Histones: should I stay or should I go?
A report on the American Society for Biochemistry and Molecular Biology symposium 'Transcriptional Regulation by Chromatin and RNA polymerase II', Lake Tahoe, USA, 29 October-1 November 2004.
Chromatin↗
Biomedical subjects
Jerry L Workman
Publications and source records attributed to Jerry L Workman.
The mammalian YL1 protein is a shared subunit of the TRRAP/TIP60 histone acetyltransferase and SRCAP complexes.
The multiprotein mammalian TRRAP/TIP60-containing histone acetyltransferase (HAT) complex performs critical functions in a variety of cellular processes including transcriptional activation, double strand DNA break repair, and apoptosis. We previously isolated the TRRAP/TIP60 complex from HeLa cells (Cai, Y., Jin, J., Tomomori-Sato, C., Sato, S., Sorokina, I., Parmely, T. J., Conaway, R. C., and Conaway, J. W. (2003) J. Biol. Chem. 278, 42733-42736). Analysis of proteins present in preparations of the TRRAP/TIP60 complex led to the identification of several new subunits, as well as several potential subunits including the YL1 protein. Here we present evidence that the YL1 protein is a previously unrecognized subunit of the TRRAP/TIP60 HAT complex. In addition, we present evidence that YL1 is also a component of a novel mammalian multiprotein complex that includes the SNF2-related helicase SRCAP and resembles the recently described Saccharomyces cerevisiae SWR1 chromatin remodeling complex. Taken together, our findings identify the YL1 protein as a new subunit of the TRRAP/TIP60 HAT complex, and they suggest that YL1 plays multiple roles in chromatin modification and remodeling in cells.
Acetyltransferases↗
Yeast recombination enhancer is stimulated by transcription activation.
Saccharomyces cerevisiae mating type switching is a gene conversion event that exhibits donor preference. MATa cells choose HMLalpha for recombination, and MATalpha cells choose HMRa. Donor preference is controlled by the recombination enhancer (RE), located between HMLalpha and MATa on the left arm of chromosome III. A number of a-cell specific noncoding RNAs are transcribed from the RE locus. Mcm1 and Fkh1 regulate RE activity in a cells. Here we show that Mcm1 binding is required for both the transcription of the noncoding RNAs and Fkh1 binding. This requirement can be bypassed by inserting another promoter into the RE. Moreover, the insertion of this promoter increases donor preference and opens the chromatin structure around the conserved domains of RE. Additionally, we determined that the level of Fkh1 binding positively correlates with the level of donor preference. We conclude that the role of Mcm1 in RE is to open chromatin around the conserved domains and activate transcription; this facilitates Fkh1 binding and the level of this binding determines the level of donor preference.
Carrier Proteins↗
The DNA repair protein yKu80 regulates the function of recombination enhancer during yeast mating type switching.
Recombination enhancer (RE) is essential for regulating donor preference during yeast mating type switching. In this study, by using minichromosome affinity purification (MAP) and mass spectrometry, we found that yeast Ku80p is associated with RE in MATa cells. Chromatin immunoprecipitation assays confirmed its occupancy in vivo. Deletion of YKU80 results in altered chromatin structure in the RE region and more importantly causes a dramatic decrease of HML usage in MATa cells. We also detect directional movement of yKu80p from the RE towards HML during switching. These results indicate a novel function of yeast Ku80p in regulating mating type switching.
Binding Sites↗
The deubiquitylation activity of Ubp8 is dependent upon Sgf11 and its association with the SAGA complex.
Covalent modifications of the histone tails and the cross talk between these modifications are hallmark features of gene regulation. The SAGA histone acetyltransferase complex is one of the most well-characterized complexes involved in these covalent modifications. The recent finding that the removal of the ubiquitin group from H2B is performed by a component of SAGA, Ubp8, is intriguing as it assigns two posttranslation modification processes to one complex. In this work, we characterize the association of Ubp8 with SAGA and the effect that acetylation and deubiquitylation have on one another in vitro and in vivo. We found not only that Ubp8 is a part of the SAGA complex, but also that its deubiquitylation activity requires Ubp8's association with SAGA. Furthermore, we found that the Ubp8 association with SAGA requires Sgf11 and that this requirement is reciprocal. We also found that the acetylation and deubiquitylation activities of SAGA are independent of one another. However, we found that preacetylating histone H2B inhibited subsequent deubiquitylation. Additionally, we found that increasing the ubiquitylation state of H2B inhibited the expression of the ARG1 gene, whose repression was previously shown to require the RAD6 ubiquitin ligase. Taken together, these data indicate that the expression of some genes, including ARG1, is regulated by a balance of histone H2B ubiquitylation in the cell.
