Nspc1 Silent Hox Genes, The Epigenetic Mechanism

Polycomb group proteins (PcG) are highly conserved from Drosophila, C. elegans to human. They are expressed in almost all cell types and play essential roles in embryonic development, cell memory and cell fate decision. PcGs are widely studied about the function in the maintenance of embryonic stem (ES) cells or adult stem cells and tumorigenesis in recent years. Novel PcG gene NSPc1 (Nervous System Polycomb 1, also named as Polycomb group RING finger 1, PCGF1) is homologous to the family members BMI1 and MEL18 in mammals, but less well studied. It was ever reported that NSPc1 is expressed higher in several types of tumors than in the normal tissues. Our previous results also demonstrated that NSPc1 promoted the tumor cell proliferation. This thesis focuses on the studies of mechanism for NSPc1 in transcriptional repression and cell proliferation regulation.We first purified NSPc1 functional repressive complex. But before we detected the purified sample by Mass Spectrometry, the results were published that Tandem Affinity Purification (TAP) showed that NSPc1 and other PcG family members like RING1, RING2 and RYBP co-eluted in the same complex. And their interactions were verified by Co-immunoprecipitation (CoIP) assay. This complex is highly similar to the human Polycomb Repressive Complex-1 Like (hPRC1L) which turned out to ubiquitinate histone H2A. Among the hPRC1L components, RING2 is the main E3 ligase, while other PcG members like BMI1 stimulate its activity. Similarly, we found that the NSPc1 complex also could ubiquitinate H2A. When NSPc1 was knocked down in HeLa cells, the global level of uH2A was reduced. On the contrary, NSPc1 overexpression promoted H2A ubiquitination. Though NSPc1 recombinant protein alone failed to ubiquitinate H2A, it dose-dependently enhanced the E3 ligase activity of RING2. CoIP assay showed that NSPc1 could interact both RING2 and H2A, probably stabilizing the ubiqutination reaction as a bridge. Therefore NSPc1 probably represses target genes transcription through mediating H2A ubiquitination. To test this hypothesis, we carried out Chromatin Immunoprecipitation (ChIP) experiment on specific target genes. Given that PcG proteins are known to regulate HOX gene expression and therefore the body segment development, we screened the potential target HOX genes for NSPc1 by RT-PCR: HOXA7 and HOXB13 were significantly derepressed in NSPc1 knockdown HeLa and NT2/D1 cells. Therefore these two target genes were chosen for further experiments. First, ChIP assay demonstrated that NSPc1 complex did bind to their promoter regions. And deficiency of NSPc1 resulted in reduced association with RING2 and significant decrease of H2A ubiquitination in the region, which supported the idea that NSPc1-mediated H2A ubiquitination was responsible for its transcriptional repression effect.PcG proteins regulate gene expression by forming large multiprotein complexes, which can at least be divided into two principal types: the maintenance complex (PRC2) and initiate complex (PRC1). PRC2 is catalytically active to trimethylate H3K27. And the H3K27 tnmethylation (me3H3K27) can recruit PRC1. Recent studies have also demonstrated that PcG proteins are closely connected with DNA methylation, which is another important transcriptionally repressive epigenetic modification. RT-PCR experiment also showed that H0XA7 expression derepression was also found in EZH2 and DNMT1 knockdown HeLa cells, as well as in 5-aza-dC (a potent DNA methyltransferase inhibitor) treated HeLa cells. Therefore EZH2-mediated H3K27 trimethylation and DNA methylation are also involved in HOX gene silencing.Then how all these epigenetic modifications coordinate to keep HOX genes silenced? ChIP experiment in HeLa cells showed that loss of NSPc1 binding and DNMT1 deficiency did not affect EZH2 recruitment and H3K27 trimethylation. But EZH2 depletion resulted in reduced EZH2 binding and H3K27 trimethylation, concomitant with a significant reduction of NSPc1 binding and H2A ubiquitination, and loss of DNMT1 recruitment. Collectively, NSPc1 and DNMT1 recruitment, H2A ubiquitination and DNA methylation are downstream events of EZH2-mediated H3K27 trimethylation. Interestingly, the knockdown of NSPc1 resulted in loss of DNMT1 binding and DNA hypomethylation examined by Bisulfite Sequencing, while the knockdown of DNMT1 or 5-aza-dCtreatment also affected NSPc1 recruitment. Therefore DNA methylation andNSPc1-mediated H2A ubiquitination are not separated, but interdependent and self-reinforce each other in HOX gene silencing.ChIP-on-chip assays have demonstrated the stem cell PcG targets are more vulnerable than non-targets to DNA hypermethylation, turning the reversible state to permanent silencing, and thereby locking stem/precursor cells into abnormal c1onal expansion which predispose to cancer. Hence our extablished epigenetic network has partially demonstrated the transcriptional repression mechanism for NSPc1 and established a functional link of NSPc1 with different chromatin modifers in tumorigenesis. The research into HOX gene expression regulation in this thesis will also help to further elucidate the function of NSPc1 in embryonic development.