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Histone variants, including histone H2A.Z, are incorporated into specific genomic sites and participate in transcription regulation. The role of H2A.Z at these sites remains poorly characterized. Our study investigates changes in the chromatin environment at the Cyclin D1 gene (CCND1) during transcriptional initiation in response to estradiol in estrogen receptor positive mammary tumour cells. We show that H2A.Z is present at the transcription start-site and downstream enhancer sequences of CCND1 when the gene is poorly transcribed. Stimulation of CCND1 expression required release of H2A.Z concomitantly from both these DNA elements. The AAA+ family members TIP48/reptin and the histone variant H2A.Z are required to remodel the chromatin environment at CCND1 as a prerequisite for binding of the estrogen receptor (ERα) in the presence of hormone. TIP48 promotes acetylation and exchange of H2A.Z, which triggers a dissociation of the CCND1 3′ enhancer from the promoter, thereby releasing a repressive intragenic loop. This release then enables the estrogen receptor to bind to the CCND1 promoter. Our findings provide new insight into the priming of chromatin required for transcription factor access to their target sequence. Dynamic release of gene loops could be a rapid means to remodel chromatin and to stimulate transcription in response to hormones., Author Summary Our study investigates changes in the chromatin environment at the Cyclin D1 gene that are a prerequisite for transcriptional initiation in response to estradiol. Gene expression is under control of chromatin structure. Histone variants, including histone H2A.Z, are incorporated into specific genomic sites and participate in transcription regulation. We show that H2A.Z is present at the transcription start-site and downstream enhancer sequences of CCND1 when the gene is poorly transcribed. Stimulation of CCND1 expression required release of H2A.Z concomitantly from both these DNA elements. The TIP48/reptin protein, which is part of several chromatin remodeling complexes, also associated with the CCND1 regulatory elements. Here, TIP48 promotes exchange of H2A.Z, which triggers a dissociation of the CCND1 enhancer from the promoter, thereby releasing a repressive intragenic loop. This release then enables estrogen receptor binding to the CCND1 promoter. Acetylation of H2A.Z is required for these processes. Our findings provide new insight into the priming of chromatin required for transcription factor access to their target sequence. Hence, we propose a new model for early events in transcription activation that were not shown before. Specifically, release of looping could be a rapid means to activate transcription efficiently in response to stimuli, in particular estrogen.

The organization of eukaryotic genomes is characterized by the presence of distinct euchromatic and heterochromatic sub-nuclear compartments. In Saccharomyces cerevisiae heterochromatic loci, including telomeres and silent mating type loci, form clusters at the nuclear periphery. We have employed live cell 3-D imaging and chromosome conformation capture (3C) to determine the contribution of nuclear positioning and heterochromatic factors in mediating associations of the silent mating type loci. We identify specific long-range interactions between HML and HMR that are dependent upon silencing proteins Sir2p, Sir3p, and Sir4p as well as Sir1p and Esc2p, two proteins involved in establishment of silencing. Although clustering of these loci frequently occurs near the nuclear periphery, colocalization can occur equally at more internal positions and is not affected in strains deleted for membrane anchoring proteins yKu70p and Esc1p. In addition, appropriate nucleosome assembly plays a role, as deletion of ASF1 or combined disruption of the CAF-1 and HIR complexes abolishes the HML-HMR interaction. Further, silencer proteins are required for clustering, but complete loss of clustering in asf1 and esc2 mutants had only minor effects on silencing. Our results indicate that formation of heterochromatic clusters depends on correctly assembled heterochromatin at the silent loci and, in addition, identify an Asf1p-, Esc2p-, and Sir1p-dependent step in heterochromatin formation that is not essential for gene silencing but is required for long-range interactions., Author Summary Chromosomes are non-randomly positioned inside cells, and this organization is relevant for genome regulation. Spatial clustering of heterochromatic loci provides a striking example of nuclear compartmentalization. In S. cerevisiae, the presence of heterochromatic sub-nuclear domains has been well established, but their mechanisms of formation are not fully understood. Here, we analyzed the DNA elements and protein complexes that are critical for formation of heterochromatic clusters. We focused on heterochromatic regions on chromosome III—the two telomeres, as well as the silent mating type loci HML and HMR, located on the left and right end of the chromosome, respectively. We employed live cell 3-D imaging and chromosome conformation capture (3C) and found that these loci specifically interact most prominently near silencer elements that flank the loci. Analysis of a panel of mutants showed that complexes involved in silencing are also involved in long-range interactions. Interestingly, we find that heterochromatic interactions are mechanistically distinct from silencing and independent of tethering to the nuclear periphery. Our results indicate that formation of heterochromatic clusters depends on correctly assembled heterochromatin, and point to a step in heterochromatin formation that is not essential for gene silencing but is required for long-range interactions between heterochromatic loci.