, Zhao Y, Chen L,Zhang H, et al. (2015) Post-transcriptional Regulationof Keratinocyte Progenitor Cell Expansion,Differentiation and Hair Follicle Regression by miR-22. PLoS Genet 11(5): e1005253. doi:10.1371/journal.pgen.1005253
Editor: Gregory S. Barsh, Stanford University Schoolof Medicine, United States of America
Received: December 16, 2014
Accepted: April 29, 2015
Published: May 28, 2015
Copyright: © 2015 Yuan et al. This is an openaccess article distributed under the terms of theCreative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in anymedium, provided the original author and source arecredited.
Data Availability Statement: All relevant data arewithin the paper and its Supporting Information files.
Funding: This work was supported by NationalNatural Science Foundation of China (NSFC,31271584), the National Transgenic ResearchProject (2011ZX08009-001-003), the National BasicResearch Program of China (973 program-2011CB944103), 2010SKLAB03-01, 2014SKLAB4-2and 2015SKLAB6-16. The funders had no role instudy design, data collection and analysis, decision topublish, or preparation of the manuscript.
Revealing Shared and Distinct Gene NetworkOrganization in Arabidopsis ImmuneResponses by Integrative Analysis1
Xiaobao Dong, Zhenhong Jiang, You-Liang Peng, and Ziding Zhang*
State Key Laboratory of Agrobiotechnology (X.D., Z.J., Y.-L.P., Z.Z.), College of Biological Sciences (X.D., Z.J., Z.Z.),and Ministry of Agriculture Key Laboratory for Plant Pathology (Y.-L.P.), China Agricultural University, Beijing100193, China
Pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) are two main plant immune responses to counterpathogen invasion. Genome-wide gene network organizing principles leading to quantitative differences between PTI and ETI haveremained elusive. We combined an advanced machine learning method and modular network analysis to systematically characterizethe organizing principles of Arabidopsis (Arabidopsis thaliana) PTI and ETI at three network resolutions. At the single network node/edge level, we ranked genes and gene interactions based on their ability to distinguish immune response from normal growth andsuccessfully identified many immune-related genes associated with PTI and ETI. Topological analysis revealed that the top-ranked geneinteractions tend to link network modules. At the subnetwork level, we identified a subnetwork shared by PTI and ETI encompassing1,159 genes and 1,289 interactions. This subnetwork is enriched in interactions linking network modules and is also a hotspot of attackby pathogen effectors. The subnetwork likely represents a core component in the coordination of multiple biological processes to favordefense over development. Finally, we constructed modular network models for PTI and ETI to explain the quantitative differences inthe global network architecture. Our results indicate that the defense modules in ETI are organized into relatively independentstructures, explaining the robustness of ETI to genetic mutations and effector attacks. Taken together, the multiscale comparisons ofPTI and ETI provide a systems biology perspective on plant immunity and emphasize coordination among network modules toestablish a robust immune response.
Plants have evolved a sophisticated immune systemthat enables each cell to monitor every possible destruc-tive invasion by microbe and to mount an appropriatedefense response when necessary (Spoel and Dong,2012). Pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) are two primary immune de-fense modes in plants (Jones and Dangl, 2006). In PTI, theimmune response is triggered when pattern-recognitionreceptors detect specific molecular patterns from patho-gens, also known as pathogen-associated molecularpatterns (PAMPs). PAMPs, such as bacterial flagellin,bacterial ELONGATION FACTOR TU (EF-Tu), lipopoly-saccharides, and peptidoglycans, are essential compo-nents of many pathogens but are lacking in plant cells.Thus, PAMPs are ideal molecular markers for detectingpathogen invasion. Pathogens can secrete virulence pro-teins called effectors into host cells to subvert the PTIprocess. Effectors usually mimic the biochemical functionof eukaryotic enzymes, such as phosphatases, proteases,and ubiquitin ligases, to efficiently block immune
signaling pathways at a low dosage (Abramovitch et al.,2006). ETI coevolved to monitor the presence of pathogeneffectors (Chisholm et al., 2006; Jones and Dangl, 2006). Incontrast to PAMPs, effectors are directly or indirectlydetected by plant resistant (R) proteins in ETI, usuallyaccompanied by a hypersensitive cell death response.After the initiation of PTI or ETI, extensive transcriptionalreprogramming occurs, one of the most remarkablephenomena in the plant immune response (Moore et al.,2011).
