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.
Highlights
Thousands of long non-coding RNAs are produced by the mammalian genome, but few are larger than 10–20 kb
Parental origin of the alleles was distinguished by crossing heterozygous mutant mice with B6(CAST7) mice, a consomic strain in which chromosome 7 is derived from M. musculus castaneus [23]
We provide strong evidence that DNA sequence elements and their relative location in the Kcnq1ot1 region are responsible for imprinted expression of Cdkn1c and, to some degree, of early embryonic Kcnq1
Summary
Thousands of long non-coding (lnc) RNAs are produced by the mammalian genome, but few are larger than 10–20 kb. It was initially assumed that Kcnq1ot silenced its sense counterpart, the Kcnq gene, two observations led us to reassess how the ncRNA regulates Kcnq: (i) studies in whole embryos and neonatal mouse tissues revealed that expression of Kcnq transitions to a biallelic mode after mid-gestation [8]; (ii) detailed expression and conformational profiles of the developing heart showed that the loss of Kcnq imprinting coincides with activation of strong cardiac-specific enhancers that physically engage with the Kcnq promoter [9] Based on these observations, we suggested that enhancer-driven expression could successfully compete and override the silencing effects of Kcnq1ot transcription on the paternal Kcnq allele
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