Madelaine Gogol

Combined Action of PHD and Chromo Domains Directs the Rpd3S HDAC to Transcribed Chromatin

Nucleosomes must be deacetylated behind elongating RNA polymerase II to prevent cryptic initiation of transcription within the coding region. RNA polymerase II signals for deacetylation through the methylation of histone H3 lysine 36 (H3K36), which provides the recruitment signal for the Rpd3S histone deacetylase complex (HDAC). The recognition of methyl H3K36 by Rpd3S requires the chromodomain of its Eaf3 subunit. Paradoxically, Eaf3 is also a subunit of the NuA4 acetyltransferase complex, yet NuA4 does not recognize methyl H3K36 nucleosomes. In Saccharomyces cerevisiae, we found that methyl H3K36 nucleosome recognition by Rpd3S also requires the plant homeobox domain (PHD) of its Rco1 subunit. Thus, the coupled chromo and PHD domains of Rpd3S specify recognition of the methyl H3K36 mark, demonstrating the first combinatorial domain requirement within a protein complex to read a specific histone code.

Global Analysis of H3K4 Methylation Defines MLL Family Member Targets and Points to a Role for MLL1-Mediated H3K4 Methylation in the Regulation of Transcriptional Initiation by RNA Polymerase II

ABSTRACT A common landmark of activated genes is the presence of trimethylation on lysine 4 of histone H3 (H3K4) at promoter regions. Set1/COMPASS was the founding member and is the only H3K4 methylase in Saccharomyces cerevisiae; however, in mammals, at least six H3K4 methylases, Set1A and Set1B and MLL1 to MLL4, are found in COMPASS-like complexes capable of methylating H3K4. To gain further insight into the different roles and functional targets for the H3K4 methylases, we have undertaken a genome-wide analysis of H3K4 methylation patterns in wild-type Mll1+/+ and Mll1−/− mouse embryonic fibroblasts (MEFs). We found that Mll1 is required for the H3K4 trimethylation of less than 5% of promoters carrying this modification. Many of these genes, which include developmental regulators such as Hox genes, show decreased levels of RNA polymerase II recruitment and expression concomitant with the loss of H3K4 methylation. Although Mll1 is only required for the methylation of a subset of Hox genes, menin, a component of the Mll1 and Mll2 complexes, is required for the overwhelming majority of H3K4 methylation at Hox loci. However, the loss of MLL3/MLL4 and/or the Set1 complexes has little to no effect on the H3K4 methylation of Hox loci or their expression levels in these MEFs. Together these data provide insight into the redundancy and specialization of COMPASS-like complexes in mammals and provide evidence for a possible role for Mll1-mediated H3K4 methylation in the regulation of transcriptional initiation.

Set2 methylation of histone H3 lysine 36 suppresses histone exchange on transcribed genes

Set2-mediated methylation of histone H3 at Lys 36 (H3K36me) is a co-transcriptional event that is necessary for the activation of the Rpd3S histone deacetylase complex, thereby maintaining the coding region of genes in a hypoacetylated state. In the absence of Set2, H3K36 or Rpd3S acetylated histones accumulate on open reading frames (ORFs), leading to transcription initiation from cryptic promoters within ORFs. Although the co-transcriptional deacetylation pathway is well characterized, the factors responsible for acetylation are as yet unknown. Here we show that, in yeast, co-transcriptional acetylation is achieved in part by histone exchange over ORFs. In addition to its function of targeting and activating the Rpd3S complex, H3K36 methylation suppresses the interaction of H3 with histone chaperones, histone exchange over coding regions and the incorporation of new acetylated histones. Thus, Set2 functions both to suppress the incorporation of acetylated histones and to signal for the deacetylation of these histones in transcribed genes. By suppressing spurious cryptic transcripts from initiating within ORFs, this pathway is essential to maintain the accuracy of transcription by RNA polymerase II.

Chromatin remodelers Isw1 and Chd1 maintain chromatin structure during transcription by preventing histone exchange

Set2-mediated methylation of histone H3 Lys36 (H3K36) is a mark associated with the coding sequences of actively transcribed genes, but it has a negative role during transcription elongation. It prevents trans-histone exchange over coding regions and signals for histone deacetylation in the wake of RNA polymerase II (RNAPII) passage. We have found that in Saccharomyces cerevisiae the Isw1b chromatin-remodeling complex is specifically recruited to open reading frames (ORFs) by H3K36 methylation through the PWWP domain of its Ioc4 subunit in vivo and in vitro. Isw1b acts in conjunction with Chd1 to regulate chromatin structure by preventing trans-histone exchange from taking place over coding regions. In this way, Isw1b and Chd1 are important in maintaining chromatin integrity during transcription elongation by RNAPII.

Histone H3 Lysine 36 Dimethylation (H3K36me2) Is Sufficient to Recruit the Rpd3s Histone Deacetylase Complex and to Repress Spurious Transcription

Histone methylation is associated with both transcription activation and repression. However, the functions of different states of methylation remain largely elusive. Here, using methyl-lysine analog technology, we demonstrate that the histone deacetylase complex, Rpd3S, can distinguish the nucleosomes methylated to different extents and that K36me2 is sufficient to target Rpd3S in vitro. Through a genome-wide survey, we identified a few mutants in which the level of K36me3 is significantly reduced, whereas the level of K36me2 is sustained. Transcription analysis and genome-wide histone modification studies on these mutants suggested that K36me2 is sufficient to target Rpd3S in vivo, thereby maintaining a functional Set2-Rpd3S pathway.

