Slide 1

Architectural TFs Slide 2 MBV4230 Odd S. Gabrielsen Overview DNA-binding TFs General principles Architectural factors Slide 3 MBV4230 Odd S. Gabrielsen Recognition of response elements Activators vrs Architectural TFs Ordinary activators with sequence specific DNA binding Sentrale rekrutteringspunkt for assembly of transcriptionscomplexer Architectural transcription factors playing a more structural role in the assembly of transcription complexes Slide 4 MBV4230 Odd S. Gabrielsen Architectural TFs - brief history Transcription activation - focus on more and more dimentions 70-ties: 1-Dimentional understanding RNAPII: TFs binding specific cis-elements required for selective transcription TFs mediate regulatory response 80-ties: 2-Dimentional understanding Promoters/enhancers: clusters of cis-elements complex regulation - Several buttons have to be pushed simultaneouly Ptashnes simplification - mixed order OK 90-ties: 3-Dimentional understanding Three-dimentional assembly of TFs required for correkt biological response Slide 5 MBV4230 Odd S. Gabrielsen 3D protein-promoter complexes - factors dedicated architecture some factors has a pure architectural function designated architectural transcription factors They lack a transactivation domain (TAD) Do not function out of their natural context (in contrast to ordinary acitvators) Their function is to confer a specific 3D structure on DNA Slide 6 MBV4230 Odd S. Gabrielsen Prokaryotes - the first discovered architectural TF Crothers 1980 - simple methode to detect bending EMSA with a series of equally long DNA probes, but where the binding site is placed in different positions along the DNA. Bending causes unequal migration of protein-DNA complexes depending on location of bend. Nash 1985 - integration host factor (IHF) Kustu 1990 - bending bringing factors together activation IHF NIFA RNApol. Slide 7 MBV4230 Odd S. Gabrielsen Classical HMG-proteins non-histone chromatin proteins - original defining criteria high mobility in PAGE soluble in 2-5% TCA small < 30 kDa High content of charged amino acids abundant: 1 per. 10-15 nucleosomes Slide 8 MBV4230 Odd S. Gabrielsen Classical HMG-proteins Three classes of HMG DNA-binding proteins HMG-box family Eks: HMG 1 and HMG 2 Bends DNA substantially Facilitators of nucleoprotein complexes HMG-AT-hook family Eks.: HMGI(Y) Antagonizing intrinsic distortions in the conformation of AT-rich DNA HMG-nucleosome binding family Eks.: HMG14 and 17 Mediates moderate destabilization of chromatin higher-order structure Not present in yeast or fly HMGB HMGA HMGN Slide 9 HMGB-proteins Slide 10 MBV4230 Odd S. Gabrielsen HMG1 and 2 3 structural domains A and B with high homology (80-90 aa) acidic C-terminal Interaction with DNA (and histones?) A and B DNA C-term histone H1 or unknown function A + + B NC ---- DNA Histon H1? Slide 11 MBV4230 Odd S. Gabrielsen HMG-boxes in architectural proteins One or two HMG-box domains acidic basic 30 Asp/Glu Slide 12 MBV4230 Odd S. Gabrielsen First eukaryotic architectural TF: LEF1 (Grosschedl 1992) LEF1: a cell type-specific TF LEF1 contains an HMG-related domain LEF1: a sequence-specific TF that binds CCTTTGAAG found in enhancer of TCR LEF1 induces strong bending of DNA - about 130 o Induced bending brings nearly TFs in contact Slide 13 MBV4230 Odd S. Gabrielsen LEF1 3D Slide 14 MBV4230 Odd S. Gabrielsen LEF1 3D Slide 15 MBV4230 Odd S. Gabrielsen A whole family of architectural TFs with HMG-domains UBF has repeated HMG-homologous repeats 4-6 ex dimer 10 HMG-like domains activator of rRNA gener UBF-DNA complex scaffold for SL-1 recruitment Interaction with 180 bp that is packed into a distinct structure DNA-motif in a series of TFs: HMG-box designate the DNA-sequence-motif HMG-domain designate the protein motif Slide 16 MBV4230 Odd S. Gabrielsen Two subclasses of HMG-domain proteins Proteins with multiple HMG-domains low sequence-specificity Ubiquitous - found in all cell types eks.: HMG1, HMG2, ABF-2, UBF Proteins with enkelt HMG-domain (moderate) sequence-specificity Cell type-specific eks.: LEF-1, SRY, TCF-1, Sox, Mat- 1, Ste11, Rox1 Slide 17 MBV4230 Odd S. Gabrielsen Characteristic DNA-binding binds minor groove induce bending of DNA has high affinity for non- canonical DNA-structures such as : cruciform DNA 4-way junctions cisplatin kinked DNA + Slide 18 MBV4230 Odd S. Gabrielsen NMR-structures HMG1 B-domain LEF-1 SRY Yeast Nhp6p Drosophila HMG-D Common: 3 helix L-form heliks II and III form an angle of about 80 o Conserved aromatic aa in kink Basic concave side interact with DNA Slide 19 MBV4230 Odd S. Gabrielsen Similar structures of HMG domains Slide 20 MBV4230 Odd S. Gabrielsen Minor groove binding, intercalation and bending Objective: shorten the distance between cis-elements facilitating interaction between bound factors DNA MBV4230 Odd S. Gabrielsen HMGA proteins heavily modified The HMGA proteins are among the most highly phosphorylated proteins in the mammalian nucleus. Cell cycle-dependent phosphorylation pga cdc2 activity in the G2/M phase of the cycle. Sites: T53 and T78 situated at the N-terminal ends of the 2. and 3. AT-hook. Phosphorylation significantly reduces (>20-fold) DNA binding. HMGA proteins are the downstream targets of a number of signal transduction pathways that lead to phosphorylation. HMGA proteins are also acetylated at Lys65 by CBP and at Lys71 by PCAF as well as methylated and poly-ADP ribosylated Hypothesis: Modifications may alter DNA-binding specificity? Slide 31 MBV4230 Odd S. Gabrielsen Architectural effects Binding of full-length HMGA proteins can bend, straighten, unwind and induce loop formation in linear DNA molecules in vitro. Multiple contact points with DNA may alter conformation of DNA A single AT-hook preferentially binds to stretches of 4-6 bp of AT-rich sequence, and partially neutralizes the negatively charged backbone phosphates on only one face of the DNA helix. The number and spacing of AT-rich binding sites in DNA influences the conformation of bound DNA and the biological effects elicited. HMGA may also induce conformational change in proteins HMGA forms protein-protein interactions with other transcription factors, which alters the 3D structure of the factors resulting in enhanced DNA binding and transcriptional activation. Slide 32 MBV4230 Odd S. Gabrielsen Maniatis: HMGI(Y) contributes to formation of enhanceosomes virus-inducible enhancer in the IFN- gene (human interferon ) cis-elements for NF-kB, IRF-1, ATF-2-c-Jun Synthetic (multiple cis-elements) enhancer natural Too high basal transcription Less induction Responds to several stimuli, while natural enhancer only responds to virus Biological function depends of HMGI(Y) as architectural component HMG I(Y) First described by Lund and Laland binds AT-rich DNA in minor groove (AT-hook) Slide 33 MBV4230 Odd S. Gabrielsen Recent verision Slide 34 MBV4230 Odd S. Gabrielsen Other functions of HMGA proteins HMGA and cancer HMGI/Y proteins are also involved in a diverse range of other cellular processes including pathologic processes such as neoplastic transformation and metastatic progression. Chromosomal translocations in a long 3.intron Intron 3 of the HMGA2 genes is extremely long (>25 kb in human and >60 kb in mouse) and separates the three exons that contain the AT hook motifs from the remainds of the 3- untranslated tail region of the gene. Translocation within the exceptionally long third intron are commonly observed in benign mesenchymal tumors. Slide 35 3. subgruppe: HMGN Slide 36 MBV4230 Odd S. Gabrielsen HMGN proteins Three functional domains of the HMGN proteins: a bipartite nuclear localization signal (NLS), a nucleosomal binding domain (NBD) and a chromatin-unfolding domain (CHUD). The CHUD domain has a net negative charge. Binding of HMGN proteins to nucleosomes decreases the compactness of chromatin, and facilitates trx Slide 37 MBV4230 Odd S. Gabrielsen HMGN: architectural elements reducing compactness of chromatin Model of the binding of HMGN proteins to chromatin HMGNs interact with both the DNA and the histone component of the nucleosome The CHUD domain interacts with the amino terminus of histone H3. May also affect H1 binding Incorporation of HMGN proteins into chromatin is believed to reduce the compactness of the chromatin fiber. Slide 38 MBV4230 Odd S. Gabrielsen Alan P. Wolffe on HMG proteins ` There must be money in proteins that control your fat, your teeth, your sex and your health (besides minor things such as transcription, cell division and DNA recombination and repair). '