Araújo, CC; (2015) Involvement of short RNAs in polycomb-mediated gene repression. Doctoral thesis , UCL (University College London).

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Abstract

Polycomb group proteins maintain cell identity by repressing developmental regulator genes specific for other cell types. There are two main complexes: Polycomb repressive complex 1 (PRC1) and 2 (PRC2). PRC2 methylates histone H3 lysine 27 (H3K27me3), creating a binding site for PRC1 that ubiquitinates H2AK119. Polycomb target genes are associated with stalled RNA polymerase II (RNAPII), and the initiation marker H3K4me3, known as bivalent chromatin. Our laboratory has demonstrated that short RNAs are transcribed from the promoter region of these genes in human T-cells, while the work carried out as part of the present thesis demonstrates that short RNAs are also transcribed in murine embryonic stem cells (ESCs). This indicates that they are conserved across different species and cell types. Northern blotting for RNAs ≤200 nucleotides extracted from murine ES cell deficient for PRC2 and PRC1 revealed that short RNA production is independent of Polycomb activity. When cells differentiate and Polycomb-target genes become activated, short RNAs are depleted. Given that PRC2 interacts with RNA, this loss of short RNAs might allow gene activation. Additionally, polycomb response elements (PRE) have been detected in Drosophila. These elements are necessary and sufficient for polycomb recruitment. A recently identified PRE, HOXD11.12, recruits PRC2 in human mesenchymal stem cells (MSC). It is hypothesized that PRE activity is due to the transcription of short RNAs. Blotting for RNA extracted from MSC identified short RNAs transcribed from D11.12. Moreover, these short RNAs can form the same secondary structure as the previously-identified short RNAs and are also located at a CpG island. Furthermore, RASL12 and YBX2 behave as PREs while D11.12 from active HOXD11 enhances gene expression, potentially also acting as a Trithorax response element (TRE).

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