Transcripon factors Once associated with the promoter, what do transcripon factors do? u Interact with/recruit the core transcriponal machinery u Recruit chroma)n-modifying ac)vi)es Transcripon factors Once associated with the promoter, what do transcripon factors do? u Interact with/recruit the core transcriponal machinery v Co-acvators Ac)vate transcrip)on but do not bind DNA v Mediator Mul)subunit complex that “mediates” between transcripon factors/coacvators and the core transcrip)on machinery (TFII family factors, RNA polymerase II) Transcripon factors Once associated with the promoter, what do transcripon factors do? u Interact with/recruit the core transcriponal machinery u Recruit chroma)n-modifying ac)vi)es v Nucleosome remodeling v Acetylases/deacetylases v Methylases/demethylases Black – DNA Blue – H3 Green – H4 Space-filling model of a core nucleosome Blue – protein Orange - DNA Nucleosome posioning around transcripon start sites is not random DNA accessibility is important for promoter func)on, this requires that nucleosomes be re-posioned (“remodeled”) during transcripon iniaon Types of Histone Modifica)ons Taken from h,ps://www.plant-epigenome.org/category/lecture-topic/histone-modificaons h,ps://www.plant-epigenome.org/teaching-tools Types of Histone Modifica)ons phosphorylaon acetylaon ubiquinaon methylaon Bhaumik, Smith, and Shilafard, 2007. Features of Histone Modifica)ons • Covalently aached groups (usually to histone tails) Methyl Acetyl Phospho Ubiqui=n SUMO • Reversible and Dynamic – Enzymes that add/remove modificaon – Signals • Have diverse biological func=ons Cell, 111:285-91, Nov. 1, 2002 " Features of Histone Modifica)ons •! Small vs. Large groups Ub = ~8.5 kDa •! One or up to three groups per residue H4 = 14 kDa Jason L J M et al. J. Biol. Chem. 2005 Histone Modifica)ons and Modifers •! Writers: enzymes that add a mark •! Readers: proteins that bind to and “interpret” the mark •! Erasers: enzymes that remove a mark Tarakhovsky, A., Nature Immunology, 2010. Roles of Acetyla)on 1. Opens up chroman: –! Reduces charge interac=ons of histones with DNA (K has a posi=ve charge) –! Prevents chroman compac=on (H4K16ac prevents 30nm fiber formaon) Robinson et al., J. Mol. Biol., 2008. 2. Recruits chroma)n proteins with bromodomains (SWI/SNF, HATs: GCN5, p300) PCAF 3. May occur at same residues as methylaon with repressive effect (compeve antagonism) H3K27ac H3K27me3 Mujtaba et al., Oncogene, 2007. Yang and Chen, Cell Research, 2011. Roles of Lysine methylation" Roles of Lysine Methyla)on 1. Recruitment of other chroma)n proteins through specific domains: Chromodomain (CHD ATPases, HP1, PC) Chromo-like Tudor (some histone demethylases) (Royal) PhD (many chroman regulators BPTF, ING2) MBT (in some polycomb proteins) WD-40 (WDR5) Roles of Lysine Methyla)on 2. Different readout depending on level of methylaon –! Methylaon status of H3K4 is recognized by different domains •! H3K4me1: chromodomain in CHD1 (ATPase) = transcrip=on ac=vaon •! H3K4me2: WD-40 domain in WDR5 in MLL (Trx) = transcrip=on ac=vaon •! H3K4me3: PhD domain of BPTF in NURF (ISWI) = transcrip=on ac=vaon •! H3K4me3: PhD ING2 recognizes during DNA damage and shuts down ac=ve transcrip=on through mSin3a-HDAC1 Promoters Enhancers Heintzman N et al., Nature Gene=cs, 2007. Roles of Lysine Methyla)on 3. Transcriponal ac)vaon –! H3K4me3: euchroman promoter, 5’ end ac=vates transcrip=on (Set1) –! H3K36me3: in gene (ORF), transcrip=onal elongaon (Set2) Li et al., Cell, 2007. Roles of Lysine Methyla)on 4. Transcriponal repression •! H3K9me (Suv39H1) •! In promoter, represses transcrip=on •! In larger domains, heterochroman formaon •! H3K27me (EZH2) •! Repression of transcrip=on •! Polycomb group silencing •! H4K20me (Suv420H1) •! Some forms of silencing and repression of gene expression •! In repe==ve elements, similar localizaon as H3K9me3 in ES cells Li et al., Cell, 2007. Histone Ubiquinaon •! Ubiqui=naon –! H2A K119: •! Func=on: Polycomb repression (Ring1a, PCG) –! H2B K123: ac=vaon (Rad6+Bre yeast; RNF20/RNF40 and UbcH6 in mam) •! Func=on: FACT recruitment, transcrip=onal elongaon –! H3 and H4: DNA repair (CUL4) •! De-ubiqui=naon –! H2A: Dub (PCAF) •! Func=on: counteract Polycomb –! H2B: Ubp8 (SAGA) •! Func=on: transcrip=on elongaon Different histone modifications are associated with different parts of the gene Chromatin and DNA methylation define chromosome domains Different histone modificaons are recognized by different families of proteins – histone H3 as an example These proteins provide connecons to downstream partners/events, and perhaps also to upstream signaling cascades ANRV413-BI79-06 ARI 27 April 2010 21:50 JARID/ LSD1/ JMJD1/ JMJD2/ JMJD3/ FBXL/ KDM5 KDM1 KDM3 KDM4 UTX/KDM6 KDM2 4 K Q T T A R G R T G 36 A 9 A V H3 K K S P A K T 27 S G K P G R H K A A A K K I P R T H R K Q L A R 20 K A Promoter or activation G G Heterochromatin or repression K G Elongation L G Enhancer K G DNA damage G H4 S G R G K Figure 3 Substrate specificity of histone demethylases described to date. Dashed lines point to the methylated residue(s) that are demethylated