The expression of p57, a critical regulator of cell proliferation and differentiation, is precisely controlled by several epigenetic mechanisms including paternal imprinting. Kcnq1ot1, a LncRNA mapping to the p57 imprinting domain, is paternally expressed and participates in the cis-silencing of the near imprinted genes through chromatin-level regulation.
Unexpectedly, we found that paternal Kcnq1ot1 depletion boosts maternal p57 expression and that Kcnq1ot1 associates with chromatin at an intragenic p57 region (p57i) on the maternal allele, recruiting the repressive chromatin mark H3K27me3. Upon differentiation, the myogenic factor MyoD binds to p57i, interacts with Kcnq1ot1 and promotes H3K27me3 displacement.
In the present project, we plan to further investigate the Kcnq1ot1-dpendent regulation of p57 in muscle cells with the following objectives:
1) Understand how paternally expressed Kcnq1ot1 regulates maternal p57.
We hypothesize that paternally transcribed Kcnq1ot1, which is unlikely to act in trans as a diffusible product, interacts with and regulate maternal p57 through a mechanism involving allele proximity. This hypothesis will be verified through RNA/DNA FISH experiments.
2) Determine the role of the functional interaction between Kcnq1ot1 and DNMT1 in p57 control in muscle cells.
Maternal p57 promoter is hypermethylated and inaccessible to transcription factors in undifferentiated myoblasts. Since Kcnq1ot1 interacts with DNMT1 during imprinting, we suppose that Kcnq1ot1 also mediates DNA methylation at maternal p57. This hypothesis will be verified by analysing:
- the physical interaction between Kcnq1ot1 and DNMT1 during differentiation
- the DNMT1 binding to p57i
- the allele-specific methylation status of p57i during differentiation and after Kcnq1ot1 depletion.
The proposed research not only will elucidate a further mechanism for fine tuning p57 expression, but also will add new insights into the biology of Kcnq1ot1, a LncRNA still poorly known.
The combination of multiple epigenetic modifications on the p57 regulatory elements reflects the necessity of an accurate dosage of its expression and of a very specific, spatial and temporal expression pattern. The skeletal muscle system, revealing the critical interplay between tissue-specific transcription factors, imprinting control regions, long-range chromatin interactions and epigenetic modifiers, offers a paradigmatic example and a useful tool to dissect the multiple mechanisms that cooperate in keeping p57 under precise control during developmental processes.
The proposed research not only will elucidate an additional mechanism involved in fine tuning the expression of p57 during a differentiation process, but also will add new insights into the biology of Kcnq1ot1, a LncRNA still poorly characterized, and into the epigenetic control of gene expression during muscle differentiation.
It is the first time that Kcnq1ot1, known to function in cis for the imprinted silencing of paternal p57, is proposed to play an imprinting-independent function by regulating the maternal, active p57 allele. The possible finding that paternal Kcnq1ot1 physically interacts with and represses maternal p57 not only would expand our knowledge about imprinting factors and their imprinting-independent roles, but also would emphasize the significance of allelic interactions in transcriptional regulation.
Kcnq1ot1 is over-expressed in different cancer types, such as colorectal carcinoma, glioma , lung adenocarcinoma and hepatoma. Moreover, the knock-down of Kcnq1ot1 exerts a tumor suppressive effect in some of these cancers (reviewed in reference 1). It is plausible that the oncogenic function of the LncRNA is mediated, at least in part, by the repression of the maternal p57 allele, frequently silenced in cancer cells, and involves a strategy similar to that observed in muscle cells. A more detailed knowledge of the complex roles of Kcnq1ot1 in mediating chromatin modifications and DNA methylation at the multiple p57 regulatory regions not only will further clarify the molecular mechanisms underlying the tight and fine regulation of p57 expression and its silencing in cancer, but also will provide a tool for devising strategies aimed at restoring the expression of the CDK inhibitor.
1) Rossi, M. N., Andresini, O., Matteini, F., and Maione, R. (2018) Transcriptional regulation of p57kip2 expression during development, differentiation and disease. Frontiers in bioscience 23, 83-108