The influence of flocculation upon global gene transcription in a yeast CYC8 mutant.

The transcriptome from a Saccharomyces cerevisiae tup1 deletion mutant was one of the first comprehensive yeast transcriptomes published. Subsequent transcriptomes from tup1 and cyc8 mutants firmly established the Tup1-Cyc8 complex as predominantly acting as a repressor of gene transcription. However, transcriptomes from tup1/cyc8 gene deletion or conditional mutants would all have been influenced by the striking flocculation phenotypes that these mutants display. In this study, we have separated the impact of flocculation from the transcriptome in a cyc8 conditional mutant to reveal those genes (i) subject solely to Cyc8p-dependent regulation, (ii) regulated by flocculation only and (iii) regulated by Cyc8p and further influenced by flocculation. We reveal a more accurate list of Cyc8p-regulated genes that includes newly identified Cyc8p-regulated genes that were masked by the flocculation phenotype and excludes genes which were indirectly influenced by flocculation and not regulated by Cyc8p. Furthermore, we show evidence that flocculation exerts a complex and potentially dynamic influence upon global gene transcription. These data should be of interest to future studies into the mechanism of action of the Tup1-Cyc8 complex and to studies involved in understanding the development of flocculation and its impact upon cell function.

Aspergillus fumigatus mitogen-activated protein kinase MpkA is involved in gliotoxin production and self-protection.

Aspergillus fumigatus is a saprophytic fungus that can cause a variety of human diseases known as aspergillosis. Mycotoxin gliotoxin (GT) production is important for its virulence and must be tightly regulated to avoid excess production and toxicity to the fungus. GT self-protection by GliT oxidoreductase and GtmA methyltransferase activities is related to the subcellular localization of these enzymes and how GT can be sequestered from the cytoplasm to avoid increased cell damage. Here, we show that GliT:GFP and GtmA:GFP are localized in the cytoplasm and in vacuoles during GT production. The Mitogen-Activated Protein kinase MpkA is essential for GT production and self-protection, interacts physically with GliT and GtmA and it is necessary for their regulation and subsequent presence in the vacuoles. The sensor histidine kinase SlnASln1 is important for modulation of MpkA phosphorylation. Our work emphasizes the importance of MpkA and compartmentalization of cellular events for GT production and self-defense.

The KdmB-EcoA-RpdA-SntB (KERS) chromatin regulatory complex controls development, secondary metabolism and pathogenicity in Aspergillus flavus.

The filamentous fungus Aspergillus flavus is a plant and human pathogen predominantly found in the soil as spores or sclerotia and is capable of producing various secondary metabolites (SM) such as the carcinogenic mycotoxin aflatoxin. Recently, we have discovered a novel nuclear chromatin binding complex (KERS) that contains the JARID1-type histone demethylase KdmB, a putative cohesion acetyl transferase EcoA, a class I type histone deacetylase RpdA and the PHD ring finger reader protein SntB in the model filamentous fungus Aspergillus nidulans. Here, we show the presence of the KERS complex in A. flavus by immunoprecipitation-coupled mass spectrometry and constructed kdmBΔ and rpdAΔ strains to study their roles in fungal development, SM production and histone post-translational modifications (HPTMs). We found that KdmB and RpdA couple the regulation of SM gene clusters with fungal light-responses and HPTMs. KdmB and RpdA have opposing roles in light-induced asexual conidiation, while both factors are positive regulators of sclerotia development through the nsdC and nsdD pathway. KdmB and RpdA are essential for the productions of aflatoxin (similar to findings for SntB) as well as cyclopiazonic acid, ditryptophenaline and leporin B through controlling the respective SM biosynthetic gene clusters. We further show that both KdmB and RpdA regulate H3K4me3 and H3K9me3 levels, while RpdA also acts on H3K14ac levels in nuclear extracts. Therefore, the chromatin modifiers KdmB and RpdA of the KERS complex are key regulators for fungal development and SM metabolism in A. flavus.

Systematic analysis of tup1 and cyc8 mutants reveals distinct roles for TUP1 and CYC8 and offers new insight into the regulation of gene transcription by the yeast Tup1-Cyc8 complex.