Acetylation↗
Acetylation by Tip60 is required for selective histone variant exchange at DNA lesions.
Phosphorylation of the human histone variant H2A.X and H2Av, its homolog in Drosophila melanogaster, occurs rapidly at sites of DNA double-strand breaks. Little is known about the function of this phosphorylation or its removal during DNA repair. Here, we demonstrate that the Drosophila Tip60 (dTip60) chromatin-remodeling complex acetylates nucleosomal phospho-H2Av and exchanges it with an unmodified H2Av. Both the histone acetyltransferase dTip60 as well as the adenosine triphosphatase Domino/p400 catalyze the exchange of phospho-H2Av. Thus, these data reveal a previously unknown mechanism for selective histone exchange that uses the concerted action of two distinct chromatin-remodeling enzymes within the same multiprotein complex.
Acetyl Coenzyme A↗
Global position and recruitment of HATs and HDACs in the yeast genome.
Chromatin regulators play fundamental roles in the regulation of gene expression and chromosome maintenance, but the regions of the genome where most of these regulators function has not been established. We explored the genome-wide occupancy of four different chromatin regulators encoded in Saccharomyces cerevisiae. The results reveal that the histone acetyltransferases Gcn5 and Esa1 are both generally recruited to the promoters of active protein-coding genes. In contrast, the histone deacetylases Hst1 and Rpd3 are recruited to specific sets of genes associated with distinct cellular functions. Our results provide new insights into the association of histone acetyltransferases and histone deacetylases with the yeast genome, and together with previous studies, suggest how these chromatin regulators are recruited to specific regions of the genome.
Acetyltransferases↗
Global nucleosome distribution and the regulation of transcription in yeast.
Recent studies show that active regulatory regions of the yeast genome have a lower density of nucleosomes than other regions, and that there is an inverse correlation between nucleosome density and the transcription rate of a gene. This may be the result of transcription factors displacing nucleosomes.
Gene Expression Regulation, Fungal↗
Histone H3 variants and modifications on transcribed genes.
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Animals↗
RPAP1, a novel human RNA polymerase II-associated protein affinity purified with recombinant wild-type and mutated polymerase subunits.
We have programmed human cells to express physiological levels of recombinant RNA polymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags. Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNAPII-associated protein 1 (RPAP1). The TAP-tagged RNAPII complex is functionally active both in vitro and in vivo. A role for RPAP1 in RNAPII transcription was established by shutting off the synthesis of Ydr527wp, a Saccharomyces cerevisiae protein homologous to RPAP1, and demonstrating that changes in global gene expression were similar to those caused by the loss of the yeast RNAPII subunit Rpb11. We also used TAP-tagged Rpb2 with mutations in fork loop 1 and switch 3, two structural elements located strategically within the active center, to start addressing the roles of these elements in the interaction of the enzyme with the template DNA during the transcription reaction.
Animals↗
In vitro targeting reveals intrinsic histone tail specificity of the Sin3/histone deacetylase and N-CoR/SMRT corepressor complexes.
The histone code is among others established via differential acetylation catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs). To unambiguously determine the histone tail specificity of HDAC-containing complexes, we have established an in vitro system consisting of nucleosomal templates reconstituted with hyperacetylated histones or recombinant histones followed by acetylation with native SAGA or NuA4. Selective targeting of the mammalian Sin3/HDAC and N-CoR/SMRT corepressor complexes by using specific chimeric repressors created a near physiological setting to assess their histone tail specificity. Recruitment of the Sin3/HDAC complex to nucleosomal templates preacetylated with SAGA or NuA4 resulted in deacetylation of histones H3 and H4, whereas recruitment of N-CoR/SMRT resulted in deacetylation of histone H3 only. These results provide solid evidence that HDAC-containing complexes display distinct, intrinsic histone tail specificities and hence may function differently to regulate chromatin structure and transcription.
Animals↗
Transcription factors prominently in Lasker Award to Roeder.
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Awards and Prizes↗
Breast cancer metastasis suppressor 1 (BRMS1) forms complexes with retinoblastoma-binding protein 1 (RBP1) and the mSin3 histone deacetylase complex and represses transcription.
Breast cancer metastasis suppressor 1 (BRMS1) suppresses metastasis of multiple human and murine cancer cells without inhibiting tumorigenicity. By yeast two-hybrid and co-immunoprecipitation, BRMS1 interacts with retinoblastoma binding protein 1 and at least seven members of the mSin3 histone deacetylase (HDAC) complex in human breast and melanoma cell lines. BRMS1 co-immunoprecipitates enzymatically active HDAC proteins and represses transcription when recruited to a Gal4 promoter in vivo. BRMS1 exists in large mSin3 complex(es) of approximately 1.4-1.9 MDa, but also forms smaller complexes with HDAC1. Deletion analyses show that the carboxyl-terminal 42 amino acids of BRMS1 are not critical for interaction with much of the mSin3 complex and that BRMS1 appears to have more than one binding point to the complex. These results further show that BRMS1 may participate in transcriptional regulation via interaction with the mSin3.HDAC complex and suggest a novel mechanism by which BRMS1 might suppress cancer metastasis.