In both PTI and ETI, the downstream immune responsemust be tightly controlled by gene networks to balanceresource allocation between normal growth and an ef-fective immune response to inhibit pathogen colonization.However, the difference in the downstream immune re-sponses of PTI and ETI remain largely unknown (Doddsand Rathjen, 2010). Genome-wide microarray studieshave demonstrated that differences in the Arabidopsis(Arabidopsis thaliana) transcriptome between PTI and ETIare largely quantitative (Tao et al., 2003; Truman et al.,2006). It has been proposed that ETI is faster and strongerthan PTI and that the signaling network components ofPTI and ETI are similar (Thomma et al., 2011). However,some studies have also suggested that distinct defenseregulation mechanisms exist between PTI and ETI (Heet al., 2006; Gao et al., 2013), while cross regulation betweenPTI and ETI has also been observed (Zhang et al., 2012).
Many immune-related genes involved in PTI or ETIhave been identified using genetic screens in com-bination with biochemistry and molecular biology
1 This work was supported by the National Natural Science Foun-dation of China (grant nos. 31271414 and 31471249).
* Address correspondence to [email protected] author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Ziding Zhang ([email protected]).
www.plantphysiol.org/cgi/doi/10.1104/pp.114.254292
1186 Plant Physiology�, March 2015, Vol. 167, pp. 1186–1203, www.plantphysiol.org � 2014 American Society of Plant Biologists. All Rights Reserved. www.plant.org on February 27, 2015 - Published by www.plantphysiol.orgDownloaded from
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research papers
2970 doi:10.1107/S1399004714019762 Acta Cryst. (2014). D70, 2970–2982
Acta Crystallographica Section D
BiologicalCrystallography
ISSN 1399-0047
Structural insights into the substrate specificity andtransglycosylation activity of a fungal glycosidehydrolase family 5 b-mannosidase
Peng Zhou,a‡ Yang Liu,b‡
Qiaojuan Yan,c Zhongzhou
Chen,b Zhen Qina and
Zhengqiang Jianga*
aDepartment of Biotechnology, College of Food
Science and Nutritional Engineering, China
Agricultural University, Beijing 100083,
People’s Republic of China, bState Key
Laboratory of Agrobiotechnology, College of
Biological Sciences, China Agricultural
University, Beijing 100193, People’s Republic
of China, and cBioresource Utilization
Laboratory, College of Engineering, China
Agricultural University, Beijing 100083,
People’s Republic of China
‡ These authors contributed equally to this
work.
Correspondence e-mail: [email protected]
# 2014 International Union of Crystallography
�-Mannosidases are exo-acting glycoside hydrolases (GHs)
that catalyse the removal of the nonreducing end �-d-mannose
from manno-oligosaccharides or mannoside-substituted mole-
cules. They play important roles in fundamental biological
processes and also have potential applications in various
industries. In this study, the first fungal GH family 5
�-mannosidase (RmMan5B) from Rhizomucor miehei was
functionally and structurally characterized. RmMan5B exhib-
ited a much higher activity against manno-oligosaccharides
than against p-nitrophenyl �-d-mannopyranoside (pNPM) and
had a transglycosylation activity which transferred mannosyl
residues to sugars such as fructose. To investigate its substrate
specificity and transglycosylation activity, crystal structures of
RmMan5B and of its inactive E202A mutant in complex with
mannobiose, mannotriose and mannosyl-fructose were deter-
mined at resolutions of 1.3, 2.6, 2.0 and 2.4 A, respectively.
In addition, the crystal structure of R. miehei �-mannanase
(RmMan5A) was determined at a resolution of 2.3 A. Both
RmMan5A and RmMan5B adopt the (�/�)8-barrel architec-
ture, which is globally similar to the other members of GH
family 5. However, RmMan5B shows several differences in the
loop around the active site. The extended loop between strand
�8 and helix �8 (residues 354–392) forms a ‘double’ steric
barrier to ‘block’ the substrate-binding cleft at the end of the
�1 subsite. Trp119, Asn260 and Glu380 in the �-mannosidase,
which are involved in hydrogen-bond contacts with the �1
mannose, might be essential for exo catalytic activity. More-
over, the structure of RmMan5B in complex with mannosyl-
fructose has provided evidence for the interactions between
the �-mannosidase and d-fructofuranose. Overall, the present
study not only helps in understanding the catalytic mechanism
of GH family 5 �-mannosidases, but also provides a basis
for further enzymatic engineering of �-mannosidases and
�-mannanases.