The Tup1-Cyc8 complex in Saccharomyces cerevisiae was one of the first global co-repressors of gene transcription discovered. However, despite years of study, a full understanding of the contribution of Tup1p and Cyc8p to complex function is lacking. We examined TUP1 and CYC8 single and double deletion mutants and show that CYC8 represses more genes than TUP1, and that there are genes subject to (i) unique repression by TUP1 or CYC8, (ii) redundant repression by TUP1 and CYC8, and (iii) there are genes at which de-repression in a cyc8 mutant is dependent upon TUP1, and vice-versa. We also reveal that Tup1p and Cyc8p can make distinct contributions to commonly repressed genes most likely via specific interactions with different histone deacetylases. Furthermore, we show that Tup1p and Cyc8p can be found independently of each other to negatively regulate gene transcription and can persist at active genes to negatively regulate on-going transcription. Together, these data suggest that Tup1p and Cyc8p can associate with active and inactive genes to mediate distinct negative and positive regulatory roles when functioning within, and possibly out with the complex.

Vacuoles and peroxisomes are involved in Aspergillus fumigatus gliotoxin production and self-protection.

Aspergillus fumigatus is a saprophytic fungus that can cause a variety of human diseases known as aspergillosis. Mycotoxin gliotoxin (GT) production is important for its virulence and must be tightly regulated to avoid excess production and toxicity to the fungus. GT self-protection by GliT oxidoreductase and GtmA methyltransferase activities is related to the subcellular localization of these enzymes and how GT can be sequestered from the cytoplasm to avoid increased cell damage. Here, we show that GliT:GFP and GtmA:GFP are localized in the cytoplasm and in vacuoles during GT production. Peroxisomes are also required for proper GT production and self-defense. The Mitogen-Activated Protein (MAP) kinase MpkA is essential for GT production and self-protection, interacts physically with GliT and GtmA and it is necessary for their regulation and subsequent presence in the vacuoles. Our work emphasizes the importance of dynamic compartmentalization of cellular events for GT production and self-defense.

The KdmB-EcoA-RpdA-SntB chromatin complex binds regulatory genes and coordinates fungal development with mycotoxin synthesis.

Chromatin complexes control a vast number of epigenetic developmental processes. Filamentous fungi present an important clade of microbes with poor understanding of underlying epigenetic mechanisms. Here, we describe a chromatin binding complex in the fungus Aspergillus nidulans composing of a H3K4 histone demethylase KdmB, a cohesin acetyltransferase (EcoA), a histone deacetylase (RpdA) and a histone reader/E3 ligase protein (SntB). In vitro and in vivo evidence demonstrate that this KERS complex is assembled from the EcoA-KdmB and SntB-RpdA heterodimers. KdmB and SntB play opposing roles in regulating the cellular levels and stability of EcoA, as KdmB prevents SntB-mediated degradation of EcoA. The KERS complex is recruited to transcription initiation start sites at active core promoters exerting promoter-specific transcriptional effects. Interestingly, deletion of any one of the KERS subunits results in a common negative effect on morphogenesis and production of secondary metabolites, molecules important for niche securement in filamentous fungi. Consequently, the entire mycotoxin sterigmatocystin gene cluster is downregulated and asexual development is reduced in the four KERS mutants. The elucidation of the recruitment of epigenetic regulators to chromatin via the KERS complex provides the first mechanistic, chromatin-based understanding of how development is connected with small molecule synthesis in fungi.

Putative Membrane Receptors Contribute to Activation and Efficient Signaling of Mitogen-Activated Protein Kinase Cascades during Adaptation of Aspergillus fumigatus to Different Stressors and Carbon Sources.

