HR Studies On The Regulatory Mechanism Of Nuclear Receptor VDR Target Genes

A number of JmjC-domain-containing proteins were identified as histone lysine demethylases (KDMs), all of which were also chromatin-associated proteins. They play important roles in regulating gene expression and other processes within an epigenetic modification system. Hailess (HR), another member of this family, is not a promising candidate of histone lysine demethylase based on bioinformatics analysis. However, Mutations in the Hr gene cause hair loss both in mice and human beings, which indicates its master roles in controlling hair growth. It's been reported that HR has a functional relationship with nuclear receptors such as vitamin D receptor(VDR) and TR and Wnt pathway, while the details are largely unkown. It's known that expression defect of VDR or?-catenin in mice produced by genetic method also results in hair loss either, which imply they function by a common pathway. It will be of great help for us to find ways to cure hair deseases to clarify the details of the roles HR plays in hair growth and the relationship among them. It will also provide us another model to learn the roles of the JmjC-domain-containing proteins.Here we studied the way by which HR regulated transcription of VDR target genes.1. A high mRNA level of Hairless gene was detected by RT-PCR in HaCaT and HeLa cells which are derived from skin, SH-SY5Y cells derived from nerve tissue and 293T cells from embryonic kidney tissue relative to mRNA level in muscle derived RD cells.2. Exogenous expressed flag tagged full length human HR showed a intranuclear discrete dot pattern observed in 293T by immnofluorescence analysis. GFP tagged HR showed mainly three patterns in MCF7, homogeneously distributed in the nucleus, intranuclear discrete dot patterns or an intranuclear discrete dot structure on a diffuse fluorescence background. Sometimes, the fluorescence signal was found located outside of the nucleus and look like a spindle.3. We cloned rat and human CYP24A1 promoters from their genomic DNA respectively. The promoter activity assay in HeLa cells showed that overexpression of HR greatly decreased both the rat and human CYP24A1 promoter activity with or without 1,25(OH)2D3 treatment.4. The promoter activity assay in HeLa cells showed that HR overexpression mildly increase human RARB promoter activity with or without 10uM ATRA treatment.5. We found that the VDRE sequence on rat CYP24A1 promoter was related to the effect of HR on the promoter activity of CYP24A1 by truncated mutants experiment. 6. It was observed that flag tagged human WT HR coimmunoprecipitated with endogenous VDR in HeLa cell.7. The promoter activity assay in HeLa cells showed that the treatment such as 20nM trichostatin A (TSA, a class I and?HDAC inhibitor),20mM Nicotinamide (NAM, a natural inhibitor of sir2-like enzymes),20uM adenosine dialdehyde (Adox, an indirect methyltransferase inhibitor), and lOuM 5-Aza-dC (5-Aza-2'-deoxycytidine, inhibitor of DNA methylation) did not change the action of HR on the promoter activity of CYP24A1. It suggested that HR might founction by other ways than through changing either the modification state of histones or methylation state of DNA.8. ChIP analysis of transcriptional start region(-83/142) and upstream (-1056/-863) region within the CYP24A1 promoter performed on chromatin of HeLa cells showed that HR overexpression did not change the trimethylation modification state of H3K4, H3K9 and H3K27 and dimethylation of H3K4, providing another evidence that HR founction by other ways.9. Exogenously expressed flag tagged HR coimmunoprecipitated with endogenous NcoRl and msin3A protein in HeLa cells. EGFP-HR colocalized with Cherry-NcoRl(756-2528aa) in 293T cells. NcoRl peptides (a 13.4% coverage by amino acid count) were found in the MS data of proteins coimmunoprecipitated with flag-HR. All data suggest HR and NcoRl exist in the same protein complex.10. It was discovered that EGFP-hHR had a exclusive distribution pattern against Cherry-ASH2L and Cherry-HP l?in nucleus observed under a laser confocal microscope in 293T cells.11. Exogenously expressed EGFP-HR and Cherry-VDR completely colocalized in incleus in 293T and MCF7 cells pointed out a new model of HR founction.12. HR mildly increased the promoter activity of super TOPflash, while had no effect on CyclinDl or c-Myc observed by dual luciferase reporter assay in HeLa cells.