Methylation of histone H3K4 correlates with active transcription, but it is not clear how this modification affects gene expression. Here we show that one function of H3K4 methylation is to recruit an HDAC complex to 5′ ends of genes via the PHD domain of the Set3 protein. Set3C contains two histone deacetylase subunits, Hos2 and Hst1. This pathway is distinct from the pathway used further downstream, where Set2 methylation of H3K36 targets histone deacetylation by the Rpd3C(S) complex (Carrozza et al., 2005
; Keogh et al., 2005
). We propose that transcribed genes can be divided into at least three distinct chromatin zones (). Promoter regions are marked by high acetylation levels and H3K4me3, but low nucleosome density. A region characterized by H3K4me2 lies just downstream, where acetylation levels are suppressed by Set3C. More distal transcribed regions have high H3K36 methylation levels and Rpd3C(S) deacetylation activity. How the boundaries between these regions are established and whether they are sharp or fluid will be important to explore.
Model for regulation of histone acetylation by H3K4 and K36 methylation
Both H3K4me3 and H3K4me2 localize near transcription start sites, but H3K4me2 peaks slightly downstream of H3K4me3 (Barski et al., 2007
; Bernstein et al., 2005
; Liu et al., 2005
; Pokholok et al., 2005
). The transition between these modifications appears to be regulated by multiple factors and our findings indicate that these two methylation levels define distinct chromatin states. Deletion of the Set1 RRM, which specifically abrogates H3K4me3 while preserving H3K4me2, does not increase 5′ acetylation levels and has no effect on Set3C recruitment. In contrast, cells lacking both H3K4me2 and me3 (rad6Δ
) lose Set3C binding and have increased levels of 5′ acetylation. Therefore, H3K4me2 must be the relevant mark for recruiting Set3C. Recently, loss of the Bur1-Bur2 kinase or PAF complex were shown to cause increased histone acetylation at 5′ ends of genes independently of the Set2/Rpd3C(S) pathway (Chu et al., 2007
). Both the Bur and PAF complexes promote H2B ubiquitylation and thereby H3K4me2 and me3 (Laribee et al., 2005
; Wood et al., 2005
), so their effects on acetylation may be mediated by Set3C.
Multiple findings suggest the physiological role of Set3C is in transcription regulation. Set3C gene deletions show synthetic negative genetic interactions with deletions of many transcription related genes, including those for Set2, the SWR/Htz1 complex, Rpd3(L)C, and many components of the SAGA, THO, PAF, and basal transcription complexes (Collins et al., 2007
; Krogan et al., 2003
). Loss of Set3C derepresses meiotic gene transcription (Pijnappel et al., 2001
) but impairs efficient transcription of GAL
genes (Wang et al., 2002
). Deletion of SET3
reduces RNApII crosslinking to GAL1
() and loss of Set3C or a Set3 PHD finger mutation results in inefficient induction of GAL1
(). These mutations also confer sensitivity to MPA, a phenotype often correlated with inefficient transcription elongation (). The experiments presented here strongly suggest that Set3C functions near 5′ ends of genes to affect transcription.
At the molecular level, deacetylation of 5′ transcribed regions could affect transcription in several ways. One important function of the Set1-Set3C pathway may be to limit histone acetylation from spreading beyond promoters into transcribed regions. Transcription activators bound upstream recruit HAT and chromatin remodeling complexes to promoters to remove or displace nucleosomes that would otherwise occlude the promoter. The Set1-Set3C pathway may “sharpen” this zone of nucleosome acetylation and remodeling. In the absence of Set1 or Set3C, we observe increased acetylation at 5′ ends that is often accompanied by lower histone H3 density (, , ). Although Set3C is not essential for laboratory growth, it may be critical for expressing particular genes or under more stringently selective growth conditions. An increasing number of genes exhibit regulation at the level of RNApII escape into elongation phase (so called “paused” polymerases, see Margaritis and Holstege, 2008
). It is interesting to speculate that early stages of elongation could be regulated through modifications of the first transcribed nucleosome (Morillon et al., 2005
Another function for the Set1-Set3C pathway could be to repress transcription initiation from cryptic promoters within transcribed regions. The Set2-Rpd3C(S) pathway deacetylates histones near 3′ ends of genes and this inhibits transcription initiation from cryptic promoters in that region (Carrozza et al., 2005
; Keogh et al., 2005
). However, this pathway appears to be biased towards cryptic promoters in long, infrequently transcribed genes (Li et al., 2007c
). Loss of Set3 did not lead to the same internal initiations reported for loss of Rpd3C(S) (data not shown). Unlike loss of the Set1-Set3C pathway, deletion of SET2
does not affect acetylation at two shorter genes, PYK1
(). The Set1-Set3C pathway may specifically be used for repression of cryptic transcription initiation near 5′ ends of long genes and at relatively short genes, but so far we have not observed such cryptic transcripts.