Received 14 April 2014
Accepted 2 September 2014
PDB references: RmMan5A,
4qp0; RmMan5B, 4lyp;
RmMan5B/E202A,
mannobiose complex, 4nrs;
RmMan5B/E202A,
mannotriose complex, 4lyq;
RmMan5B/E202A, mannosyl-
fructose complex, 4nrr;
RmMan5B/E301A, 4lyr
1. Introduction
Mannans are important constituents of higher plant cell walls,
and also display a storage function as nonstarch carbohydrate
reserves in vegetative tissues. They are the dominant consti-
tuents of hemicelluloses, which represent the second most
abundant source of organic carbon within the biosphere
(Moreira & Filho, 2008). Mannans have been classified into
two major groups depending on whether the �-1,4-linked
backbone contains only d-mannose residues (mannans) or a
combination of mannose and d-glucose residues (gluco-
mannans). Each of these groups can be subdivided based on
the number of �-1,6-linked galactose side groups (galacto-
mannans and galactoglucomannans; Van Zyl et al., 2010).
Owing to their complex structures, the mannan-degrading
enzymes are composed of �-mannanase (EC 3.2.1.78),
ARTICLE
Received 7 Aug 2014 | Accepted 20 Oct 2014 | Published 9 Jan 2015
Structural basis for preferential avian receptorbinding by the human-infecting H10N8 avianinfluenza virusMin Wang1,2,*, Wei Zhang2,*, Jianxun Qi2, Fei Wang1,2, Jianfang Zhou3, Yuhai Bi2, Ying Wu2, Honglei Sun1,
Jinhua Liu1, Chaobin Huang4, Xiangdong Li4, Jinghua Yan1, Yuelong Shu3, Yi Shi2,5 & George F. Gao1,2,3,5,6
Since December 2013, at least three cases of human infections with H10N8 avian influenza
virus have been reported in China, two of them being fatal. To investigate the epidemic
potential of H10N8 viruses, we examined the receptor binding property of the first human
isolate, A/Jiangxi-Donghu/346/2013 (JD-H10N8), and determined the structures of its
haemagglutinin (HA) in complex with both avian and human receptor analogues. Our results
suggest that JD-H10N8 preferentially binds the avian receptor and that residue
R137—localized within the receptor-binding site of HA—plays a key role in this preferential
binding. Compared with the H7N9 avian influenza viruses, JD-H10N8 did not exhibit
the enhanced binding to human receptors observed with the prevalent H7N9 virus isolate
Anhui-1, but resembled the receptor binding activity of the early-outbreak H7N9 isolate
(Shanghai-1). We conclude that the H10N8 virus is a typical avian influenza virus.
DOI: 10.1038/ncomms6600
1 College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. 2 CAS Key Laboratory of Pathogenic Microbiology and Immunology,Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. 3 National Institute for Viral Disease Control and Prevention, Chinese Centerfor Disease Control and Prevention (China CDC), Beijing 102206, China. 4 State Key Laboratory of Agro-biotechnology, China Agricultural University, Beijing100193, China. 5 Research Network of Immunity and Health (RNIH), Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing 100101, China.6 Office of Director-General, Chinese Center for Disease Control and Prevention (China CDC), Beijing 102206, China. * These authors contributed equally tothis work. Correspondence and requests for materials should be addressed to G.F.G. (email: [email protected]).
NATURE COMMUNICATIONS | 6:5600 | DOI: 10.1038/ncomms6600 | www.nature.com/naturecommunications 1
& 2015 Macmillan Publishers Limited. All rights reserved.
Nucleic Acids Research, 2014 1doi: 10.1093/nar/gku1351
Structural basis of DNA recognition by PCG2 reveals anovel DNA binding mode for winged helix-turn-helixdomainsJunfeng Liu1,†, Jinguang Huang1,2,3,†, Yanxiang Zhao1,†, Huaian Liu1, Dawei Wang1,2,Jun Yang1,2, Wensheng Zhao1,2, Ian A. Taylor4 and You-Liang Peng1,2,*
1MOA Key Laboratory of Plant Pathology, China Agricultural University, Beijing 100193, China, 2State key Laboratoryof Agrobiotechnology, China Agricultural University, Beijing 100193, China, 3College of Agronomy and PlantProtection, Qingdao Agricultural University, Qingdao, Shandong 266109, China and 4Division of Molecular Structure,MRC-NIMR, London, NW7 1AA, UK
Received September 18, 2014; Revised December 11, 2014; Accepted December 15, 2014
ABSTRACT
The MBP1 family proteins are the DNA binding sub-units of MBF cell-cycle transcription factor com-plexes and contain an N terminal winged helix-turn-helix (wHTH) DNA binding domain (DBD). Althoughthe DNA binding mechanism of MBP1 from Saccha-romyces cerevisiae has been extensively studied,the structural framework and the DNA binding modeof other MBP1 family proteins remains to be dis-closed. Here, we determined the crystal structure ofthe DBD of PCG2, the Magnaporthe oryzae ortho-logue of MBP1, bound to MCB–DNA. The structurerevealed that the wing, the 20-loop, helix A and he-lix B in PCG2–DBD are important elements for DNAbinding. Unlike previously characterized wHTH pro-teins, PCG2–DBD utilizes the wing and helix-B tobind the minor groove and the major groove of theMCB–DNA whilst the 20-loop and helix A interactnon-specifically with DNA. Notably, two glutaminesQ89 and Q82 within the wing were found to recog-nize the MCB core CGCG sequence through makinghydrogen bond interactions. Further in vitro assaysconfirmed essential roles of Q89 and Q82 in the DNAbinding. These data together indicate that the MBP1homologue PCG2 employs an unusual mode of bind-ing to target DNA and demonstrate the versatility ofwHTH domains.
