Regulatory Mechanisms And Roles Of Epigenetic Modifications In HOXB13 Gene Transcription

HOXB13 is a homeodomain protein implicated to play a role in growth arrest in prostate cancer cells. Expression of HOXB13 is restricted to the AR (androgen receptor)-expressing prostate cells; however, the mechanism underlying this silencing has not been fully understood. In this study, we investigated the possible regulatory mechanisms and roles of epigenetic modifications in HOXB13 gene transcription. We demonstrate that the HDAC inhibitor sodium butyrate (NaB) was able to induce cell growth arrest and to increase HOXB13 expression in AR-negative prostate cancer cells. Results arising our study revealed that both histone deacetylase HDAC4 and transcript factor YY1 could regulate HOXB13 gene at the levels of promoter activity, mRNA and protein expression. Moreover, we show that both HDAC4 and YY1 participated in the repression of HOXB13 expression through an epigenetic mechanism involving histone acetylation modification. Specifically, co-immuno- precipitation (CoIP) assays revealed that HDAC4 and YY1 formed a complex. The chromatin immunoprecipitation (ChIP) assays verified that HDAC4 was recruited to HOXB13 promoter by YY1. Additionally, promoter truncation and point mutation studies determined that the two proximal YY1 binding sites on the HOXB13 promoter were essential for the recruitments of YY1 and HDAC4. Results from MTT assays suggested that YY1 and HDAC4 affected cell growth through repressing the transcription of HOXB13 gene. Meanwhile, we found that the siRNA-induced partial silencing of Polycomb group proteins EZH2 and BMI1 resulted in a significant upregulation of both HOXB13 mRNA and protein levels in AR-negative prostate cancer cells. The ChIP assays demonstrated that H3K27me3 played an important role in this process. Also, we showed that the methyltransferase inhibitor 5-aza-dC induced the recovery of HOXB13 mRNA and protein expression, and we further manifested that almost all the CpG sites at HOXB13 promoter were densely methylated in AR-negative prostate cancer cells. Our evidence also showed that DNMT3b was recruited to the HOXB13 promoter by EZH2 to inhibit HOXB13 expression. Altogether, data included in this thesis will contribute to elucidating the unique silencing mechanisms of HOXB13 transcription, as well as its effects on prostate cancer cell growth. These findings may also implicate the possibility of HOXB13 as a potential molecular therapeutic target for prostate cancer.