Study On The Functional Binding Of Brpf1 And Its Novel Transcript Brpf2 To Rhox5 Protein

Brpfl (Bromodomain and PHD finger containing 1), also called Br140 or Peregrin, is a novel TrxG (Trithorax group) member with a central role during development. Brpf1 is a multidomain protein. It contains a unique combination of domains typically found in chromatin-associated factors, including PHD domain, bromodomain and PWWP domain. Brpf1 functions in association with the histone acetyltransferase Moz and promotes histone acetylation. Brpfl recruits Moz to distinct sites of active chromatin and remains at chromosomes during mitosis by binding to histone directly with its bromodomain and PWWP domain.Rhox5 is the founding member of the reproductive homeobox on the X chromosome (Rhox) gene cluster. It is selectively expressed in male and female reproductive tissues; moreover, mutational inactivation of Rhox5 in mice caused aberrant germ cell apoptosis, decreased sperm cell count, impaired sperm motility and subfertility. These observations together indicate a crucial role of RHOX5 in the development of reproductive tissues and spermatogenesis. However, the molecular mechanisms underlying RHOX5 functions remained unclear. In our previous research, we screened mouse 7 days embryo cDNA library using Rhox5 protein as a bait and discovered a novel clone 2-31. In this thesis, we carried out the following assays:1. Identification of a noval transcript of Brpf1, Brpf2, and study on its expression mapThe plasmid was extracted from yeast clone No.2-31 and then transfected into E.coli. After sequencing, the sequence was blast in NCBI and the results indicated that it was a novel transcript of Brpfl gene. This sequence was submitted to the GenBank and got the GenBank accession No. DQ288860。In order to map the expression level of Brpf1 and its noval transcript Brpf2, total RNA was isolated from seven types of mouse tissues separately. RT-PCR and Northern blotting results indicated that the expression of both Brpf1 and Brpf2 could be detected in most of the mouse tissue including liver, embryo, epididymis, testis, ovary and muscle. However, only Brpf2 transcript could be detected in spleen. This suggested that both Brpf1 and Brpf2 transcripts have some kind of physiological functions. Among the seven types of tissues, both Brpfl and Brpfs represented a higher expression level in epididymis, testis and ovary, while a lower expression level in liver, embryo, spleen and muscle. This suggested that Brpfl and Brpf2 may play an important role in the reproductive tisses. In order to understand the function of Brpf2, we analyzed the biological property of Brpf1 and Brpf2 by bioinformatics methods. The results indicated that the full length of Brpf1 mRNA is 4443 bp, encoding 1246aa. It is a multidomain protein, including two PHD domain, bromodomain and PWWP domain. The predicted Molecular Mass is 140 kDa. However, the full length of Brpf2 mRNA is 2747 bp and it only encodes 442 aa, partly owing to the creation of a new stop condon. Different from Brpf1, Brpf2 only has two PHD domains and the predicted Molecular Mass is 50 kDa. In order to confirm the actual Molecular Mass of Brpfl and Brpf2 protein, we amplified the full length of Brpf1 and Brpf2 mRNA and then obtained the pure protein of Brpfl and Brpf2 using a TNT T7 Quick Coupled transcription/translation reaction kit. The results demonstrated that the actual Molecular Mass of Brpfl and Brpf2 protein are 140 kDa and 50 kDa respectively.2. Study on the interaction of Brpf1/Brpf2 and Rhox5In order to confim the interaction between Brpf2 and Rhox5, the CDS sequence of Brpf2 was amplified and subcloned into the pGBKT7 vector. Yeast two hybrid assay indicated that Brpf2 could interact with Rhox5 directly in yeast. GST-RHOX5 fusion proteins were expressed in E. coli RosettaTM2 (DE3) cells induced with 1 mM IPTG at 37℃for 6 h and were purified effectively using the Glutathione-Sepharose beads. In Vitro GST-pull down assay, the beads coupled with purified GST-Rhox5 fusion protein can pull down the Brpf2 protein, which suggested that Brpf2 could bind Rhox5 in vitro.In order to confim the interaction between Brpfl and Rhox5, the CDS sequence of Brpfl was amplified and subcloned into the pGBKT7 and pGADT7 vector. Yeast two hybrid assay indicated that Brpf2 could interact with Rhox5 directly in yeast. In Vitro GST-pull down assay, the glutathione-Sepharose beads coupled with purified GST-Rhox5 fusion protein can pull down the Brpfl protein, which suggested that Brpf1 could bind Rhox5 in vitro.3. The critical structure for the interaction between Brpf1/Brpf2 and Rhox5 proteinCompared with Brpfl transcript, Brp2 loses the functional domain Bromodomain and PWWP domain. However, it keeps the two conserved PHD domain. Based on our previous study, both Brpfl and Brpf2 protein could interact with Rhox5 protein. Therefore, we postulate that one of the two PHD domains mediate their interaction with Rhox5.In order to verify whether the PHD domain mediates the Brpfl/Brpf2 interation with Rhox5, three types of Brpf2 trunctated mutants named Brpf2 A, Brpf 2B and Brpf2C were constructed. Brpf2 A includes two PHD domains; Brpf2 B includes the first PHD domain, while Brpf2C includes the second PHD domain. Two-ways yeast two-hybrid assay and GST-pull down assay suggested the second PHD domain is the critical domain for Brpfl/Brpf2 and Rhox5 interaction.In the meantime, we used two types of Rhxo5 tructated mutants, Rhox5N (N-terminal of Rhox5 protien, without homeobox domain) and Rhox5C (C-terminal of Rhox5 protein, including homeobox domain). Furthure yeast two-hybrid assay and GST-pull down assay suggested that the homeodomain of Rhox5 is the critical domain for Brpfl/Brpf2 and Rhox5 interaction.Based on the above results, we postulate:The second PHD domain of both Brpfl and Brpf2 mediates their interaction with Rhox5. When Rhox5 interacts with Brpf1, the bromodomain and PWWP domain of Brpfl will be shaded, which make Brpfl loses the ability of binding with nucleosomes. Wheras, when Brpf2 interacts with Rhox5, Brpf1 protein will be released. Therefore, the bromodomain and PWWP domain can intact with histone directly recruit the other transcriptional factors and then activate the gene expression.