Enhancers compete with a long non-coding RNA for regulation of the Kcnq1 domain

Abstract

The imprinted Kcnq1 domain contains a differentially methylated region (KvDMR) in intron 11 of Kcnq1. The Kcnq1ot1 non-coding RNA emerges from the unmethylated paternal KvDMR in antisense direction, resulting in cis-repression of neighboring genes. The KvDMR encompasses the Kcnq1ot1 promoter, CTCF sites and other DNA elements, whose individual contribution to regulation of the endogenous domain is unknown. We find that paternal inheritance of a deletion of the minimal Kcnq1ot1 promoter derepresses the upstream Cdkn1c gene. Surprisingly, Kcnq1ot1 transcripts continue to emerge from alternative sites, evidence that silencing depends, not on the ncRNA, but on the promoter sequence. Detailed analyses of Kcnq1ot during cardiogenesis show substantial chromatin reorganization coinciding with discontinuous RNA production in both wild-type and mutant mice, with loss of imprinting. We show that CTCF binds to both methylated and unmethylated alleles of the KvDMR. Furthermore, we report a multitude of enhancers within the Kcnq1ot1 region, and present conformational dynamics of a novel heart enhancer engaged in Kcnq1 expression. Our results have important implications on tissue-specific imprinting patterns and how transcriptional mechanisms compete to maximize the expression of vital genes, in addition to shifting our perception on the role of the long ncRNA in regulating this imprinted domain.

Blocked transcription through KvDMR1 results in absence of methylation and gene silencing resembling Beckwith-Wiedemann syndrome

“…S6). This could indicate that insertion of the truncation cassette disrupted a currently undefined negative regulatory element for these two genes; in this regard, it is notable that recent evidence supports the existence of multiple enhancer elements in the KvDMR1/Kcnq1ot1 region (Korostowski et al, 2011;Schultz et al, 2015). Disruption of a gene repressor/silencer at this insertion site may also explain the apparently less efficient silencing effect that insertion of the YJ11 cassette has on Cdkn1c and Kcnq1 (Figs S5 and S6).…”

Non-coding RNAs in muscle differentiation and musculoskeletal disease

“…Expression of Kcnq1ot1 inversely correlates with that of Kcnq1. The altered expression is due not to the formation of repressive chromatin, but to changes in the three-dimensional conformation of chromatin that results in the inactivation of heart-specific enhancers (102). Proper Kcnq1 levels are essential for maintaining cardiac rhythm and heart function (103).…”

LncRNAs in vertebrates: Advances and challenges

“…Because this strategy is likely not applicable to the majority of lncRNAs, several alternative strategies to perturb in vivo lncRNA expression are employed routinely including the deletion of whole lncRNA loci [66,76,141], the deletion of promoter regions [142][143][144][145], the insertion of a premature polyadentylation (polyA) termination signal [74,146,147], the replacement of a lncRNA locus with a reporter gene [65,73,148], and the deletion of putative functional domains in cases where lncRNA function has been characterized. Recently, using these different strategies to achieve loss of lncRNA function, several genetic lncRNA mouse mutants, as well as numerous lncRNA mutant cell lines have been generated and analyzed (reviewed in [136,149]).…”

“…This distinction is particularly important because at least for some lncRNA loci, such as Airn [150], the act of transcription and not the lncRNA product per se is an important M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT regulatory element for at least a subset of its functions. However, caution is warranted when implementing this promoter deletion strategy as some lncRNAs have alternative promoter usage, and, thus, incomplete deletion of promoter sequences may lead to only partial loss of lncRNA function due to the retention of limited transcription, as has recently been shown for the lncRNA Kcnq1ot1 [144]. In addition to alternative promoter usage, there are some examples where the promoters of lncRNA genes overlap with the promoters or regulatory elements of their adjacent genes.…”