Effects of Sin- versions of histone H4 on yeast chromatin structure and function

Wechser, Mark A.; Kladde, Michael P.; Alfieri, Jennifer A.; Peterson, Craig L.

doi:10.1093/emboj/16.8.2086

Cited by 78 publications

(76 citation statements)

References 47 publications

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“…Previous studies have suggested that residual levels of Pho4 are present in the nucleus in high P i (38)(39)(40), despite its predominant cytoplasmic localization under these conditions (25). The presence of marked targeted 5me C at PHO5, PHO8, and PHO84 provides direct evidence of a low level of Pho4 binding in the presence of P i .…”

Section: Discussionmentioning

confidence: 77%

Targeted cytosine methylation for in vivo detection of protein–DNA interactions

Carvin

Dhasarathy

Friesenhahn

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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We report a technique, named targeted gene methylation (TAGM), for identifying in vivo protein-binding sites in chromatin. M.CviPI, a cytosine-5 DNA methyltransferase recognizing GC sites, is fused to a DNA-binding factor enabling simultaneous detection of targeted methylation, factor footprints, and chromatin structural changes by bisulfite genomic sequencing. Using TAGM with the yeast transactivator Pho4, methylation enrichments of up to 34-fold occur proximal to native Pho4-binding sites. Additionally, significant selective targeting of methylation is observed several hundred nucleotides away, suggesting the detection of long-range interactions due to higher-order chromatin structure. In contrast, at an extragenic locus lacking Pho4-binding sites, methylation levels are at the detection limit at early times after Pho4 transactivation. Notably, substantial amounts of methylation are targeted by Pho4-M.CviPI under repressive conditions when most of the transactivator is excluded from the nucleus. Thus, TAGM enables rapid detection of DNA-protein interactions even at low occupancies and has potential for identifying factor targets at the genome-wide level. Extension of TAGM from yeast to vertebrates, which use methylation to initiate and propagate repressed chromatin, could also provide a valuable strategy for heritable inactivation of gene expression.T he interaction of proteins with chromosomal target sites, either directly or through recruitment by DNA-bound factors, is central to many processes, including transcriptional activation and repression, replication and repair of DNA, recombination, and chromosome segregation. Therefore, strategies are needed that can efficiently identify specific chromosomal sites at which factors act. Few techniques are capable of demonstrating these interactions in the context of native chromatin in living cells, and these methods have limitations (1). For example, with footprinting techniques, protection against chemical (e.g., dimethyl sulfate) or enzymatic probes expressed in cells, e.g., DNA methyltransferases (MTases) (2-6) or DNase I (7), requires close proximity of the interacting factor to DNA sites that are modified or cleaved by the footprinting agent. Footprinting methods also require that the factor resists displacement by the enzymatic or chemical probe. Moreover, because many proteins can exclude probe access, a footprint does not provide an unequivocal identity of the bound protein (8). To circumvent this latter problem, proteins have been fused to an endonuclease (9); however, the resulting DNA damage alters chromatin structure and activates checkpoint controls. Another method, chromatin immunoprecipitation (ChIP), uses in situ fixation with formaldehyde followed by immunoselection of DNA-bound complexes. The requirements for large numbers of cells and highly specific antibodies as well as low fixation efficiencies (Ϸ0.1-0.5%) (10, 11) present distinct disadvantages of ChIP analysis. The approach of tethering chromatin proteins to the Dam MTase, which methylates ...

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Section: Discussionmentioning

confidence: 77%

Targeted cytosine methylation for in vivo detection of protein–DNA interactions

Carvin

Dhasarathy

Friesenhahn

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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“…However, when we examined GAL10-MEL1 expression in a yeast strain carrying the hhf2-13 mutation in histone H4, we did not see any increase in stimulation by GAL4-TBP (Ryan and Morse, unpublished). Thus, although this sin mutation alleviates the requirement for SWI-SNF for transcriptional activation of an HO-lacZ reporter gene (58,75) and increases accessibility of nucleosomal DNA to MNase and Escherichia coli Dam methyltransferase in yeast (75), it evidently does not sufficiently perturb the local chromatin structure of the GAL10 promoter to allow activation by GAL4-TBP. We also employed an altered GAL10-MEL1 reporter having a LexA binding site between the GAL4 binding sites and the TATA element to ask whether artificial depletion of histone H4 would allow activation by LexATBP.…”

Section: Discussionmentioning

confidence: 94%

Artificially Recruited TATA-Binding Protein Fails To Remodel Chromatin and Does Not Activate Three Promoters That Require Chromatin Remodeling

