Eaf5/7/3 form a functionally independent NuA4 submodule linked to RNA polymerase II-coupled nucleosome recycling.

The NuA4 histone acetyltransferase complex is required for gene regulation, cell cycle progression, and DNA repair. Dissection of the 13-subunit complex reveals that the Eaf7 subunit bridges Eaf5 with Eaf3, a H3K36me3-binding chromodomain protein, and this Eaf5/7/3 trimer is anchored to NuA4 through Eaf5. This trimeric subcomplex represents a functional module, and a large portion exists in a native form outside the NuA4 complex. Gene-specific and genome-wide location analyses indicate that Eaf5/7/3 correlates with transcription activity and is enriched over the coding region. In agreement with a role in transcription elongation, the Eaf5/7/3 trimer interacts with phosphorylated RNA polymerase II and helps its progression. Loss of Eaf5/7/3 partially suppresses intragenic cryptic transcription arising in set2 mutants, supporting a role in nucleosome destabilization. On the other hand, loss of the trimer leads to an increase of replication-independent histone exchange over the coding region of transcribed genes. Taken together, these results lead to a model where Eaf5/7/3 associates with elongating polymerase to promote the disruption of nucleosomes in its path, but also their refolding in its wake.

Histone H3K4 demethylation is negatively regulated by histone H3 acetylation in Saccharomyces cerevisiae.

Histone H3 lysine 4 trimethylation (H3K4me3) is a hallmark of transcription initiation, but how H3K4me3 is demethylated during gene repression is poorly understood. Jhd2, a JmjC domain protein, was recently identified as the major H3K4me3 histone demethylase (HDM) in Saccharomyces cerevisiae. Although JHD2 is required for removal of methylation upon gene repression, deletion of JHD2 does not result in increased levels of H3K4me3 in bulk histones, indicating that this HDM is unable to demethylate histones during steady-state conditions. In this study, we showed that this was due to the negative regulation of Jhd2 activity by histone H3 lysine 14 acetylation (H3K14ac), which colocalizes with H3K4me3 across the yeast genome. We demonstrated that loss of the histone H3-specific acetyltransferases (HATs) resulted in genome-wide depletion of H3K4me3, and this was not due to a transcription defect. Moreover, H3K4me3 levels were reestablished in HAT mutants following loss of JHD2, which suggested that H3-specific HATs and Jhd2 serve opposing functions in regulating H3K4me3 levels. We revealed the molecular basis for this suppression by demonstrating that H3K14ac negatively regulated Jhd2 demethylase activity on an acetylated peptide in vitro. These results revealed the existence of a general mechanism for removal of H3K4me3 following gene repression.

Histone H3 lysine 36 methylation targets the Isw1b remodeling complex to chromatin.

Histone H3 lysine 36 methylation is a ubiquitous hallmark of productive transcription elongation. Despite the prevalence of this histone posttranslational modification, however, the downstream functions triggered by this mark are not well understood. In this study, we showed that H3K36 methylation promoted the chromatin interaction of the Isw1b chromatin-remodeling complex in Saccharomyces cerevisiae. Similar to H3K36 methylation, Isw1b was found at the mid- and 3' regions of transcribed genes genome wide, and its presence at active genes was dependent on H3K36 methylation and the PWWP domain of the Isw1b subunit, Ioc4. Moreover, purified Isw1b preferentially interacted with recombinant nucleosomes that were methylated at lysine 36, and this interaction also required the Ioc4 PWWP domain. While H3K36 methylation has been shown to regulate the binding of numerous factors, this is the first time that it has been shown to facilitate targeting of a chromatin-remodeling complex.

CHROMATRA: a Galaxy tool for visualizing genome-wide chromatin signatures.

UNLABELLED: CHROMATRA (CHROmatin Mapping Across TRAnscripts) is a visualization tool available as plug-in for the Galaxy platform. It allows detailed yet concise presentations of data derived from ChIP-chip or ChIP-seq experiments by visualizing enrichment scores across genes or other genomic features while accounting for their length and additional characteristics such as gene expression. It integrates into typical analysis workflows and enables rapid graphical assessment and comparison of genome-wide data at a glance. AVAILABILITY: https://github.com/cmmt/chromatra.

Splitting the task: Ubp8 and Ubp10 deubiquitinate different cellular pools of H2BK123.