INTRODUCTION
The START point, also referred to as the restriction check-point in the mammalian cell cycle, is the period betweenlate G1 to S in the cell cycle (1–3). During this stage, many
genes required for DNA replication are induced in prepara-tion for DNA synthesis in the S-phase. In the mammaliancell cycle, heterodimeric E2F/DP complexes regulate tran-scription at the restriction checkpoint (4). In comparison,transcription of the START genes in Saccharomyces cere-visiae is controlled by the MBF (MCB-Binding Factor) andSBF (SCB-Binding Factor) transcription factor complexes(1–3) and by the two functionally homologous complexes,Res1/Cdc10 and Res2/Cdc10 in the fission yeast Schizosac-charomyces pombe (5–9).
In the yeast heteromeric transcription factor complexes,a common subunit such as Swi6 or Cdc10 is required fortranscription activation. However, these subunits lack anyDNA binding activity (2,10 and 11). Instead, DNA recog-nition is provided by the other subunit of the MBF and SBFcomplexes. In S. cerevisiae, Swi6 combines with MBP1 toform MBF and with Swi4 to form SBF (1). In S. pombe,Res1 or Res2 provides the DNA binding activity for theRes1/Cdc10 and Res2/Cdc10 protein complexes, respec-tively (7). The four DNA binding proteins, MBP1, Swi4,Res1 and Res2 all have the same arrangement of domains,each consisting of an N-terminal DNA binding domain(DBD), a C-terminal heteromerization domain and a cen-tral ANK(Ankyrin) repeat region (11). In each of the fourproteins, the N-terminal DBD is responsible for recognizingdifferent DNA sequences. Swi4 binds to the SCB (Swi6/4dependent cell cycle box) motif, 5′-CACGAAA-3′, whileMBP1, Res1 and Res2 binding to the MCB (Mlu I cell cyclebox) sequences with consensus 5′-ACGCGTNA-3′.
In the past decade, a number of studies have character-ized the interaction between MBP1 and MCB–DNA se-quences (12–20). The structure of the DBD of MBP1 hasbeen determined by both X-ray crystallography and nu-clear magnetic resonance (NMR) methods and revealedthat MBP1–DBD has the same topology as the winged
*To whom correspondence should be addressed. Tel: +86 106 273 3607; Fax: +86 106 273 3607; Email: [email protected]†The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors.
C© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), whichpermits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acids Research Advance Access published December 29, 2014 at U
niversidad Polité
cnica de Madrid on January 13, 2015
http://nar.oxfordjournals.org/D
ownloaded from
Structure of human MDM2 complexedwith RPL11 reveals the molecular basisof p53 activationJiangge Zheng,1 Yue Lang,1,2 Qi Zhang,1 Di Cui,1,2 Haili Sun,1,2 Lun Jiang,1 Zhenhang Chen,1
Rui Zhang,1,2 Yina Gao,1 Wenli Tian,3 Wei Wu,1 Jun Tang,1,2 and Zhongzhou Chen1
1State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100193, China; 2College of VeterinaryMedicine, China Agricultural University, Beijing 100193, China; 3Institute of Apicultural Research, Chinese Academy ofAgricultural Sciences, Beijing 100093, China
The central regionofMDM2 is critical for p53 activation and tumor suppression.Upon ribosomal stress, this region isbound by ribosomal proteins, particularly ribosomal protein L11 (RPL11), leading to MDM2 inactivation and sub-sequent p53 activation.Here,we solved the complex structure of humanMDM2–RPL11 at 2.4Å.MDM2extensivelyinteracts with RPL11 through an acidic domain and two zinc fingers. Formation of the MDM2–RPL11 complex in-duces substantial conformational changes inbothproteins.RPL11,unable tobindMDM2mutants, fails to induce theactivation of p53 in cells.MDM2mimics 28S rRNAbinding to RPL11. TheC4 zinc finger determines RPL11 bindingtoMDM2 but not its homolog, MDMX. Our results highlight the essential role of the RPL11–MDM2 interaction inp53 activation and tumor suppression and provide a structural basis for potential new anti-tumor drug development.