Ryan

Stafford

et al. 2000

Molecular and Cellular Biology

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Transcriptional activators are believed to work in part by recruiting general transcription factors, such as TATA-binding protein (TBP) and the RNA polymerase II holoenzyme. Activation domains also contribute to remodeling of chromatin in vivo. To determine whether these two activities represent distinct functions of activation domains, we have examined transcriptional activation and chromatin remodeling accompanying artificial recruitment of TBP in yeast (Saccharomyces cerevisiae). We measured transcription of reporter genes with defined chromatin structure by artificial recruitment of TBP and found that a reporter gene whose TATA element was relatively accessible could be activated by artificially recruited TBP, whereas two promoters, GAL10 and CHA1, that have accessible activator binding sites, but nucleosomal TATA elements, could not. A third reporter gene containing the HIS4 promoter could be activated by GAL4-TBP only when a RAP1 binding site was present, although RAP1 alone could not activate the reporter, suggesting that RAP1 was needed to open the chromatin structure to allow activation. Consistent with this interpretation, artificially recruited TBP was unable to perturb nucleosome positioning via a nucleosomal binding site, in contrast to a true activator such as GAL4, or to perturb the TATA-containing nucleosome at the CHA1 promoter. Finally, we show that activation of the GAL10 promoter by GAL4, which requires chromatin remodeling, can occur even in swi gcn5 yeast, implying that remodeling pathways independent of GCN5, the SWI-SNF complex, and TFIID can operate during transcriptional activation in vivo.Transcriptional activators are thought to stimulate transcription of TATA-containing promoters in part by recruiting TFIID, a multiprotein complex consisting of the TATA-binding protein (TBP) and TBP-associated factors (TAFs), to the TATA box (25,60). Several in vitro and in vivo studies support this model. For example, transcription initiated at a mutated TATA element by induced synthesis of TBP with altered specificity was enhanced in both rate and extent in the presence of an activator that could bind upstream of the relevant promoter, consistent with activator-mediated recruitment (40). Recruitment has also been inferred from the results of "activator bypass" experiments in which artificial recruitment of TBP to promoter sites near the TATA element resulted in transcriptional activation, implying that TBP recruitment is a rate-limiting step in transcriptional activation in vivo (10,39,79). Most convincingly, chromatin immunoprecipitation experiments revealed TBP to be physically associated with promoters of numerous genes under activated, but not nonactivated, conditions, implying that recruitment accompanies activation (43,46).A potential obstacle to recruitment of TBP in vivo is posed by chromatin. Binding of TBP to nucleosomal TATA elements is greatly impeded in vitro (23, 32). Transcription is also strongly repressed by chromatin, but this repression can be largely alleviated by binding of act...

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“…Whereas the Val111 of H3T does not directly interact with H2A/H2B and DNA, a mutation at Arg116 (to His) close to Val111 reportedly destabilized the nucleosome (14,15). This H3-R116H mutation in Saccharomyces cerevisiae has been identified as a Sin mutation that alleviates the requirement for the nucleosome-remodeling factor, Swi/Snf, which activates transcription (14,16). A structural study revealed that the H3-Arg116 residue may not directly interact with the DNA backbone and H2A/H2B, but instead forms a salt bridge with H3-Asp123 (17).…”

Section: Discussionmentioning

confidence: 99%

Structural basis of instability of the nucleosome containing a testis-specific histone variant, human H3T

Tachiwana

Kagawa

Osakabe

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

203

236

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A histone H3 variant, H3T, is highly expressed in the testis, suggesting that it may play an important role in the chromatin reorganization required for meiosis and/or spermatogenesis. In the present study, we found that the nucleosome containing human H3T is significantly unstable both in vitro and in vivo, as compared to the conventional nucleosome containing H3.1. The crystal structure of the H3T nucleosome revealed structural differences in the H3T regions on both ends of the central α2 helix, as compared to those of H3.1. The H3T-specific residues (Met71 and Val111) are the source of the structural differences observed between H3T and H3.1. A mutational analysis revealed that these residues are responsible for the reduced stability of the H3T-containing nucleosome. These physical and structural properties of the H3T-containing nucleosome may provide the basis of chromatin reorganization during spermatogenesis.

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Effects of Sin- versions of histone H4 on yeast chromatin structure and function

Cited by 78 publications

References 47 publications

Targeted cytosine methylation for in vivo detection of protein–DNA interactions

Targeted cytosine methylation for in vivo detection of protein–DNA interactions

Artificially Recruited TATA-Binding Protein Fails To Remodel Chromatin and Does Not Activate Three Promoters That Require Chromatin Remodeling

Structural basis of instability of the nucleosome containing a testis-specific histone variant, human H3T

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