The high-osmolarity glycerol (HOG) response pathway is a multifunctional signal transduction pathway that specifically transmits ambient osmotic signals. Saccharomyces cerevisiae Hog1p has two upstream signaling branches, the sensor histidine kinase Sln1p and the receptor Sho1p. The Sho1p branch includes two other proteins, the Msb2p mucin and Opy2p. Aspergillus fumigatus is the leading cause of pulmonary fungal diseases. Here, we investigated the roles played by A. fumigatus SlnASln1p, ShoASho1p, MsbAMsb2p, and OpyAOpy2p putative homologues during the activation of the mitogen-activated protein kinase (MAPK) HOG pathway. The shoA, msbA, and opyA singly and doubly null mutants are important for the cell wall integrity (CWI) pathway, oxidative stress, and virulence as assessed by a Galleria mellonella model. Genetic interactions of ShoA, MsbA, and OpyA are also important for proper activation of the SakAHog1p and MpkASlt2 cascade and the response to osmotic and cell wall stresses. Comparative label-free quantitative proteomics analysis of the singly null mutants with the wild-type strain upon caspofungin exposure indicates that the absence of ShoA, MsbA, and OpyA affects the osmotic stress response, carbohydrate metabolism, and protein degradation. The putative receptor mutants showed altered trehalose and glycogen accumulation, suggesting a role for ShoA, MsbA, and OpyA in sugar storage. Protein kinase A activity was also decreased in these mutants. We also observed genetic interactions between SlnA, ShoA, MsbA, and OpyA, suggesting that both branches are important for activation of the HOG/CWI pathways. Our results help in the understanding of the activation and modulation of the HOG and CWI pathways in this important fungal pathogen.IMPORTANCEAspergillus fumigatus is an important human-pathogenic fungal species that is responsible for a high incidence of infections in immunocompromised individuals. A. fumigatus high-osmolarity glycerol (HOG) and cell wall integrity pathways are important for the adaptation to different forms of environmental adversity such as osmotic and oxidative stresses, nutrient limitations, high temperatures, and other chemical and mechanical stresses that may be produced by the host immune system and antifungal drugs. Little is known about how these pathways are activated in this fungal pathogen. Here, we characterize four A. fumigatus putative homologues that are important for the activation of the yeast HOG pathway. A. fumigatus SlnASln1p, ShoASho1p, MsbAMsb2p, and OpyAOpy2p are genetically interacting and are essential for the activation of the HOG and cell wall integrity pathways. Our results contribute to the understanding of A. fumigatus adaptation to the host environment.

The Pheromone Module SteC-MkkB-MpkB-SteD-HamE Regulates Development, Stress Responses and Secondary Metabolism in Aspergillus fumigatus.

In order for eukaryotes to efficiently detect and respond to environmental stimuli, a myriad of protein signaling pathways are utilized. An example of highly conserved signaling pathways in eukaryotes are the mitogen-activated protein kinase (MAPK) pathways. In fungi, MAPK pathways have been shown to regulate a diverse array of biological processes, such as asexual and sexual development, stress responses and the production of secondary metabolites (SMs). In the model fungus Aspergillus nidulans, a MAPK pathway known as the pheromone module is utilized to regulate both development and SM production. This signaling cascade consists of the three kinases SteC, MkkB, and MpkB, as well as the SteD adaptor protein and the HamE scaffold. In this study, homologs of each of these proteins have been identified in the opportunistic human pathogen A. fumigatus. By performing epitope tagging and mass spectrometry experiments, we have shown that these proteins form a pentameric complex, similar to what is observed in A. nidulans. This complex has been shown to assemble in the cytoplasm and MpkB enters the nucleus, where it would presumably interact with various transcription factors. Pheromone module mutant strains exhibit drastic reductions in asexual sporulation, vegetative growth rate and production of SMs, such as gliotoxin. Mutants also display increased sensitivity to cell wall and oxidative stress agents. Overall, these data provide evidence of the existence of a conserved MAP kinase signaling pathway in Aspergillus species and suggest that this pathway is critical for the regulation of fungal development and secondary metabolism.

The tetrameric pheromone module SteC-MkkB-MpkB-SteD regulates asexual sporulation, sclerotia formation and aflatoxin production in Aspergillus flavus.

For eukaryotes like fungi to regulate biological responses to environmental stimuli, various signalling cascades are utilized, like the highly conserved mitogen-activated protein kinase (MAPK) pathways. In the model fungus Aspergillus nidulans, a MAPK pathway known as the pheromone module regulates development and the production of secondary metabolites (SMs). This pathway consists five proteins, the three kinases SteC, MkkB and MpkB, the adaptor SteD and the scaffold HamE. In this study, homologs of these five pheromone module proteins have been identified in the plant and human pathogenic fungus Aspergillus flavus. We have shown that a tetrameric complex consisting of the three kinases and the SteD adaptor is assembled in this species. It was observed that this complex assembles in the cytoplasm and that MpkB translocates into the nucleus. Deletion of steC, mkkB, mpkB or steD results in abolishment of both asexual sporulation and sclerotia production. This complex is required for the positive regulation of aflatoxin production and negative regulation of various SMs, including leporin B and cyclopiazonic acid (CPA), likely via MpkB interactions in the nucleus. These data highlight the conservation of the pheromone module in Aspergillus species, signifying the importance of this pathway in regulating fungal development and secondary metabolism.