[Keywords: cancer; RPL11—MDM2; p53 activation; molecular basis; complex structure]
Supplemental material is available for this article.
Received March 9, 2015; revised version accepted June 30, 2015.
The tumor suppressor p53 plays a critical role in tumorsuppression, and its inactivation has been linked to tu-morigenesis. In >50% of human cancers, p53 loss-of-func-tion mutations occur (Ozaki and Nakagawara 2011). Forcancers in which p53 is not mutated, its functions arecompromised because of defects that occur in othercomponents of the pathway. p53 is a transcription factorthat can up-regulate many downstream genes that are in-volved in cell cycle arrest, apoptosis, and senescence in re-sponse to a wide range of stress stimuli (Vousden andPrives 2009). Under normal physiological conditions, theanti-proliferative functions of p53 are strictly inhibited,mainly by the actions of an E3 ubiquitin ligase, MDM2,which mediates p53 ubiquitination and degradation.The MDM2 gene is also a p53 target, which forms a feed-back loop that restrains p53 activity (Pant et al. 2013). Thenegative regulation of p53 byMDM2 has been geneticallyconfirmed, as the early embryonic lethality caused bydeletion of theMDM2 gene can be rescued by p53 deletion(Montes de Oca Luna et al. 1995).
MDM2 is a focal point for p53 activation in response tovarious stress signals, such asDNAdamage, oncogenic ac-tivation, and ribosomal stresses (Zhou et al. 2012). Ribo-somal stresses, which are caused by impaired ribosome
biogenesis, can activate p53 through the inhibition ofMDM2 by ribosomal proteins (RPs), notably RPL11 andRPL5 (Lohrum et al. 2003; Zhang et al. 2003; Fumagalliet al. 2012). Earlier studies have suggested that ribosomalstress can affect the integrity of the nucleolus, resultingin the passive release of RPs into the nucleoplasm, wherethey bind MDM2 and inhibit its activity (Zhang and Lu2009; Miliani de Marval and Zhang 2011). Recently, ithas been shown that RPL11 can actively accumulate inthe nucleoplasm via the specific up-regulation of RPL11mRNA translation induced by the inhibition of 40Sribosome biogenesis (Fumagalli et al. 2009; Bursac et al.2014). Among the RPs that can bind to MDM2, RPL11has been reported to play the most significant role inp53 activation and ribosomal stress sensing (Fumagalliet al. 2009; Zhou et al. 2012; Kimet al. 2014). The accumu-lation of RPL11 in the nucleoplasm appears to be the keymechanism for both MDM2 inactivation and p53 activa-tion. Further studies have suggested that RPL11 in the nu-cleoplasm can be stabilized by forming a complex withRPL5 and 5S rRNA (Donati et al. 2013; Sloan et al. 2013).
Corresponding authors: [email protected], [email protected] is online at http://www.genesdev.org/cgi/doi/10.1101/gad.261792.115.
© 2015 Zheng et al. This article is distributed exclusively by Cold SpringHarbor Laboratory Press for the first six months after the full-issuepublication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml).After six months, it is available under a Creative Commons License(Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
1524 GENES & DEVELOPMENT 29:1524–1534 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/15; www.genesdev.org
ARTICLE
Received 25 Aug 2014 | Accepted 11 Feb 2015 | Published 23 Mar 2015
The amino-terminal structure of human fragileX mental retardation protein obtained usingprecipitant-immobilized imprinted polymersYufeng Hu1,*, Zhenhang Chen2,*, Yanjun Fu2, Qingzhong He3, Lun Jiang2, Jiangge Zheng2, Yina Gao2,
Pinchao Mei3, Zhongzhou Chen2 & Xueqin Ren1
Flexibility is an intrinsic property of proteins and essential for their biological functions.
However, because of structural flexibility, obtaining high-quality crystals of proteins with
heterogeneous conformations remain challenging. Here, we show a novel approach to
immobilize traditional precipitants onto molecularly imprinted polymers (MIPs) to facilitate
protein crystallization, especially for flexible proteins. By applying this method, high-quality
crystals of the flexible N-terminus of human fragile X mental retardation protein are obtained,
whose absence causes the most common inherited mental retardation. A novel KH domain
and an intermolecular disulfide bond are discovered, and several types of dimers are found in
solution, thus providing insights into the function of this protein. Furthermore, the precipitant-
immobilized MIPs (piMIPs) successfully facilitate flexible protein crystal formation for five
model proteins with increased diffraction resolution. This highlights the potential of piMIPs
for the crystallization of flexible proteins.
