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The structure and dynamics of promoter chromatin have a profound effect on the expression levels of genes. Yet, the contribution of DNA sequence, histone post-translational modifications, histone variant usage and other factors in shaping the architecture of chromatin, and the mechanisms by which this architecture modulates expression of specific genes are not yet completely understood. Here we use optical tweezers to study the roles that DNA sequence and the histone variant H2A.Z have in shaping the chromatin landscape at the promoters of two model genes, Cga and Lhb. Guided by MNase mapping of the promoters of these genes, we reconstitute nucleosomes that mimic those located near the transcriptional start site and immediately downstream (+1), and measure the forces required to disrupt these nucleosomes, and their mobility along the DNA sequence. Our results indicate that these genes are basally regulated by two distinct strategies, making use of H2A.Z to modulate separate phases of transcription, and highlight how DNA sequence, alternative histone variants and remodelling machinery act synergistically to modulate gene expression.

Since the discovery that many transcriptional enhancers are transcribed into long noncoding RNAs termed "enhancer RNAs" (eRNAs), their putative role in enhancer function has been debated. Very recent evidence has indicted that some eRNAs play a role in initiating or activating transcription, possibly by helping recruit and/or stabilize binding of the general transcription machinery to the proximal promoter of their target genes. The distal enhancer of the gonadotropin hormone α-subunit gene, chorionic gonadotropin alpha (Cga), is responsible for Cga cell-specific expression in gonadotropes and thyrotropes, and we show here that it encodes two bidirectional nonpolyadenylated RNAs whose levels are increased somewhat by exposure to gonadotropin-releasing hormone but are not necessarily linked to Cga transcriptional activity. Knockdown of the more distal eRNA led to a drop in Cga mRNA levels, initially without effect on the forward eRNA levels. With time, however, the repression on the Cga increased, and the forward eRNA levels were suppressed also. We demonstrate that the interaction of the enhancer with the promoter is lost after eRNA knockdown. Dramatic changes also were seen in the chromatin, with an increase in total histone H3 occupancy throughout this region and a virtual loss of histone H3 Lys 4 trimethylation at the promoter following the eRNA knockdown. Moreover, histone H3 Lys 27 (H3K27) acetylation, which was found at both enhancer and promoter in wild-type cells, appeared to have been replaced by H3K27 trimethylation at the enhancer. Thus, the Cga eRNA mediates the physical interaction between these genomic regions and determines the chromatin structure of the proximal promoter to allow gene expression.

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