Accelerated nuclei preparation and methods for analysis of histone modifications in yeast

Kizer, Kelby O.; Xiao, Tiaojiang; Strahl, Brian D.

doi:10.1016/j.ymeth.2006.06.022

Cited by 27 publications

(22 citation statements)

References 40 publications

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“…Purification of chromatin in yeast and spores. The preparation of nuclei was based on published methods (18,29). A dit1⌬ mutant was used to make possible the digestion of the spore wall by Zymolyase.…”

Section: Methodsmentioning

confidence: 99%

The Linker Histone Plays a Dual Role during Gametogenesis in Saccharomyces cerevisiae

Bryant

Govin

Zhang

et al. 2012

Molecular and Cellular Biology

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fThe differentiation of gametes involves dramatic changes to chromatin, affecting transcription, meiosis, and cell morphology. Sporulation in Saccharomyces cerevisiae shares many chromatin features with spermatogenesis, including a 10-fold compaction of the nucleus. To identify new proteins involved in spore nuclear organization, we purified chromatin from mature spores and discovered a significant enrichment of the linker histone (Hho1). The function of Hho1 has proven to be elusive during vegetative growth, but here we demonstrate its requirement for efficient sporulation and full compaction of the spore genome. Hho1 chromatin immunoprecipitation followed by sequencing (ChIP-seq) revealed increased genome-wide binding in mature spores and provides novel in vivo evidence of the linker histone binding to nucleosomal linker DNA. We also link Hho1 function to the transcription factor Ume6, the master repressor of early meiotic genes. Hho1 and Ume6 are depleted during meiosis, and analysis of published ChIP-chip data obtained during vegetative growth reveals a high binding correlation of both proteins at promoters of early meiotic genes. Moreover, Ume6 promotes binding of Hho1 to meiotic gene promoters. Thus, Hho1 may play a dual role during sporulation: Hho1 and Ume6 depletion facilitates the onset of meiosis via activation of Ume6-repressed early meiotic genes, whereas Hho1 enrichment in mature spores contributes to spore genome compaction. Gametogenesis is a complex, highly regulated differentiation program that is integral to the survival of higher eukaryotes. Mammalian spermatogenesis begins with DNA replication and recombination, ultimately generating four genetically unique haploid cells. The postmeiotic differentiation of spermatozoa involves a complete reorganization of the chromatin and a dramatic compaction of the nucleus that prepare the genome for passage to a new generation (16,30). In fact, specific combinations of histone posttranslational modifications (PTMs) are found at genes encoding important developmental regulators (5,20).Gametogenesis in Saccharomyces cerevisiae, or sporulation, shares several chromatin features with mammalian spermatogenesis, namely, drastic compaction of the nucleus and stage-specific appearance of histone PTMs such as phosphorylation of histone H3 at serine 10 (H3S10ph), H3T11ph, and H4S1ph (17, 31). Sporulation takes place in diploid yeast deprived of nitrogen and a fermentable carbon source (15,39). Under these starvation conditions, yeast cells enter a meiotic program similar to that found in mammalian spermatogenesis. During the postmeiotic phase, cell and nuclear volumes are substantially decreased, the genome is compacted, transcription is silenced, and a protective spore wall is synthesized. The resultant quiescent spore is able to survive unfavorable growth conditions of heat, dehydration, and chemical insult. When a fermentable carbon source is reintroduced, spores are induced to reenter the cell cycle through a process called germination, characterized by a spec...

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Section: Methodsmentioning

confidence: 99%

The Linker Histone Plays a Dual Role during Gametogenesis in Saccharomyces cerevisiae

Bryant

Govin

Zhang

et al. 2012

Molecular and Cellular Biology

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“…Interactions were detected using enhanced chemiluminescence (GE Healthcare or Pierce) and a horseradish peroxidase anti-species secondary antibody. Nuclear extracts were prepared as described previously (17).…”

Section: Methodsmentioning

confidence: 99%

“…Aliquots of the reaction mixture were analyzed by Western blotting using antibodies raised against Set2, H3K36me2, H3K36me3, and histone H3. Due to the challenges in the use of the H3K36me2 antibody as described previously (17), representative blots from more than three experiments are shown. In each case, the independent replicates yielded equivalent results.…”

mentioning

confidence: 99%

Roles for Ctk1 and Spt6 in Regulating the Different Methylation States of Histone H3 Lysine 36