Animals↗
Opposite role of yeast ING family members in p53-dependent transcriptional activation.
The inhibitor-of-growth (ING) family of proteins was founded by human ING1, a tumor suppressor interacting with p53 in vivo and required for its function in transcription/apoptosis. There are five different ING genes in humans, three of which have been linked to p53 function. In this study, we analyzed the three ING family members present in yeast. We demonstrate that each one is purified as a key component of a specific histone-modifying complex. Pho23 is part of Rpd3/Sin3 histone deacetylase complex, while Yng1 and Yng2 are subunits of the NuA3 and NuA4 histone acetyltransferase complexes, respectively. We also show that the three different ING proteins have opposite roles in transcriptional activation by p53 in vivo. These effects are linked to the presence of each ING in its respective chromatin modifying complex, since mutation of the corresponding catalytic subunit gave similar results. Depletion of Pho23/Rpd3 leads to increased p53-dependent transcription in vivo while depletion of Yng2 abrogates it. Surprisingly, deletion of YNG1 or SAS3 leads to increased transcriptional activation by p53. These data suggest that the NuA3 complex can function in gene-specific repression, an unusual role for a histone acetyltransferase complex. They also demonstrate the key specific role of ING proteins in different chromatin modifying complexes and their opposite functions in p53-dependent transcription.
Acetyltransferases↗
Sas4 and Sas5 are required for the histone acetyltransferase activity of Sas2 in the SAS complex.
The SAS2 gene is involved in transcriptional silencing in Saccharomyces cerevisiae. Based on its primary sequence, the Sas2 protein is predicted to be a member of the MYST family of histone acetyltransferases (HATs). Sas2 forms a complex with Sas4 and Sas5, which are required for its silencing function. Here we show that recombinant Sas2 has HAT activity that absolutely requires Sas4 and is stimulated by Sas5. The recombinant SAS complex acetylates H4 lysine 16 and H3 lysine 14. Furthermore, a purified SAS complex from yeast shows similar activity and specificity. In contrast to other MYST HATs, neither the recombinant nor the native SAS complex acetylated nucleosomal histones under conditions that were optimum for acetylating free histones. Finally, although the SAS subunits interact genetically and physically with Asf1, a histone deposition factor, association of H3 and H4 with Asf1 blocks their acetylation by the SAS complex, raising the possibility that the SAS HAT complex may acetylate free histones prior to their deposition onto DNA by Asf1 or CAF-I.
Acetylation↗
The Set2 histone methyltransferase functions through the phosphorylated carboxyl-terminal domain of RNA polymerase II.
The histone methyltransferase Set2, which specifically methylates lysine 36 of histone H3, has been shown to repress transcription upon tethering to a heterologous promoter. However, the mechanism of targeting and the consequence of Set2-dependent methylation have yet to be demonstrated. We sought to identify the protein components associated with Set2 to gain some insights into the in vivo function of this protein. Mass spectrometry analysis of the Set2 complex, purified using a tandem affinity method, revealed that RNA polymerase II (pol II) is associated with Set2. Immunoblotting and immunoprecipitation using antibodies against subunits of pol II confirmed that the phosphorylated form of pol II is indeed an integral part of the Set2 complex. Gst-Set2 preferentially binds to CTD synthetic peptides phosphorylated at serine 2, and to a lesser extent, serine 5 phosphorylated peptides, but has no affinity for unphosphorylated CTD, suggesting that Set2 associates with the elongating form of the pol II. Furthermore, we show that set2Delta ppr2Delta double mutants (PPR2 encodes TFIIS, a transcription elongation factor) are synthetically hypersensitive to 6-azauracil, and that deletions in the CTD reduce in vivo levels of H3 lysine 36 methylation. Collectively, these results suggest that Set2 is involved in regulating transcription elongation through its direct contact with pol II.
Antimetabolites↗
Assay of activator recruitment of chromatin-modifying complexes.
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Biochemistry↗
The diverse functions of histone acetyltransferase complexes.
Although histone acetylation has historically been linked to transcription activation, recent studies indicate that this modification and the enzymes that catalyze it have much broader and diverse functions. Histone acetyltransferase complexes are involved in such diverse processes as transcription activation, gene silencing, DNA repair and cell-cycle progression. The high conservation of the acetyltransferase complexes and their functions illustrates their central role in cell growth and development.
Acetylation↗