H2A.Z is an evolutionary conserved histone variant involved in transcriptional regulation, antisilencing, silencing, and genome stability. The mechanism(s) by which H2A.Z regulates these various biological functions remains poorly defined, in part due to the lack of knowledge regarding its physical location along chromosomes and the bearing it has in regulating chromatin structure. Here we mapped H2A.Z across the yeast genome at an approximately 300-bp resolution, using chromatin immunoprecipitation combined with tiling microarrays. We have identified 4,862 small regions—typically one or two nucleosomes wide—decorated with H2A.Z. Those “Z loci” are predominantly found within specific nucleosomes in the promoter of inactive genes all across the genome. Furthermore, we have shown that H2A.Z can regulate nucleosome positioning at the GAL1 promoter. Within HZAD domains, the regions where H2A.Z shows an antisilencing function, H2A.Z is localized in a wider pattern, suggesting that the variant histone regulates a silencing and transcriptional activation via different mechanisms. Our data suggest that the incorporation of H2A.Z into specific promoter-bound nucleosomes configures chromatin structure to poise genes for transcriptional activation. The relevance of these findings to higher eukaryotes is discussed.

Transcription by RNA polymerase II (RNAPII) is coupled to mRNA processing and chromatin modifications via the C-terminal domain (CTD) of its largest subunit, consisting of multiple repeats of the heptapeptide YSPTSPS. Pioneering studies showed that CTD serines are differentially phosphorylated along genes in a prescribed pattern during the transcription cycle. Genome-wide analyses challenged this idea, suggesting that this cycle is not uniform among different genes. Moreover, the respective role of enzymes responsible for CTD modifications remains controversial. Here, we systematically profiled the location of the RNAPII phosphoisoforms in wild-type cells and mutants for most CTD modifying enzymes. Together with results of in vitro assays, these data reveal a complex interplay between the modifying enzymes, and provide evidence that the CTD cycle is uniform across genes. We also identify Ssu72 as the Ser7 phosphatase and show that proline isomerization is a key regulator of CTD dephosphorylation at the end of genes.

Nuclear receptors can activate diverse biological pathways within a target cell in response to their cognate ligands, but how this compartmentalization is achieved at the level of gene regulation is poorly understood. We used a genome-wide analysis of promoter occupancy by the estrogen receptor ␣ (ER␣) in MCF-7 cells to investigate the molecular mechanisms underlying the action of 17␤-estradiol (E2) in controlling the growth of breast cancer cells. We identified 153 promoters bound by ER␣ in the presence of E2. Motif-finding algorithms demonstrated that the estrogen response element (ERE) is the most common motif present in these promoters whereas conventional chromatin immunoprecipitation assays showed E2-modulated recruitment of coactivator AIB1 and RNA polymerase II at these loci. The promoters were linked to known ER␣ targets but also to many genes not directly associated with the estrogenic response, including the transcriptional factor FOXA1, whose expression correlates with the presence of ER␣ in breast tumors. We found that ablation of FOXA1 expression in MCF-7 cells suppressed ER␣ binding to the prototypic TFF1 promoter (which contains a FOXA1 binding site), hindered the induction of TFF1 expression by E2, and prevented hormone-induced reentry into the cell cycle. Taken together, these results define a paradigm for estrogen action in breast cancer cells and suggest that regulation of gene expression by nuclear receptors can be compartmentalized into unique transcriptional domains by means of licensing of their activity to cofactors such as FOXA1.ChIP-on-chip ͉ forkhead box ͉ transcription ͉ cell cycle E stradiol (17␤-estradiol, E 2 ) is a potent growth factor of human breast cancer cells that exerts its action mainly through estrogen receptor ␣ (NR3A1, ER␣), a member of the superfamily of nuclear receptors (1). Despite significant advancement into our understanding of the molecular mechanisms of ER␣ action (2), little is known about mediators of the estrogen pathway that assist in the initiation, compartmentalization, and propagation of its signal at the level of gene expression. Delineation of how ER␣ induces precise biological responses in breast cancer cells and other cell types has clearly been limited by the lack of data on the transcriptional regulatory regions of ER␣ direct target genes.ER␣ regulates the expression of target genes by binding to specific sites in the chromatin, referred to as estrogen response elements (EREs) (3), or by interacting with other transcription factors bound to their own specific recognition sites (4-6). Determination of ER␣ target genes has recently been undertaken by using DNA microarrays, identifying hundreds of genes with altered expression upon E 2 treatment of human breast cancer cells (7-17). However, while providing information of the global action of E 2 in these cells, gene expression profiling can rarely discriminate between direct and indirect ER␣ targets. In addition, bioinformatic and comparative genomics have also been used successfully to identify high-...