Monoubiquitination of H2BK123 (H2BK123ub), catalyzed by Rad6/Bre1, is a transient histone modification with roles in transcription and is essential for establishing H3K4 and H3K79 trimethylations (H3K4me3 and H3K79me3). Here, we investigated the chromatin network around H2BK123ub by examining its localization and co-occurrence with its dependent marks as well as the transcription elongation mark H3K36me3 across the genome of Saccharomyces cerevisiae. In yeast, H2BK123ub is removed by the deubiquitinases Ubp8 and Ubp10, but their genomic target regions remain to be determined. Genome-wide maps of H2BK123ub in the absence of Ubp8 and Ubp10 revealed their distinct target loci, which were genomic sites enriched for H3K4me3 and H3K79me3, respectively. We propose an extended model of the H2BK123ub cross-talk by integrating existing relationships with the substrate specificities of Ubp8 and Ubp10 reported here.

The specificity and topology of chromatin interaction pathways in yeast.

Packaging of DNA into chromatin has a profound impact on gene expression. To understand how changes in chromatin influence transcription, we analyzed 165 mutants of chromatin machinery components in Saccharomyces cerevisiae. mRNA expression patterns change in 80% of mutants, always with specific effects, even for loss of widespread histone marks. The data are assembled into a network of chromatin interaction pathways. The network is function based, has a branched, interconnected topology, and lacks strict one-to-one relationships between complexes. Chromatin pathways are not separate entities for different gene sets, but share many components. The study evaluates which interactions are important for which genes and predicts additional interactions, for example between Paf1C and Set3C, as well as a role for Mediator in subtelomeric silencing. The results indicate the presence of gene-dependent effects that go beyond context-dependent binding of chromatin factors and provide a framework for understanding how specificity is achieved through regulating chromatin.

Dot1 and histone H3K79 methylation in natural telomeric and HM silencing.

The expression of genes residing near telomeres is attenuated through telomere position-effect variegation (TPEV). By using a URA3 reporter located at TEL-VII-L of Saccharomyces cerevisiae, it was proposed that the disruptor of telomeric silencing-1 (Dot1) regulates TPEV by catalyzing H3K79 methylation. URA3 reporter assays also indicated that H3K79 methylation is required for HM silencing. Surprisingly, a genome-wide expression analysis of H3K79 methylation-defective mutants identified only a few telomeric genes, such as COS12 at TEL-VII-L, to be subject to H3K79 methylation-dependent natural silencing. Consistently, loss of Dot1 did not globally alter Sir2 or Sir3 occupancy in subtelomeric regions, but only led to some telomere-specific changes. Furthermore, H3K79 methylation by Dot1 did not play a role in the maintenance of natural HML silencing. Therefore, commonly used URA3 reporter assays may not report on natural PEV, and therefore, studies concerning the epigenetic mechanism of silencing in yeast should also employ assays reporting on natural gene expression patterns.

Reading chromatin: insights from yeast into YEATS domain structure and function.

Chromatin-modifying complexes typically contain signature domains that either have catalytic activity or recognize and bind to specific histone modifications such as acetylation, methylation, and phosphorylation. Despite tremendous progress in this area, much remains to be learned in particular about the mechanistic functions of less well characterized signature domains. One such module is the evolutionary conserved YEATS domain, found in a variety of chromatin-modifying and transcription complexes from yeast to human. Three yeast proteins contain a YEATS domain, including Yaf9, a subunit of both the histone variant H2A.Z deposition complex SWR1-C and the histone acetyltransferase complex NuA4. The three-dimensional structure of the YEATS domain from Yaf9 was solved recently, revealing the existence of three distinct structural regions. One region is characterized by a shallow groove that might constitute a potential acetyl-lysine binding pocket, raising questions about potential protein interaction partners of the Yaf9 YEATS domain.

The YEATS domain of Taf14 in Saccharomyces cerevisiae has a negative impact on cell growth.

