Many genes are recruited to the nuclear periphery upon transcriptional activation. The mechanism and functional significance of this recruitment is unclear. We find that recruitment of the yeast INO1 and GAL1 genes to the nuclear periphery is rapid and independent of transcription. Surprisingly, these genes remain at the periphery for generations after they are repressed. Localization at the nuclear periphery serves as a form of memory of recent transcriptional activation, promoting reactivation. Previously expressed GAL1 at the nuclear periphery is activated much more rapidly than long-term repressed GAL1 in the nucleoplasm, even after six generations of repression. Localization of INO1 at the nuclear periphery is necessary and sufficient to promote more rapid activation. This form of transcriptional memory is chromatin based; the histone variant H2A.Z is incorporated into nucleosomes within the recently repressed INO1 promoter and is specifically required for rapid reactivation of both INO1 and GAL1. Furthermore, H2A.Z is required to retain INO1 at the nuclear periphery after repression. Therefore, H2A.Z-mediated localization of recently repressed genes at the nuclear periphery represents an epigenetic state that confers memory of transcriptional activation and promotes reactivation.
Many genes are recruited to the nuclear periphery upon transcriptional activation in Saccharomyces cerevisiae. We have identified two Gene Recruitment Sequences (GRS I and II) from the promoter of the INO1 gene that target the gene to the nuclear periphery. These GRSs function as DNA zip codes; they are sufficient to target a nucleoplasmic locus to the nuclear periphery. Targeting requires components of the nuclear pore complex (NPC) and a GRS is sufficient to confer a physical interaction with the NPC. GRS I elements are enriched in promoters of genes that interact with the NPC and genes that are induced by protein folding stress. Full transcriptional activation of INO1 and another GRS-containing gene requires GRS-mediated targeting of the promoter to the nuclear periphery. Finally, GRS I also functions as a DNA zip code in Schizosaccharomyces pombe, suggesting that this mechanism of targeting to the nuclear periphery has been conserved over approximately one billion years of evolution.
SUMMARYSeveral lines of evidence suggest that long non-coding RNA (lncRNA)-dependent mechanisms regulate transcription and CpG DNA methylation. Whereas CpG island methylation has been studied in detail, the significance of enhancer DNA methylation and its relationship with lncRNAs is relatively unexplored. Previous experiments proposed that the ultraconserved lncRNA Evf2 represses transcription through Dlx6 antisense (Dlx6as) transcription and methyl-CpG binding protein (MECP2) recruitment to the Dlx5/6 ultraconserved DNA regulatory enhancer (Dlx5/6ei) in embryonic day 13.5 medial ganglionic eminence (E13.5 MGE). Here, genetic epistasis experiments show that MECP2 transcriptional repression of Evf2 and Dlx5, but not Dlx6, occurs through antagonism of DLX1/2 in E13.5 MGE. Analysis of E13.5 MGE from mice lacking Evf2 and of partially rescued Evf2 transgenic mice shows that Evf2 prevents site-specific CpG DNA methylation of Dlx5/6ei in trans, without altering Dlx5/6 expression. Dlx1/2 loss increases CpG DNA methylation, whereas Mecp2 loss does not affect Dlx5/6ei methylation. Based on these studies, we propose a model in which Evf2 inhibits enhancer DNA methylation, effectively modulating competition between the DLX1/2 activator and MECP2 repressor. Evf2 antisense transcription and Evf2-dependent balanced recruitment of activator and repressor proteins enables differential transcriptional control of adjacent genes with shared DNA regulatory elements.
Transcription-regulating long non-coding RNAs (lncRNAs) have the potential to control the site-specific expression of thousands of target genes. Previously, we showed that Evf2, the first described ultraconserved lncRNA, increases the association of transcriptional activators (DLX homeodomain proteins) with key DNA enhancers but represses gene expression. In this report, mass spectrometry shows that the Evf2-DLX1 ribonucleoprotein (RNP) contains the SWI/SNFrelated chromatin remodelers Brahma-related gene 1 (BRG1, SMARCA4) and Brahma-associated factor (BAF170, SMARCC2) in the developing mouse forebrain. Evf2 RNA colocalizes with BRG1 in nuclear clouds and increases BRG1 association with key DNA regulatory enhancers in the developing forebrain. While BRG1 directly interacts with DLX1 and Evf2 through distinct binding sites, Evf2 directly inhibits BRG1 ATPase and chromatin remodeling activities. In vitro studies show that both RNA-BRG1 binding and RNA inhibition of BRG1 ATPase/remodeling activity are promiscuous, suggesting that context is a crucial factor in RNAdependent chromatin remodeling inhibition. Together, these experiments support a model in which RNAs convert an active enhancer to a repressed enhancer by directly inhibiting chromatin remodeling activity, and address the apparent paradox of RNAmediated stabilization of transcriptional activators at enhancers with a repressive outcome. The importance of BRG1/RNA and BRG1/homeodomain interactions in neurodevelopmental disorders is underscored by the finding that mutations in Coffin-Siris syndrome, a human intellectual disability disorder, localize to the BRG1 RNAbinding and DLX1-binding domains.
Gene regulation requires selective targeting of DNA regulatory enhancers over megabase distances. Here we show that Evf2, a cloud-forming Dlx5/6 ultraconserved enhancer (UCE) lncRNA, simultaneously localizes to activated (Umad1, 1.6 Mb distant) and repressed (Akr1b8, 27 Mb distant) chr6 target genes, precisely regulating UCE-gene distances and cohesin binding in mouse embryonic forebrain GABAergic interneurons (INs). Transgene expression of Evf2 activates Lsm8 (12 Mb distant) but fails to repress Akr1b8, supporting trans activation and long-range cis repression. Through both short-range (Dlx6 antisense) and long-range (Akr1b8) repression, the Evf2-5'UCE links homeodomain and mevalonate pathway-regulated enhancers to IN diversity. The Evf2-3' end is required for long-range activation but dispensable for RNA cloud localization, functionally dividing the RNA into 3'-activator and 5'UCE repressor and targeting regions. Together, these results support that Evf2 selectively regulates UCE interactions with multi-megabase distant genes through complex effects on chromosome topology, linking lncRNA-dependent topological and transcriptional control with interneuron diversity and seizure susceptibility.
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