DOI: 10.1038/ncomms7634
1 Department of Environmental Sciences and Engineering, College of Resources and Environmental Sciences, China Agricultural University, Beijing 100193,China. 2 State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100193, China. 3 Department of Biochemistry and MolecularBiology, National Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking UnionMedical College, Beijing 100005, China. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed toZ.C. (email: [email protected]) or to X.R. (email: [email protected]).
NATURE COMMUNICATIONS | 6:6634 | DOI: 10.1038/ncomms7634 | www.nature.com/naturecommunications 1
& 2015 Macmillan Publishers Limited. All rights reserved.
JCB: Article
JCB 209
The Rockefeller University Press $30.00J. Cell Biol. Vol. 210 No. 2 209–224www.jcb.org/cgi/doi/10.1083/jcb.201503039
Introduction
The mitotic spindle is a bipolar array of microtubules (MTs) required for the symmetrical distribution of chromosomes to each daughter cell (Merdes et al., 2000; Silk et al., 2009). The process of bipolar spindle formation is controlled by both the centrosome- and chromatin-mediated pathways. Whereas the minus ends of spindle MTs cluster together at the spindle poles, their plus ends grow toward the cell equator and capture the kinetochores (Gadde and Heald, 2004; Wong et al., 2006; Radulescu and Cleveland, 2010). Ubiquitination is a wide-spread modification that ensures fidelity of mitotic progression (Fournane et al., 2012). Ubiquitination is highly dynamic and reversible, and is determined by ubiquitin ligases and deubiq-uitinating enzymes (DUBs) (Komander et al., 2009; Komander and Rape, 2012). Despite recent advances in our understanding of the E3 ubiquitin ligases, the precise roles and substrate spec-ificity of DUBs in the regulation of mitosis are only beginning to be understood (Fournane et al., 2012).
BRCC36 was identified as a component of the BRCA1–BRCA2-containing complex (BRCC) (Dong et al., 2003). It is
a JAMM/MPN+-containing DUB that preferentially cleaves K63-linked polyubiquitin chains (K63Ubs) (Cooper et al., 2009) and exists in at least two distinct complexes, the Rap80 complex (also called the BRCA1-A complex) and the BRCC36 isopeptidase complex (BRISC) (Feng et al., 2010; Hu et al., 2011). The Rap80 complex consists of five proteins (Rap80, BRCC36, MERIT40/NBA1, BRE/BRCC45, and Abraxas) and has been shown to disassemble K63Ub upon targeting to DNA double-strand breaks (Sobhian et al., 2007; Feng et al., 2009; Shao et al., 2009b; Wang et al., 2009). The BRISC complex contains four stoichiometric subunits: ABRO1/KIAA0157, BRCC36, MERIT40/NBA1, and BRCC45/BRE (Cooper et al., 2009; Feng et al., 2010; Hu et al., 2011). BRCC36 and ABRO1 are the two most important components, as they control BRISC DUB activity and cytoplasmic localization, whereas the other two contribute to the integrity and stability of the complex (Cooper et al., 2010; Feng et al., 2010; Hu et al., 2011). The biochemical activity of BRISC has been well characterized, and it has been shown to function as a DUB that specifically cleaves K63Ubs (Cooper et al., 2009, 2010). BRISC was re-cently shown to deubiquitinate IFNAR1 and thereby regulate
Deubiquitinating enzymes (DUBs) negatively regulate protein ubiquitination and play an important role in diverse phys-iological processes, including mitotic division. The BRCC36 isopeptidase complex (BRISC) is a DUB that is specific for lysine 63–linked ubiquitin hydrolysis; however, its biological function remains largely undefined. Here, we identify a critical role for BRISC in the control of mitotic spindle assembly in cultured mammalian cells. BRISC is a microtubule (MT)-associated protein complex that predominantly localizes to the minus ends of K-fibers and spindle poles and di-rectly binds to MTs; importantly, BRISC promotes the assembly of functional bipolar spindle by deubiquitinating the es-sential spindle assembly factor nuclear mitotic apparatus (NuMA). The deubiquitination of NuMA regulates its interaction with dynein and importin-β, which are required for its function in spindle assembly. Collectively, these results uncover BRISC as an important regulator of the mitotic spindle assembly and cell division, and have important implications for the development of anticancer drugs targeting BRISC.