The role of a highly conserved YEATS protein motif is explored in the context of the Taf14 protein of Saccharomyces cerevisiae. In S. cerevisiae, Taf14 is a protein physically associated with many critical multisubunit complexes including the general transcription factors TFIID and TFIIF, the chromatin remodeling complexes SWI/SNF, Ino80 and RSC, Mediator and the histone modification enzyme NuA3. Taf14 is a member of the YEATS superfamily, conserved from bacteria to eukaryotes and thought to have a transcription stimulatory activity. However, besides its ubiquitous presence and its links with transcription, little is known about Taf14's role in the nucleus. We use structure-function and mutational analysis to study the function of Taf14 and its well conserved N-terminal YEATS domain. We show here that the YEATS domain is not necessary for Taf14's association with these transcription and chromatin remodeling complexes, and that its presence in these complexes is dependent only on its C-terminal domain. Our results also indicate that Taf14's YEATS domain is not necessary for complementing the synthetic lethality between TAF14 and the general transcription factor TFIIS (encoded by DST1). Furthermore, we present evidence that the YEATS domain of Taf14 has a negative impact on cell growth: its absence enables cells to grow better than wild-type cells under stress conditions, like the microtubule destabilizing drug benomyl. Moreover, cells expressing solely the YEATS domain grow worser than cells expressing any other Taf14 construct tested, including the deletion mutant. Thus, this highly conserved domain should be considered part of a negative regulatory loop in cell growth.

Asf1-like structure of the conserved Yaf9 YEATS domain and role in H2A.Z deposition and acetylation.

Chromatin can be modified by posttranslational modifications of histones, ATP-dependent remodeling, and incorporation of histone variants. The Saccharomyces cerevisiae protein Yaf9 is a subunit of both the essential histone acetyltransferase complex NuA4 and the ATP-dependent chromatin remodeling complex SWR1-C, which deposits histone variant H2A.Z into euchromatin. Yaf9 contains a YEATS domain, found in proteins associated with multiple chromatin-modifying enzymes and transcription complexes across eukaryotes. Here, we established the conservation of YEATS domain function from yeast to human, and determined the structure of this region from Yaf9 by x-ray crystallography to 2.3 A resolution. The Yaf9 YEATS domain consisted of a beta-sandwich characteristic of the Ig fold and contained three distinct conserved structural features. The structure of the Yaf9 YEATS domain was highly similar to that of the histone chaperone Asf1, a similarity that extended to an ability of Yaf9 to bind histones H3 and H4 in vitro. Using structure-function analysis, we found that the YEATS domain was required for Yaf9 function, histone variant H2A.Z chromatin deposition at specific promoters, and H2A.Z acetylation.

Linking cell cycle to histone modifications: SBF and H2B monoubiquitination machinery and cell-cycle regulation of H3K79 dimethylation.

To identify regulators involved in determining the differential pattern of H3K79 methylation by Dot1, we screened the entire yeast gene deletion collection by GPS for genes required for normal levels of H3K79 di- but not trimethylation. We identified the cell cycle-regulated SBF protein complex required for H3K79 dimethylation. We also found that H3K79 di- and trimethylation are mutually exclusive, with M/G1 cell cycle-regulated genes significantly enriched for H3K79 dimethylation. Since H3K79 trimethylation requires prior monoubiquitination of H2B, we performed genome-wide profiling of H2BK123 monoubiquitination and showed that H2BK123 monoubiquitination is not detected on cell cycle-regulated genes and sites containing H3K79me2, but is found on H3K79me3-containing regions. A screen for genes responsible for the establishment/removal of H3K79 dimethylation resulted in identification of NRM1 and WHI3, both of which impact the transcription by the SBF and MBF protein complexes, further linking the regulation of methylation status of H3K79 to the cell cycle.

YEATS domain proteins: a diverse family with many links to chromatin modification and transcription.

Chromatin modifications play crucial roles in various biological processes. An increasing number of conserved protein domains, often found in multisubunit protein complexes, are involved in establishing and recognizing different chromatin modifications. The YEATS domain is one of these domains, and its role in chromatin modifications and transcription is just beginning to be appreciated. The YEATS domain family of proteins, conserved from yeast to human, contains over 100 members in more than 70 eukaryotic species. Yaf9, Taf14, and Sas5 are the only YEATS domain proteins in Saccharomyces cerevisiae. Human YEATS domain family members, such as GAS41, ENL, and AF9, have a strong link to cancer. GAS41 is amplified in glioblastomas and astrocytomas; ENL and AF9 are among the most frequent translocation partners of the mixed lineage leukemia (MLL) gene. This review will focus on the best characterized YEATS proteins, discuss their diverse roles, and reflect potential functions of the YEATS domain.