Chromatin Remodeling and Gene Activation

基因激活涉及到一系列因子被募集到启动子中，以响应适当的信号，最终通过RNA聚合酶II和转录形成起始复合物。这些事件与启动子核小体的去除产生核小体耗尽区（NDR）相吻合。这一观察结果导致了普遍接受的模型，即启动子核小体物理上阻断转录起始，通过阻止进入特定的转录因子结合位点而起到抑制因子的作用。核小体是一种非常稳定的结构，含有紧密缠绕的DNA，序列特异性DNA结合蛋白在很大程度上是无法接近的。如果序列特异性的“先锋”转录因子存在（这些蛋白质以高亲和力结合核小体位点），和/或通常被核小体阻断的“经典”转录因子招募依赖atp的染色质重塑子移动或驱逐启动子核小体，从而促进起始复合物的形成，就会发生激活。

Gene activation involves the recruitment of a set of factors to a promoter in response to appropriate signals, ultimately resulting in the formation of an initiation complex by RNA polymerase II and transcription. These events coincide with the removal of promoter nucleosomes to create a nucleosome-depleted region (NDR). This observation has led to the generally accepted model that promoter nucleosomes physically block transcript initiation, acting as repressors by preventing access to specific transcription factor binding sites. The nucleosome is a very stable structure containing tightly wound DNA that is largely inaccessible to sequence-specific DNA binding proteins. Activation occurs if sequence-specific 'pioneer' transcription factors are present (these proteins bind nucleosomal sites with high affinity), and/or if 'classical' transcription factors, which are normally blocked by nucleosomes, recruit ATP-dependent chromatin remodelers to move or evict promoter nucleosomes, thus facilitating initiation complex formation. The ATP-dependent chromatin remodelers variously move nucleosomes along DNA, or remove the histones altogether, or form arrays of regularly spaced nucleosomes. Examples include the SWI/SNF and RSC complexes, which remodel nucleosomes on genes and at promoters, and the CHD and ISWI complexes, which are often involved in determining nucleosome spacing. The Ino80C complex is unusual because it has both properties. We have written a review of the ATP-dependent remodelers and their roles in chromatin organization (1). Human diseases have been linked to chromatin remodeling enzymes. For example, mutations in the hSNF5 subunit of the SWI/SNF complex are strongly linked to pediatric rhabdoid tumors, and the CHD remodelers have been linked to cancer and autism. Therapies and drugs aimed at epigenetic targets are being tested. A full understanding of chromatin structure and the mechanisms by which it is manipulated is therefore vital. Chromatin may block access to the DNA at several levels: the nucleosome itself, the higher order coiling of the nucleosomal fiber, and further condensation of nucleosomal fibers into heterochromatin. We have begun to test the hypothesis that DNA accessibility is of major importance in regulating gene expression (2). To do this, we needed a fully quantitative measure of DNA accessibility. Accordingly, we used the restriction enzyme AluI as a probe of chromatin structure and as a proxy for transcription factors, since both bind to specific DNA sequences. We measured the digestion rate and the fraction of accessible DNA accurately at all genomic AluI sites in budding yeast nuclei and in mouse liver cell nuclei. We observe that mouse liver DNA is significantly more accessible than yeast DNA, which can be explained simply by recognizing that the linker DNA between nucleosomes is very much more accessible than nucleosomal DNA and that nucleosomes are spaced farther apart in mouse chromatin, resulting in longer linkers than in yeast chromatin. These data indicate that nucleosome spacing is a major determinant of accessibility. More remarkable is the fact that DNA accessibility is binary, such that each site is accessible in some cells (i.e. in a linker and cut by AluI) and essentially completely inaccessible in the remaining cells (i.e. nucleosomal and resistant to AluI). In fact, we find no sites that are accessible in every cell or inaccessible in every cell. This observation indicates that nucleosome positioning is generally imperfect, even at promoters, such that nucleosomes never reliably block a specific site. For example, AluI sites in inactive mouse promoters are accessible in some cells, even though the gene is inactive, implying that the nucleosome is insufficient to block transcription factor binding in all cells and suggesting that the simple promoter nucleosome block model is incorrect. Surprisingly, in mouse nuclei, the relatively decondensed euchromatin (which contains mostly active genes) and the highly condensed heterochromatin (which contains mostly inactive genes) have very similar accessibilities to AluI, suggesting that transcription factors could also penetrate heterochromatin. Overall, our observations suggest that DNA accessibility is not likely to be the primary determinant of gene regulation. Most eukaryotic cells have a characteristic average nucleosome spacing of 190 bp, corresponding to a 45 bp linker. However, cortical neurons have a shorter average spacing of 165 bp, similar to that of the yeasts. The significance of this atypical global chromatin organization is unclear. In a collaboration with Doug Fields (NICHD) (3), we compared the chromatin structures of purified mouse dorsal root ganglia (DRG) neurons, cortical oligodendrocyte precursor cells (OPCs) and cortical astrocytes. We find that DRG neurons have short average spacing (165 bp), whereas OPCs (182 bp) and astrocytes (183 bp) have longer spacing. Most genes in all three cell types have a promoter chromatin organization typical of active genes: a promoter NDR flanked by regularly spaced nucleosomes. In DRG neurons, nucleosome spacing downstream of promoters is longer than expected from the genomic average, whereas nucleosome spacing in OPCs and astrocytes is similar to the global average for these cells. Thus, the atypical nucleosome spacing of neuronal chromatin does not extend to promoter-proximal regions. In a collaboration with Jeff Hayes (University of Rochester), we developed a method to assess exposure of histone protein surfaces in yeast chromatin (4). A histone amino acid residue is substituted with cysteine and its exposure is measured by reaction with a thiol-specific reagent. Nuclei are treated with biotin-maleimide and then the chromatin is digested to nucleosomes by micrococcal nuclease. Modified nucleosomes are purified using streptavidin beads. Nucleosomal DNA from input and modified samples are sequenced and compared. Currently, we are assessing the exposure of several different histone surfaces within active and inactive chromatin. We continued our collaboration with Alan Hinnebusch (NICHD). We have published several papers together concerning the roles of the SWI/SNF and RSC chromatin remodelers in promoter nucleosome eviction. We have now studied the role of the Ino80 remodeling complex (Ino80C) in transcriptional activation (5). We show that Ino80C is important for promoter nucleosome eviction and transcriptional activation. Compared to SWI/SNF, Ino80C generally functions over a wider region, spanning the promoter-flanking nucleosomes and NDR, at genes highly dependent on its function. Nucleosome eviction defects in cells lacking the Ino80 ATPase subunit are frequently accompanied by reduced promoter occupancies of TATA-binding protein (TBP), and diminished transcription. We conclude that Ino80C acts widely in the yeast genome, together with RSC and SWI/SNF, to evict promoter nucleosomes and enhance transcription. In a collaboration with Munira Basrai's Lab (National Cancer Institute), we investigated the mechanism by which yeast cells prevent mislocalization of the centromeric histone H3 variant (Cse4 in yeast; CENP-A in humans). Correct localization of Cse4 to centromeres is vital for faithful chromosome segregation. Overexpression and mislocalization of CENP-A has been observed in many cancers and correlates with increased invasiveness and poor prognosis. We identified yeast genes required to prevent Cse4 mislocalization using a genetic screen (6). They include two F-box proteins, Met30 and Cdc4, which interact and cooperatively regulate Cse4 proteolysis, preventing its incorporation into non-centromeric nucleosomes and consequent chromosome instability. The Dbf4-dependent kinase complex also plays a role in Cse4 proteolysis, probably through the Psh1 E3 ubiquitin ligase (7).