Draft Genome Sequence and Annotation of Acinetobacter junii MHI21018, Isolated from Bovine Colostrum.

We report here the draft genome sequence of Acinetobacter junii MHI21018, isolated in 2009 from bovine colostrum. The draft genome sequence is composed of 3,267,995 bp, has a GC content of 38.54%, and was assembled into 114 contigs (contig size, >500 bp) with an N 50 value of 72,566 bp.

A role for histone acetylation in regulating transcription elongation.

Recently, we reported that a major function of histone acetylation at the yeast FLO1 gene was to regulate transcription elongation. Here, we discuss possible mechanisms by which histone acetylation might regulate RNA polymerase II processivity, and comment on the contribution to transcription of chromatin remodelling at gene coding regions and promoters.

Sas3 and Ada2(Gcn5)-dependent histone H3 acetylation is required for transcription elongation at the de-repressed FLO1 gene.

The Saccharomyces cerevisiae FLO1 gene encodes a cell wall protein that imparts cell-cell adhesion. FLO1 transcription is regulated via the antagonistic activities of the Tup1-Cyc8 co-repressor and Swi-Snf co-activator complexes. Tup1-Cyc8 represses transcription through the organization of strongly positioned, hypoacetylated nucleosomes across gene promoters. Swi-Snf catalyzes remodeling of these nucleosomes in a mechanism involving histone acetylation that is poorly understood. Here, we show that FLO1 de-repression is accompanied by Swi-Snf recruitment, promoter histone eviction and Sas3 and Ada2(Gcn5)-dependent histone H3K14 acetylation. In the absence of H3K14 acetylation, Swi-Snf recruitment and histone eviction proceed, but transcription is reduced, suggesting these processes, while essential, are not sufficient for de-repression. Further analysis in the absence of H3K14 acetylation reveals RNAP II recruitment at the FLO1 promoter still occurs, but RNAP II is absent from the gene-coding region, demonstrating Sas3 and Ada2-dependent histone H3 acetylation is required for transcription elongation. Analysis of the transcription kinetics at other genes reveals shared mechanisms coupled to a distinct role for histone H3 acetylation, essential at FLO1, downstream of initiation. We propose histone H3 acetylation in the coding region provides rate-limiting control during the transition from initiation to elongation which dictates whether the gene is permissive for transcription.

Distinct histone methylation and transcription profiles are established during the development of cellular quiescence in yeast.

BACKGROUND: Quiescent cells have a low level of gene activity compared to growing cells. Using a yeast model for cellular quiescence, we defined the genome-wide profiles of three species of histone methylation associated with active transcription between growing and quiescent cells, and correlated these profiles with the presence of RNA polymerase II and transcripts. RESULTS: Quiescent cells retained histone methylations normally associated with transcriptionally active chromatin and had many transcripts in common with growing cells. Quiescent cells also contained significant levels of RNA polymerase II, but only low levels of the canonical initiating and elongating forms of the polymerase. The RNA polymerase II associated with genes in quiescent cells displayed a distinct occupancy profile compared to its pattern of occupancy across genes in actively growing cells. Although transcription is generally repressed in quiescent cells, analysis of individual genes identified a period of active transcription during the development of quiescence. CONCLUSIONS: The data suggest that the transcript profile and histone methylation marks in quiescent cells were established both in growing cells and during the development of quiescence and then retained in these cells. Together, this might ensure that quiescent cells can rapidly adapt to a changing environment to resume growth.

Telomeric ORFs (TLOs) in Candida spp. Encode mediator subunits that regulate distinct virulence traits.

