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?miRNAs and Histone Modifications
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By CourteneyLai, Section Biology
Posted on Fri Apr 30th, 2010 at 11:56:04 AM PST
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In recent years, two important concepts have emerged in the understanding of how genes controlling normal biological processes are regulated. Both the small molecule microRNAs (miRNAs) and structural changes to genetic material, epigenetics, have the ability to greatly alter gene expression, the former through cleavage or translational repression of target mRNAs and the latter through post-translational modifications of histone proteins or cytosine DNA bases that cause structural changes in chromatin, modifying access to DNA target sequences.[1, 2] Specifically the Polycomb group proteins in repressive complex 2 (PRC2) induce gene silencing through methylation of histone 3 on lysine 27 (H3K27).[3] These two fields have merged through discoveries that they interact, and inappropriate activity can lead to development of diseases like cancer.
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miR-29 is often down-regulated in lung cancer, specifically non-small cell lung cancer (NSCLC), and has complementary binding sites in the 3' UTRs of de novo DNMT3a and 3b.[4] Fabbri and colleagues demonstrated that the miR-29 family is down-regulated in lung cancer cell lines while DNMT3a and DNMT3b is up-regulated, resulting in DNA methylation and silencing of the tumour suppressor genes FHIT and WWOX.[4] In addition, the miR-29 family members bind to their target sequences in the DNMT3b promoter, resulting in decreased DNMT3b protein levels in the cytoplasm of these cells.[4] This inverse correlation demonstrated between DNMT3b and miR-29b was confirmed through the observation that enforced expression of miR-29 in lung cancer cell lines restored the DNA methylation patterns reminiscent of those seen in wildtype cell lines and decreased tumourigenicity and tumour size in mice.[4] Despite this link, however, the activating mechanism of miR-29 remains a mystery.
NF-κB, which plays a role in innate immunity, inflammation, and cell proliferation, has been associated with numerous forms of cancer, most notably in tumour survival via interactions with antiapoptotic genes like Bcl-2.[5] NF-κB has also been linked to chromatin regulation.[6] Specifically, the Rel-B component of NF-κB is reported to activate YY1, a multifunctional transcription factor coding for an initiator element binding protein that acts as both a repressor and activator.[7, 8] YY1 can direct and initiate transcription of cellular and viral genes or act as a Polycomb protein to mediate silencing depending on its environmental cues.[7, 9] As a part of PRC2, YY1 recruits Polycomb proteins to DNA, resulting in histone H3 deacetylation and H3K27 methylation.[10] YY1 activation or overexpression is associated with unchecked cellular proliferation, resistance to apoptosis, tumourigenesis, and metastasis, strengthening its link to the NF-κB pathway.[7]
Studies have also uncovered a link between miR-29 and chromatin regulation, with the former being epigenetically suppressed by NF-κB via the transcription factor YY1 and the Polycomb protein complex in myoblasts but expressed during differentiation.[6] In dysregulated myogenesis, such as in rhabdomyosarcoma, miR-29 expression remains suppressed, suggesting that this non-coding RNA plays an important role in normal differentiation.[6]
It would, therefore, be interesting to determine if the downregulation of miR-29 seen in NSCLC occurs through H3K27 methylation-induced repression, brought on by increased NF-κB signalling. To investigate this possibility, we would first determine that an upregulation of the NF-κB pathway is seen in NSCLC using gene (qRT-PCR) and protein (Western blot) expression assays. We would then need to establish that YY1 interacts with miR-29. This could be accomplished through the use of bimolecular fluorescence complementation, an in vivo screen used to validate protein-protein interactions. Here, a fragment of the Venus fluorescent protein would be fused to miR-10a with the complementary Venus portion fused to the Polycomb protein YY1. These complementary fragments would be co-tranfected into cells of a NSCLC cell line and observed under an inverted fluorescence microscope, with a fluorescence signal indicating an interaction.[11] The existence of such an interaction would be the first step in establishing an association between miR-29 expression and H3K27 methylation-induced repression in NSCLC.
REFERENCES
1. Bartel, D.P., MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 2004. 116(2): p. 281-97.
2. Sharma, S., T.K. Kelly, and P.A. Jones, Epigenetics in cancer. Carcinogenesis. 31(1): p. 27-36.
3. Simon, J.A. and R.E. Kingston, Mechanisms of polycomb gene silencing: knowns and unknowns. Nat Rev Mol Cell Biol, 2009. 10(10): p. 697-708.
4. Fabbri, M., et al., MicroRNA-29 family reverts aberrant methylation in lung cancer by targeting DNA methyltransferases 3A and 3B. Proc Natl Acad Sci U S A, 2007. 104(40): p. 15805-10.
5. Naugler, W.E. and M. Karin, NF-kappaB and cancer-identifying targets and mechanisms. Curr Opin Genet Dev, 2008. 18(1): p. 19-26.
6. Wang, H., et al., NF-kappaB-YY1-miR-29 regulatory circuitry in skeletal myogenesis and rhabdomyosarcoma. Cancer Cell, 2008. 14(5): p. 369-81.
7. Gordon, S., et al., Transcription factor YY1: structure, function, and therapeutic implications in cancer biology. Oncogene, 2006. 25(8): p. 1125-42.
8. Hyde-DeRuyscher, R.P., E. Jennings, and T. Shenk, DNA binding sites for the transcriptional activator/repressor YY1. Nucleic Acids Res, 1995. 23(21): p. 4457-65.
9. Atchison, L., et al., Transcription factor YY1 functions as a PcG protein in vivo. EMBO J, 2003. 22(6): p. 1347-58.
10. Wilkinson, F.H., K. Park, and M.L. Atchison, Polycomb recruitment to DNA in vivo by the YY1 REPO domain. Proc Natl Acad Sci U S A, 2006. 103(51): p. 19296-301.
11. Kerppola, T.K., Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells. Nat Protoc, 2006. 1(3): p. 1278-86.
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