If H3K4me2 recruits Set3C, what is the role of H3K4me3? Cells expressing Set1ΔRRM lack H3K4me3 (Fingerman et al., 2005
; Schlichter and Cairns, 2005
) and have increased histone acetylation near telomeres (Fig S2
), consistent with a previous report that H3K4me3 is required for telomeric silencing (Fingerman et al., 2005
). This effect was not seen in cells lacking Set3C, arguing that telomeric deacetylation is carried out by another HDAC, most likely Sir2/3/4. How H3K4me3 promotes telomeric deacetylation is unclear, but it appears that loss of H3K4me3 allows Sir2/3/4 to bind to other regions of the genome, thereby titrating it away from telomeres (Santos-Rosa et al., 2004
; van Leeuwen and Gottschling, 2002
; Venkatasubrahmanyam et al., 2007
). H3K4 methylation levels at telomeres are much lower than that seen around transcribed regions (), making it unlikely that H3K4me3 directly promotes telomeric silencing.
The role of H3K4me3 in transcription remains unclear. We did not observe major changes in acetylation of genes in Set1ΔRRM cells. Several groups (Fingerman et al., 2005
; Schlichter and Cairns, 2005
) saw no transcription defects at several test genes in this mutant. While one analysis of gene expression in a set1Δ
strain indicated a widespread role in transcription (Boa et al., 2003
), a recent microarray analysis found that loss of Set1 only resulted in partial derepression of genes near telomeres (Venkatasubrahmanyam et al., 2007
). This study suggested that Set1 functions redundantly with Htz1 to antagonize ectopic silencing of euchromatic genes by Sir2/3/4 (Venkatasubrahmanyam et al., 2007
). H3K4me3 may directly block binding of the Sir2 complex to nucleosomes or instead antagonize repression by recruiting HATs and remodelers to promoters.
Several HAT complexes could fit this second model (). NuA3 complex contains a PHD finger protein called Yng1 that binds to H3K4me3, helping recruit the complex to 5′ ends of genes where the Sas3 subunit acetylates H3K14 (Taverna et al., 2006
). NuA3 contains a second PHD finger protein called Nto1, which binds methylated H3K36 in vitro (Shi et al., 2007
), that is also necessary for NuA3 recruitment (Martin et al., 2006
). A second HAT that may bind H3K4me3 is NuA4, which contains two chromodomain proteins, Esa1 and Eaf3, and a PHD finger protein, Yng2. The specific binding site for the Esa1 chromodomain is unknown, but Eaf3 and Yng2 bind to methylated H3K36 and H3K4, respectively. For NuA3 and NuA4, both H3K4 and H3K36 methylation could promote recruitment either together or separately. Interestingly, a slight decrease in promoter acetylation is seen upon deletion of SET2
() or EAF3
(Reid et al., 2004
), indicating that H3K36 methylation may be more important near promoters than current models predict.
Given the overall conservation of co-transcriptional H3K4 and H3K36 methylation, it is likely that higher eukaryotes also contain a deacetylase complex that is recruited by H3K4me2. Based on computational analysis of protein sequence and domain architecture, the mammalian protein that most closely resembles Set3 is MLL5, which also has a single PHD finger and SET domain. Little is known about MLL5, but it is located in a region of chromosome 7 that is often deleted in myeloid leukemias (Emerling et al., 2002
). It has also been suggested that the Set3 complex may be related to the NcoR-SMRT complexes, both of which contain an Rpd3-like protein (HDAC3 in mammals, Hos2 in yeast), a WD40 protein (TBL1 in mammals, Sif2 in yeast), and a SANT domain protein (NcoR/SMRT and yeast Snt1) (Pijnappel et al., 2001
; Yang and Seto, 2008
). It is also possible that a non-homologous complex performs an analogous role to the yeast Set3C. The mammalian ING1 and ING2 proteins contain PHD fingers and are subunits of mSin3A histone deacetylase complexes (Yang and Seto, 2008
). These ING proteins can bind to methylated H3K4, but it remains to be seen whether they target histone deacetylation to specific transcribed regions of genes.
In yeast and mammalian genomes, there are multiple SET domain and PHD finger proteins that remain to be characterized. Given the connections between the aberrant gene expression in cancer cells and the complexes containing Set1/MLL and Set2, understanding the functions of Set3 and the other Set/MLL proteins remains an important goal.