The long non-coding RNA Kcnq1ot1 controls maternal p57 expression in muscle cells by promoting H3K27me3 accumulation to an intragenic MyoD-binding region

Abstract

Background: The cell-cycle inhibitor p57 kip2 plays a critical role in mammalian development by coordinating cell proliferation and differentiation in many cell types. p57 kip2 expression is finely regulated by several epigenetic mechanisms, including paternal imprinting. Kcnq1ot1, a long non-coding RNA (LncRNA), whose gene maps to the p57 Kip2 imprinting domain, is expressed exclusively from the paternal allele and participates in the cis-silencing of the neighboring imprinted genes through chromatin-level regulation. In light of our previous evidence of a functional interaction between myogenic factors and imprinting control elements in the regulation of the maternal p57 Kip2 allele during muscle differentiation, we examined the possibility that also Kcnq1ot1 could play an imprinting-independent role in the control of p57 Kip2 expression in muscle cells. Results: We found that Kcnq1ot1 depletion by siRNA causes the upregulation of the maternal and functional p57 Kip2 allele during differentiation, suggesting a previously undisclosed role for this LncRNA. Consistently, Chromatin Oligo-affinity Precipitation assays showed that Kcnq1ot1 physically interacts not only with the paternal imprinting control region of the locus, as already known, but also with both maternal and paternal alleles of a novel p57 Kip2 regulatory region, located intragenically and containing two binding sites for the muscle-specific factor MyoD. Moreover, chromatin immunoprecipitation assays after Kcnq1ot1 depletion demonstrated that the LncRNA is required for the accumulation of H3K27me3, a chromatin modification catalyzed by the histone-methyl-transferase EZH2, at the maternal p57 kip2 intragenic region. Finally, upon differentiation, the binding of MyoD to this region and its physical interaction with Kcnq1ot1, analyzed by ChIP and RNA immunoprecipitation assays, correlate with the loss of EZH2 and H3K27me3 from chromatin and with p57 Kip2 de-repression. Conclusions: These findings highlight the existence of an imprinting-independent role of Kcnq1ot1, adding new insights into the biology of a still mysterious LncRNA. Moreover, they expand our knowledge about the molecular mechanisms underlying the tight and fine regulation of p57 Kip2 during differentiation and, possibly, its aberrant silencing observed in several pathologic conditions.