A general method for purification of H1 histones that are active for repression of basal RNA polymerase II transcription

Croston, Glenn; Lira, Lucy M.; Kadonaga, James T.

doi:10.1016/1046-5928(91)90066-r

Cited by 33 publications

(20 citation statements)

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“…Moreover, ume6⌬ yeast shows major meiotic defects due to the deregulation of the tightly controlled meiotic transcriptional program (47,54). Similar to Ume6, linker histones in other eukaryotes have generally been associated with transcriptional repression (7).…”

Section: Resultsmentioning

confidence: 99%

“…One complicating factor is that several isoforms of H1 are present in humans and mice, and knockout studies have proven to be difficult due to functional redundancy (21). The linker histone represses transcription in vitro, but its transcriptional role in vivo has been the subject of debate (7,24). Although H1 has historically been associated with chromatin compaction and general repression, several recent studies have implicated the linker histone in the repression of specific genes via recruitment by transcription factors (10,33,37,45,48,52).…”

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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: Resultsmentioning

confidence: 99%

mentioning

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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“…Biochemical studies using in vitro assembly of a variety of promoter sequences into chromatin have demonstrated a general inhibitory role for nucleosomes in transcription (15,23,27,56). However, the prebinding of transcription factors, such as the transcription factor IID (TFIID) complex, is sufficient to overcome this effect (55).…”

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“…The significance of a repressive function of chromatin structure has been amply demonstrated with genetic and biochemical experiments (15,19,37). Genetic studies demonstrated that production of yeast strains deleted of specific histones and/or with specific mutations to prevent posttranslational modifications results in profound effects on regulated gene expression.…”

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confidence: 99%

“…A particularly well studied group are the inducible promoters as exemplified by the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) (6, 57), the PH05 gene in Saccharomyces cerevisiae (1), and the tyrosine aminotransferase gene in liver (12,42). While recent experiments analyzing the Drosophila HSP26 and the chicken vitellogenin genes have suggested a positive potentiation for chromatin (29,44), in most cases its effect has been to inhibit transcription (1,6,50).The significance of a repressive function of chromatin structure has been amply demonstrated with genetic and biochemical experiments (15,19,37). Genetic studies demonstrated that production of yeast strains deleted of specific histones and/or with specific mutations to prevent posttranslational modifications results in profound effects on regulated gene expression.…”

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Nucleosome-mediated disruption of transcription factor-chromatin initiation complexes at the mouse mammary tumor virus long terminal repeat in vivo.

Lee¹,

Archer²

1994

Mol. Cell. Biol.

115

156

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Glucocorticoid induction of mouse mammary tumor virus (MMTV) is short lived, returning to base levels within 24 h despite the continued presence of hormone. MMTV DNA sequences assembled as chromatin require hormone for binding by nuclear factor 1 (NF1) and octamer proteins (OCT). However, in the same cells, NF1 and OCT factors are bound to transiently introduced DNA in the absence of hormone. In contrast, recruitment of the TATA-binding protein and a novel DNA-binding protein, which we have designated FDT, for factor downstream of the TATA-binding protein, is hormone dependent for both stable and transient templates. Furthermore, transient DNA templates, but not nucleosomal templates, retain these transcription factors over the course of 24 h. This finding suggests that MMTV chromatin structure contributes to activation and cessation of transcription in vivo.The regulation of transcription requires the complex interaction of sequence-specific transactivating proteins as well as the extensive array of basal transcription factors without sequence-specific binding capacity (24, 58). These interactions are knit together by an increasingly large number of coactivator proteins which appear to facilitate contact between the enhancer or repressing proteins on this basal machinery (16,30,40,59). In the nucleus of eukaryotes, this process is further complicated by the fact that the target sites of trans-acting factors are often assembled as chromatin (18,51). Not surprisingly, this compaction of DNA sequences produced by the association of histones has been shown to be relevant to the regulation of transcription for several eukaryotic genes in vivo (17,18,51). A particularly well studied group are the inducible promoters as exemplified by the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) (6, 57), the PH05 gene in Saccharomyces cerevisiae (1), and the tyrosine aminotransferase gene in liver (12,42). While recent experiments analyzing the Drosophila HSP26 and the chicken vitellogenin genes have suggested a positive potentiation for chromatin (29,44), in most cases its effect has been to inhibit transcription (1,6,50).The significance of a repressive function of chromatin structure has been amply demonstrated with genetic and biochemical experiments (15,19,37). Genetic studies demonstrated that production of yeast strains deleted of specific histones and/or with specific mutations to prevent posttranslational modifications results in profound effects on regulated gene expression. In one case, Simpson and colleagues demonstrated that the ability of certain transactiving factors such as the a2 repressor to influence nucleosomal positions * Corresponding author. Mailing address:

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In Vitro Transcription and Immobilized Template Analysis of Preinitiation Complexes

Lin

Carey

2012

CP Molecular Biology

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In the study of gene regulation, it is often necessary to employ functional assays that investigate the action or mechanism of specific promoters or enhancer binding factors and their role in transcription by RNA Polymerase II. There are many assays that measure the transcription of a gene under the control of an endogenous or model activator in vivo. However, to study specific regulatory mechanisms it can be useful to recreate transcription in vitro. In this unit, we will present protocols and techniques that will allow the investigator to perform transcription in vitro using preinitiation complexes (PICs) assembled from cellular extracts on either naked DNA or chromatin templates.

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A general method for purification of H1 histones that are active for repression of basal RNA polymerase II transcription

Cited by 33 publications

References 35 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

Nucleosome-mediated disruption of transcription factor-chromatin initiation complexes at the mouse mammary tumor virus long terminal repeat in vivo.

In Vitro Transcription and Immobilized Template Analysis of Preinitiation Complexes

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