The TLO genes are a family of telomere-associated ORFs in the fungal pathogens Candida albicans and C. dubliniensis that encode a subunit of the Mediator complex with homology to Med2. The more virulent pathogen C. albicans has 15 copies of the gene whereas the less pathogenic species C. dubliniensis has only two (CdTLO1 and CdTLO2). In this study we used C. dubliniensis as a model to investigate the role of TLO genes in regulating virulence and also to determine whether TLO paralogs have evolved to regulate distinct functions. A C. dubliniensis tlo1Δ/tlo2Δ mutant is unable to form true hyphae, has longer doubling times in galactose broth, is more susceptible to oxidative stress and forms increased levels of biofilm. Transcript profiling of the tlo1Δ/tlo2Δ mutant revealed increased expression of starvation responses in rich medium and retarded expression of hypha-induced transcripts in serum. ChIP studies indicated that Tlo1 binds to many ORFs including genes that exhibit high and low expression levels under the conditions analyzed. The altered expression of these genes in the tlo1Δ/tlo2Δ null mutant indicates roles for Tlo proteins in transcriptional activation and repression. Complementation of the tlo1Δ/tlo2Δ mutant with TLO1, but not TLO2, restored wild-type filamentous growth, whereas only TLO2 fully suppressed biofilm growth. Complementation with TLO1 also had a greater effect on doubling times in galactose broth. The different abilities of TLO1 and TLO2 to restore wild-type functions was supported by transcript profiling studies that showed that only TLO1 restored expression of hypha-specific genes (UME6, SOD5) and galactose utilisation genes (GAL1 and GAL10), whereas TLO2 restored repression of starvation-induced gene transcription. Thus, Tlo/Med2 paralogs encoding Mediator subunits regulate different virulence properties in Candida spp. and their expansion may account for the increased adaptability of C. albicans relative to other Candida species.

The yeast Cyc8-Tup1 complex cooperates with Hda1p and Rpd3p histone deacetylases to robustly repress transcription of the subtelomeric FLO1 gene.

We demonstrate that the yeast flocculation gene, FLO1, is representative of a distinct subset of subtelomeric genes that are robustly repressed by the Cyc8-Tup1 complex. We have examined Cyc8-Tup1 localisation, histone acetylation and long-range chromatin remodelling within the extensive FLO1 upstream region. We show that Cyc8-Tup1 is localised in a DNase I hypersensitive site within an ordered array of strongly positioned nucleosomes around -700 base pairs upstream of the transcription start site. In cyc8 deletion mutant strains, Tup1p localisation is absent, with concomitant histone hyperacetylation of adjacent regions at the FLO1 promoter. This is accompanied by extensive histone depletion across the upstream region and gene activation. The yeast histone deacetylases, Hda1p and Rpd3p, occupy the repressed FLO1 promoter region in a Cyc8-Tup1 dependent manner and coordinate histone deacetylation, nucleosome stabilisation and gene repression. Moreover, we show that the ATP-dependent chromatin remodelling complex Swi-Snf occupies the site vacated by Cyc8-Tup1 in a cyc8 mutant. These data suggest that distinctly bound Cyc8-Tup1 cooperates with Hda1p and Rpd3p to establish or maintain an extensive array of strongly positioned, deacetylated nucleosomes over the FLO1 promoter and upstream region which inhibit histone acetylation, block Swi-Snf binding and prevent transcription.

H2B ubiquitylation is part of chromatin architecture that marks exon-intron structure in budding yeast.

BACKGROUND: The packaging of DNA into chromatin regulates transcription from initiation through 3' end processing. One aspect of transcription in which chromatin plays a poorly understood role is the co-transcriptional splicing of pre-mRNA. RESULTS: Here we provide evidence that H2B monoubiquitylation (H2BK123ub1) marks introns in Saccharomyces cerevisiae. A genome-wide map of H2BK123ub1 in this organism reveals that this modification is enriched in coding regions and that its levels peak at the transcribed regions of two characteristic subgroups of genes. First, long genes are more likely to have higher levels of H2BK123ub1, correlating with the postulated role of this modification in preventing cryptic transcription initiation in ORFs. Second, genes that are highly transcribed also have high levels of H2BK123ub1, including the ribosomal protein genes, which comprise the majority of intron-containing genes in yeast. H2BK123ub1 is also a feature of introns in the yeast genome, and the disruption of this modification alters the intragenic distribution of H3 trimethylation on lysine 36 (H3K36me3), which functionally correlates with alternative RNA splicing in humans. In addition, the deletion of genes encoding the U2 snRNP subunits, Lea1 or Msl1, in combination with an htb-K123R mutation, leads to synthetic lethality. CONCLUSION: These data suggest that H2BK123ub1 facilitates cross talk between chromatin and pre-mRNA splicing by modulating the distribution of intronic and exonic histone modifications.

Pol II and its associated epigenetic marks are present at Pol III-transcribed noncoding RNA genes.

