| Summary: | 碩士 === 國立陽明大學 === 醫學生物技術暨檢驗學系暨研究所 === 97 === Rnf33 is a mouse TRIM/RBCC protein gene expressed in the unfertilized egg, the pre-implantation embryo and in the testis, and may play an important biological role in early development and in the testis. In this study, we aim to investigate transcriptional regulation of Rnf33.
In order to analyze transcriptional regulation, we used luciferase assays to dissect the Rnf33 promoter functions. We showed that a putative atypical TATA box and a transcription initiator (Inr) element act as the core promoter elements of Rnf33. Our data also indicate that removal of a 305-bp sequence, designated as R4, located at the 3’-end of the 1,187-bp intron sequence resulted in 80% loss of transcription activity indicating the presence of a crucial transcriptional cis element in this region. Further, we demonstrated the intronic R4 sequence acts as a positive regulator. In addition, we searched for putative transcription factor binding sites (TFBS) in the R4 fragment using TFBS’s databases, R4 could be divided into three parts: R4-1 contains a NF-kB, a hypoxia response element (HRE) and a GATA protein binding site; R4-2 contains an AT-rich fragment; R4-3 contains one N-box and two E-boxes. Luciferase assays indicated that R4-1 is crucial to Rnf33 regulation. Mutation and deletion analysis of these TFBS in R4-1 indicated that NF-�羠 could affect Rnf33 transcription.
To demonstrate Rnf33 is targeted by the NF-kB subunits p65 and p50, EMSA were performed using a R4-1 NF-kB element probe; supershift assays were also performed using anti-p65 and anti-p50 antibodies. The results of EMSA indicated that R4-1 sequence is targeted by the p65 and p50 in vitro. Chromatin immunoprecipitation experiments (ChIP) further demonstrated in vivo Rnf33-NF-kB association in the testis, TM3 and TM4 mouse testicular cell lines. We further demonstrate TNF�� would not induce NF-kB activation in the TM3 and TM4 mouse testicular cell lines. In conclusion, our results show that the Rnf33 expression in the testis is regulated by the ubiquitous NF-kB subunits p65 & p50 binding at an intronic cis-element and further understanding of transcriptional regulation of an active retrogene via a cis sequence present in the intron. My work also expands the biological roles of NF-kB to regulation of gene expression in the testis and maybe in pre-implantation development.
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