by the indicated enzymes. The embedded numbers refer to the methylated amino acid residue on each histone. The general function of each mono-, di-, and trimethylation state is depicted in dots of distinct colors as shown in the figure key. Abbreviations: JARID, Jumonji and ARID domain protein (also known as KDM5); LSD1, Lysine-specific demethylase, also known as KDM1A; JMJD1, Jumonji domain protein 1, also known as KDM3; JMJD2, Jumonji domain protein 2, also known as KDM4; UTX, Ubiquitously transcribed tetratricopeptide repeat, X chromosome protein, also known as KDM6A; JMJD3, Jumonji domain protein 3, also known as KDM6B; FBXL, F-box and leucine-rich repeat protein, also known as KDM2. Recent structural efforts have provided sig- globular halves of the amine oxidase active site. nificant insights into some of these issues, as This so-called tower domain serves as the bind- by University of Kentucky on 08/16/10. For personal use only. discussed below. ing interface between LSD1 and its protein co- factor, CoREST,and distinguishes it from other amine oxidases (75, 76). Although nucleosomal LSD1-Mediated Demethylation substrates are refractory to recombinant LSD1, Annu. Rev. Biochem. 2010.79:155-179. Downloaded from arjournals.annualreviews.org and Chemical Inhibitors addition of recombinant CoREST endows nu- Analysis of the LSD1 structure has led to signif- cleosomal demethylation by LSD1, indicating icantly greater insight into its function. Over- that the primary function of CoREST is to en- all, the structure of LSD1 consists of an N- able LSD1 demethylation of nucleosomal sub- terminal SWIRM domain (74), and an amine strates (77, 78). Consistently, mutations that oxidase domain split into two halves, consist- inhibit the ability of CoREST to bind to DNA ing of a substrate-binding half and an FAD- inhibit the ability of the complex to demethy- binding half, which come together to form a late nucleosomes (76). globular domain (Figure 4a) (75, 76). The How does LSD1 achieve specificity for active site of the enzyme is located in be- H3K4me2/me1? According to these structural tween these two halves. Two long, antiparal- studies, much of the specificity of LSD1 is lel α-helices divide and project away from the intrinsic to its enzymatic pocket. The amine www.annualreviews.org • Histone Demethylases 161 ANRV413-BI79-06 ARI 27 April 2010 21:50 abcATP Mi2 ADP + Pi Coactivator? HDAC LSD1 HDAC ? 1/2 LSD1 1/2 LSD1 BHC80 BRAF CoREST ? 35 PHD RbAp MTA MBD AR/ER LSD1-CoREST LSD1-NuRD LSD1-AR/ER de PTIP Ash2L Activation-associated methyl marks SUZ12 Repression-associated methyl marks EED MOF MLL Histone acetylation RBBP2 UTX EZH2 RbBP5 Add modification WDR5 Remove modification ActivatorActivator MLL-UTX PRC2-RBBP2 Figure 5 Key histone demethylase complexes and their activities described to date. Green and red circles represent activation- and repression- associated methyl marks, respectively, and green triangles represent histone acetylation. Solid green lines and dashed red lines represent enzymatic activities that add or remove specific modifications, respectively. (a) The lysine-specific demethylase 1 (LSD1)-corepressor of RE1-silencing transcription factor (CoREST) complex. (b) The LSD1-nucleosome remodeling and deacetylase (NuRD) complex. (c) The LSD1-nuclear hormone receptor (AR/ER) complex. (d ) The mixed-lineage leukemia (MLL) protein-ubiquitously transcribed tetratricopeptide repeat, X chromosome protein (UTX) complex. (e) The Polycomb repressive complex 2 (PRC2)-retinoblastoma- binding protein 2 (RBBP2) complex. Abbreviations: AR/ER, androgen receptor/estrogen receptor; Ash2L, absent, small, or homeotic like 2; BHC80, BRAF and histone deacetylase complex 80; EED, embryonic ectoderm development; EZH2, enhancer of zeste homolog 2; HDAC1-2, histone deacetylase 1-2; MBD, methyl-CpG-binding domain protein 2; Mi2, myositis autoantigen 2; by University of Kentucky on 08/16/10. For personal use only. MOF, males absent on first; MTA, metastasis associated; PTIP, PAX interacting protein; RbAp, retinoblastoma-associated protein; RbBP, retinoblastoma-binding protein; SUZ12, suppressor of zeste homolog 12; WDR5, WD repeat domain protein 5. activity of LSD1 can also be coupled to Although human LSD1 has a clear role in Annu. Rev. Biochem. 2010.79:155-179. Downloaded from arjournals.annualreviews.org chromatin-remodeling Swi/Snf type ATPases repression as an H3K4 demethylase, there is important for transcriptional repression also evidence that it functions as an activator (Figure 5b). In the NuRD complex, the when present in other complexes, thus playing metastasis-associated (MTA) proteins function opposing roles in different physiological con- in place of CoREST and play an analogous texts. LSD1 has been demonstrated to associate function to allow LSD1 demethylation of directly with androgen receptor (AR), and in nucleosomal substrates (102). Like CoREST, this molecular context, LSD1 can demethy- the MTA proteins each contain a SANT do- late H3K9me2/me1 but not H3K4me2/1 main, which serve as Myb-like DNA-binding (Figure 5c) (103).