The deubiquitinating enzyme complex BRISC is required for proper mitotic spindle assembly in mammalian cells
Kaowen Yan,1,3* Li Li,1* Xiaojian Wang,4 Ruisha Hong,1,5 Ying Zhang,1 Hua Yang,1 Ming Lin,1 Sha Zhang,1 Qihua He,2 Duo Zheng,5 Jun Tang,4 Yuxin Yin,3 and Genze Shao1,3
1Department of Cell Biology, School of Basic Medical Sciences, 2Center of Medical and Health Analysis, and 3Institute of Systems Biomedicine, Beijing Key Laboratory of Tumor Systems Biology, Peking University, Beijing 100191, China
4State Key Laboratory of Agrobiotechnology, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China5School of Medicine, Shenzhen University, Shenzhen, Guangdong 518060, China
© 2015 Yan et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
*K. Yan and L. Li contributed equally to this paper.Correspondence to Genze Shao: [email protected]; or Li Li: [email protected] used in this paper: BRISC, BRCC36 isopeptidase complex; DUB, deubiquitinating enzyme; K63Ub, K63-linked polyubiquitin chain; MAP, microtu-bule-associated protein; MT, microtubule; NEBD, nuclear envelope breakdown; NOC, nocodazole; NuMA, nuclear mitotic apparatus; pH3, phospho-histone H3; SAF, spindle assembly factor; STLC, S-trityl-l-cysteine; WT, wild type.
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Published July 20, 2015
http://jcb.rupress.org/content/suppl/2015/07/20/jcb.201503039.DC1.html Supplemental Material can be found at:
Cell Reports
Article
The Msi Family of RNA-Binding Proteins FunctionRedundantly as Intestinal OncoproteinsNing Li,1,13 Maryam Yousefi,9,13 Angela Nakauka-Ddamba,13 Fan Li,5,8 Lee Vandivier,5,9 Kimberly Parada,13
Dong-Hun Woo,13 Shan Wang,1,13 Ammar S. Naqvi,5 Shilpa Rao,6 John Tobias,6 Ryan J. Cedeno,9,13 Gerard Minuesa,3
Katz Y,2 Trevor S. Barlowe,3 Alexander Valvezan,9,10 Sheila Shankar,13 Raquel P. Deering,2 Peter S. Klein,9,10,12
Shane T. Jensen,7 Michael G. Kharas,3 Brian D. Gregory,5,8 Zhengquan Yu,1,* and Christopher J. Lengner4,9,11,12,13,*1State Key Laboratories for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100194, China2Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA3Molecular Pharmacology and Chemistry Program, Experimental Therapeutics Center and Center for Stem Cell Biology, Memorial
Sloan-Kettering Cancer Center, New York, NY 10065, USA4Center for Molecular Studies in Digestive and Liver Diseases5Department of Biology, School of Arts and Sciences6PENN Molecular Profiling Facility7Department of Statistics, The Wharton School8Genomics and Computational Biology Graduate Program9Cell and Molecular Biology Graduate Program10Department of Medicine, School of Medicine11Department of Cell and Developmental Biology, School of Medicine12Institute for Regenerative Medicine13Department of Biomedical Sciences, School of Veterinary Medicine
University of Pennsylvania, Philadelphia, PA 19104, USA
*Correspondence: [email protected] (Z.Y.), [email protected] (C.J.L.)http://dx.doi.org/10.1016/j.celrep.2015.11.022
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
SUMMARY
Members of the Msi family of RNA-binding proteinshave recently emerged as potent oncoproteins in arange of malignancies. MSI2 is highly expressed inhematopoietic cancers, where it is required for dis-ease maintenance. In contrast to the hematopoieticsystem, colorectal cancers can express both Msifamily members, MSI1 and MSI2. Here, we demon-strate that, in the intestinal epithelium, Msi1 andMsi2 have analogous oncogenic effects. Further,comparison of Msi1/2-induced gene expression pro-grams and transcriptome-wide analyses of Msi1/2-RNA-binding targets reveal significant functionaloverlap, including induction of the PDK-Akt-mTORC1 axis. Ultimately, we demonstrate thatconcomitant loss of function of both MSI familymembers is sufficient to abrogate the growth of hu-man colorectal cancer cells, and Msi gene deletioninhibits tumorigenesis in several mousemodels of in-testinal cancer. Our findings demonstrate that MSI1andMSI2 act as functionally redundant oncoproteinsrequired for the ontogeny of intestinal cancers.
INTRODUCTION
Mammalian orthologs of the Drosophila melanogaster Musashi
RNA-binding protein include Msi1/MSI1 and Msi2/MSI2.