Epigenetic control is an important aspect of gene regulation. Despite detailed understanding of protein-coding gene expression, the transcription of noncoding RNA genes by RNA polymerase III (Pol III) is less well characterized. Here we profile the epigenetic features of Pol III target genes throughout the human genome. This reveals that the chromatin landscape of Pol III-transcribed genes resembles that of Pol II templates in many ways, although there are also clear differences. Our analysis also uncovered an entirely unexpected phenomenon: namely, that Pol II is present at the majority of genomic loci that are bound by Pol III.

H2B ubiquitylation plays a role in nucleosome dynamics during transcription elongation.

The monoubiquitylation of histone H2B has been associated with transcription initiation and elongation, but its role in these processes is poorly understood. We report that H2B ubiquitylation is required for efficient reassembly of nucleosomes during RNA polymerase II (Pol II)-mediated transcription elongation in yeast. This role is carried out in cooperation with the histone chaperone Spt16, and in the absence of H2B ubiquitylation and functional Spt16, chromatin structure is not properly restored in the wake of elongating Pol II. Moreover, H2B ubiquitylation and Spt16 play a role in each other's regulation. H2B ubiquitylation is required for the stable accumulation of Spt16 at the GAL1 coding region, and Spt16 regulates the formation of ubiquitylated H2B both globally and at the GAL1 gene. These data provide a mechanism linking H2B ubiquitylation to Spt16 in the regulation of nucleosome dynamics during transcription elongation.

Tup1-Ssn6 and Swi-Snf remodelling activities influence long-range chromatin organization upstream of the yeast SUC2 gene.

The traditional model for chromatin remodelling during transcription has focused upon the remodelling of nucleosomes at gene promoters. However, in this study, we have determined that Tup1-Ssn6 and Swi-Snf chromatin remodelling activities extend far upstream of the SUC2 gene promoter into the intergenic region of the Saccharomyces cerevisiae chromosome. We mapped the nucleosomal array over a 7.5 kb region that encompassed the SUC2 gene promoter and upstream region but was devoid of other transcriptionally active genes. Nucleosome positioning over this region was determined under conditions of glucose repression and derepression, and in snf2, ssn6 and snf2 ssn6 mutant strains. A map detailing remodelling events extending as much as 5 kb upstream of the SUC2 gene promoter underlines the roles of the Tup1-Ssn6 and Swi-Snf complexes in respectively organizing and disrupting nucleosome arrays. The gene specificity of these events suggests a role in gene regulation. We propose that long-range chromatin remodelling activities of Swi-Snf and Tup1-Ssn6 may ultimately influence whether the chromosomal state of the SUC2 gene is proficient for transcription. These data raise the possibility that remodelling of extensive chromatin domains may be a general property of the Swi-Snf and Tup1-Ssn6 complexes.

Histone ubiquitylation and the regulation of transcription.

The small (76 amino acids) and highly conserved ubiquitin protein plays key roles in the physiology of eukaryotic cells. Protein ubiquitylation has emerged as one of the most important intracellular signaling mechanisms, and in 2004 the Nobel Prize was awarded to Aaron Ciechanower, Avram Hersko, and Irwin Rose for their pioneering studies of the enzymology of ubiquitin attachment. One of the most common features of protein ubiquitylation is the attachment of polyubiquitin chains (four or more ubiquitin moieties attached to each other), which is a widely used mechanism to target proteins for degradation via the 26S proteosome. However, it is noteworthy that the first ubiquitylated protein to be identified was histone H2A, to which a single ubiquitin moiety is most commonly attached. Following this discovery, other histones (H2B, H3, H1, H2A.Z, macroH2A), as well as many nonhistone proteins, have been found to be monoubiquitylated. The role of monoubiquitylation is still elusive because a single ubiquitin moiety is not sufficient to target proteins for turnover, and has been hypothesized to control the assembly or disassembly of multiprotein complexes by providing a protein-binding site. Indeed, a number of ubiquitin-binding domains have now been identified in both polyubiquitylated and monoubiquitylated proteins. Despite the early discovery of ubiquitylated histones, it has only been in the last five or so years that we have begun to understand how histone ubiquitylation is regulated and what roles it plays in the cell. This review will discuss current research on the factors that regulate the attachment and removal of ubiquitin from histones, describe the relationship of histone ubiquitylation to histone methylation, and focus on the roles of ubiquitylated histones in gene expression.