Youdell

Kizer

Kisseleva-Romanova

et al. 2008

Molecular and Cellular Biology

146

194

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“…Nuclear fractions were prepared as in Kizer et al (2006). Proteins were transferred to PVDF membranes (Hybond) and probed with the following: anti-H4K5Ac (1:5000 dilution, Serotec), anti-H4K8Ac (1:2000 dilution, Serotec), anti-H4K12Ac (1:2000 dilution, Active Motif), anti-H4K16Ac (1:2000 dilution, Upstate), anti-H3CT (1:10,000 dilution, Millipore), anti-H3K9,K14Ac (1:10,000 dilution, Upstate), anti-H4 (1:2000 dilution, Active Motif), and anti-b-tubulin (1:20,000) (Bond et al 1986).…”

Section: Immunoblotsmentioning

confidence: 99%

Bypassing the Requirement for an Essential MYST Acetyltransferase

Torres-Machorro

Pillus

2014

Genetics

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Histone acetylation is a key regulatory feature for chromatin that is established by opposing enzymatic activities of lysine acetyltransferases (KATs/HATs) and deacetylases (KDACs/HDACs). Esa1, like its human homolog Tip60, is an essential MYST family enzyme that acetylates histones H4 and H2A and other nonhistone substrates. Here we report that the essential requirement for ESA1 in Saccharomyces cerevisiae can be bypassed upon loss of Sds3, a noncatalytic subunit of the Rpd3L deacetylase complex. By studying the esa1Δ sds3Δ strain, we conclude that the essential function of Esa1 is in promoting the cellular balance of acetylation. We demonstrate this by fine-tuning acetylation through modulation of HDACs and the histone tails themselves. Functional interactions between Esa1 and HDACs of class I, class II, and the Sirtuin family define specific roles of these opposing activities in cellular viability, fitness, and response to stress. The fact that both increased and decreased expression of the ESA1 homolog TIP60 has cancer associations in humans underscores just how important the balance of its activity is likely to be for human well-being.T HE genetic information of DNA is packed into chromatin, which is predominantly composed of the H3, H4, H2A, and H2B core histone proteins that together with DNA form nucleosomes, the basic subunits of the genome (Kornberg and Lorch 1999). Chromatin structure regulates many cellular processes including gene expression, DNA replication, DNA damage repair, and recombination (Felsenfeld and Groudine 2003). Nucleosomes themselves are tightly regulated by mobilization and positioning that are mediated by ATP-dependent chromatin-remodeling machines (Rando and Winston 2012) and by multiple types of histone posttranslational modifications that include acetylation and many other marks (Campos and Reinberg 2009).Nucleosome acetylation is a highly dynamic modification that is promoted by HATs and removed by HDACs. HATs are classified by sequence into different families (Allis et al. 2007). ESA1/KAT5 belongs to the widely conserved MYST HAT family, named for its founding members (MOZ-YBF2/ SAS3-SAS2-TIP60) (Lafon et al. 2007). Esa1 is an essential HAT in yeast (Smith et al. 1998;Clarke et al. 1999) and the catalytic subunit of two distinct multi-protein complexes: NuA4 and Piccolo (Boudreault et al. 2003). Notably, the human homolog of Esa1 is Tip60, which is also essential in vertebrates and has been linked to multiple human diseases (Squatrito et al. 2006;Avvakumov and Côté 2007;Lafon et al. 2007), thus increasing the relevance of gaining a deeper understanding of essential HAT functions.Esa1 primarily acetylates H4 and H2A in vivo (Clarke et al. 1999;Lin et al. 2008) and regulates the expression of active protein-encoding genes (Reid et al. 2000;Lin et al. 2008). It plays a crucial role in cell cycle progression and ribosomal DNA (rDNA) silencing (Clarke et al. 1999(Clarke et al. , 2006 and is recruited to DNA double-strand breaks (DSBs) to promote damage repair by acetylati...

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Accelerated nuclei preparation and methods for analysis of histone modifications in yeast

Cited by 27 publications

References 40 publications

The Linker Histone Plays a Dual Role during Gametogenesis in Saccharomyces cerevisiae

The Linker Histone Plays a Dual Role during Gametogenesis in Saccharomyces cerevisiae

Roles for Ctk1 and Spt6 in Regulating the Different Methylation States of Histone H3 Lysine 36

Bypassing the Requirement for an Essential MYST Acetyltransferase

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