Drosophila Musashi governs asymmetric cell fate determination
in neuroblasts through translational suppression of mRNAs
encoding a lineage determinant (Nakamura et al., 1994; Okabe
et al., 2001). A similar role for Msi2 in regulating asymmetric
fate determination has been proposed based on analysis of
asymmetric partitioning of the Msi RNA-binding target Numb in
hematopoietic stem cells with Msi2 gain or loss of function (Kha-
ras et al., 2010; Park et al., 2014). Besides a potential role in gov-
erning asymmetric cell division, Msi proteins act as potent onco-
proteins in a number of cancers. In particular, Msi2/MSI2 is a
cooperative oncoprotein in hematopoietic malignancies, where
it sustains a cancer stem cell self-renewal program through inter-
action with a number of mRNA-binding targets (Ito et al., 2010;
Kharas et al., 2010; Park et al., 2014, 2015). Whereas significant
progress has been made in understanding the contribution of
Msi2 to hematopoietic malignancies, very little is known about
the functional contribution of Msi proteins to oncogenic transfor-
mation in other human malignancies and murine tumor models.
In the hematopoietic system, Msi2 is the only Msi family mem-
ber expressed and its expression is largely restricted to the
hematopoietic stem cell compartment. In contrast, Msi1 and
Msi2 are coexpressed in the putative stem cell compartments
of a variety of other tissues including the hair follicle (Su-
giyama-Nakagiri et al., 2006), mammary gland (Clarke et al.,
2003; Katz et al., 2014;Wang et al., 2008), germ cells (Sutherland
et al., 2014), intestinal epithelium (Kayahara et al., 2003; Li et al.,
2014; Potten et al., 2003; Wang et al., 2015), and neural epithe-
lium (Sakakibara et al., 2002). The observation that both Msi1
and Msi2 are coexpressed in these tissues, coupled with an
absence of phenotype upon genetic ablation of either Msi1 or
2440 Cell Reports 13, 2440–2455, December 22, 2015 ª2015 The Authors
ARTICLE
Received 1 Dec 2014 | Accepted 30 Jan 2015 | Published 16 Mar 2015
Transformation of the intestinal epithelium by theMSI2 RNA-binding proteinShan Wang1,2,*, Ning Li2,*, Maryam Yousefi2,3, Angela Nakauka-Ddamba2, Fan Li4,5,6, Kimberly Parada2,
Shilpa Rao7, Gerard Minuesa8, Yarden Katz9, Brian D. Gregory4,5,6, Michael G. Kharas8, Zhengquan Yu1 &
Christopher J. Lengner2,3,10,11,12
The MSI2 RNA-binding protein is a potent oncogene playing key roles in haematopoietic stem
cell homeostasis and malignant haematopoiesis. Here we demonstrate that MSI2 is
expressed in the intestinal stem cell compartment, that its expression is elevated in colorectal
adenocarcinomas, and that MSI2 loss-of-function abrogates colorectal cancer cell growth.
MSI2 gain-of-function in the intestinal epithelium in a drug-inducible mouse model is
sufficient to phenocopy many of the morphological and molecular consequences of acute loss
of the APC tumour suppressor in the intestinal epithelium in a Wnt-independent manner.
Transcriptome-wide RNA-binding analysis indicates that MSI2 acts as a pleiotropic inhibitor
of known intestinal tumour suppressors including Lrig1, Bmpr1a, Cdkn1a and Pten. Finally, we
demonstrate that inhibition of the PDK–AKT–mTORC1 axis rescues oncogenic consequences
of MSI2 induction. Taken together, our findings identify MSI2 as a central component in an
unappreciated oncogenic pathway promoting intestinal transformation.
DOI: 10.1038/ncomms7517
1 State Key Laboratories for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100194, China. 2 Department of AnimalBiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. 3 Cell and Molecular Biology Graduate Program,University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. 4 Department of Biology, School of Arts and Sciences, University of Pennsylvania,Philadelphia, Pennsylvania 19104, USA. 5 PENN Genome Frontiers Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. 6 Genomicsand Computational Biology Graduate Program, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. 7 PENN Molecular Profiling Facility,University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. 8 Molecular Pharmacology and Chemistry Program, Experimental Therapeutics Center andCenter for Stem Cell Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA. 9 The Broad Institute of Harvard and MIT, 415 MainStreet, Cambridge, Massachusetts 02142, USA. 10 Center for Molecular Studies in Digestive and Liver Diseases, University of Pennsylvania, Philadelphia,Pennsylvania 19104, USA. 11 Department of Cell and Developmental Biology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104,USA. 12 Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. * These authors contributed equally to thiswork. Correspondence and requests for materials should be addressed to Z.Y. (email: [email protected]) or to C.J.L. (email: [email protected]).
NATURE COMMUNICATIONS | 6:6517 | DOI: 10.1038/ncomms7517 | www.nature.com/naturecommunications 1
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