Dynamic transcriptional events in embryonic stem cells mediated by the super elongation complex (SEC)

Transcriptional regulation of developmentally controlled genes is at the heart of differentiation and organogenesis. In this study, we performed global genomic analyses in murine embryonic stem (ES) cells and in human cells in response to activation signals. We identified an essential role for the ELL (eleven-nineteen lysine-rich leukemia gene)/P-TEFb (positive transcription elongation factor)-containing super elongation complex (SEC) in the regulation of gene expression, including several genes bearing paused RNA polymerase II (Pol II). Paused Pol II has been proposed to be associated with loci that respond rapidly to environmental stimuli. However, our studies in ES cells also identified a requirement for SEC at genes without paused Pol II, which also respond dynamically to differentiation signals. Our findings suggest that SEC is a major class of active P-TEFb-containing complexes required for transcriptional activation in response to environmental cues such as differentiation signals.

High conservation of transcription factor binding and evidence for combinatorial regulation across six Drosophila species

The binding of some transcription factors has been shown to diverge substantially between closely related species. Here we show that the binding of the developmental transcription factor Twist is highly conserved across six Drosophila species, revealing strong functional constraints at its enhancers. Conserved binding correlates with sequence motifs for Twist and its partners, permitting the de novo discovery of their combinatorial binding. It also includes over 10,000 low-occupancy sites near the detection limit, which tend to mark enhancers of later developmental stages. These results suggest that developmental enhancers can be highly evolutionarily constrained, presumably because of their complex combinatorial nature.

Promoter-driven splicing regulation in fission yeast

The meiotic cell cycle is modified from the mitotic cell cycle by having a pre-meiotic S phase that leads to high levels of recombination, two rounds of nuclear division with no intervening DNA synthesis and a reductional pattern of chromosome segregation. Rem1 is a cyclin that is only expressed during meiosis in the fission yeast Schizosaccharomyces pombe. Cells in which rem1 has been deleted show decreased intragenic meiotic recombination and a delay at the onset of meiosis I (ref. 1). When ectopically expressed in mitotically growing cells, Rem1 induces a G1 arrest followed by severe mitotic catastrophes. Here we show that rem1 expression is regulated at the level of both transcription and splicing, encoding two proteins with different functions depending on the intron retention. We have determined that the regulation of rem1 splicing is not dependent on any transcribed region of the gene. Furthermore, when the rem1 promoter is fused to other intron-containing genes, the chimaeras show a meiotic-specific regulation of splicing, exactly the same as endogenous rem1. This regulation is dependent on two transcription factors of the forkhead family, Mei4 (ref. 2) and Fkh2 (ref. 3). Whereas Mei4 induces both transcription and splicing of rem1, Fkh2 is responsible for the intron retention of the transcript during vegetative growth and the pre-meiotic S phase.

Transcription factors Pcr1 and Atf1 have distinct roles in stress- and Sty1-dependent gene regulation.

ABSTRACT The mitogen-activated protein kinase Sty1 is essential for the regulation of transcriptional responses that promote cell survival in response to different types of environmental stimuli in Schizosaccharomyces pombe. Upon stress activation, Sty1 reversibly accumulates in the nucleus, where it stimulates gene expression via the Atf1 transcription factor. The Atf1 protein forms a heterodimer with Pcr1, but the specific role of this association is controversial. We have carried out a comparative analysis of strains lacking these proteins individually. We demonstrate that Atf1 and Pcr1 have similar but not identical roles in S. pombe, since cells lacking Pcr1 do not share all the phenotypes reported for Δatf1 cells. Northern blot and microarray analyses demonstrate that the responses to specific stresses of cells lacking either Pcr1 or Atf1 do not fully overlap, and even though most Atf1-dependent genes induced by osmotic stress are also Pcr1 dependent, a subset of genes require only the presence of Atf1 for their induction. Whereas binding of Atf1 to most stress-dependent genes requires the presence of Pcr1, we demonstrate here that Atf1 can bind to the Pcr1-independent promoters in a Δpcr1 strain in vivo. Furthermore, these analyses show that both proteins have a global repressive effect on stress-dependent and stress-independent genes.

Serine and SAM Responsive Complex SESAME Regulates Histone Modification Crosstalk by Sensing Cellular Metabolism

Pyruvate kinase M2 (PKM2) is a key enzyme for glycolysis and catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate, which supplies cellular energy. PKM2 also phosphorylates histone H3 threonine 11 (H3T11); however, it is largely unknown how PKM2 links cellular metabolism to chromatin regulation. Here, we show that the yeast PKM2 homolog, Pyk1, is a part of a novel protein complex named SESAME (Serine-responsive SAM-containing Metabolic Enzyme complex), which contains serine metabolic enzymes, SAM (S-adenosylmethionine) synthetases, and an acetyl-CoA synthetase. SESAME interacts with the Set1 H3K4 methyltransferase complex, which requires SAM synthesized from SESAME, and recruits SESAME to target genes, resulting in phosphorylation of H3T11. SESAME regulates the crosstalk between H3K4 methylation and H3T11 phosphorylation by sensing glycolysis and glucose-derived serine metabolism. This leads to auto-regulation of PYK1 expression. Thus, our study provides insights into the mechanism of regulating gene expression, responding to cellular metabolism via chromatin modifications.