Unfortunately, among them, CCND1 is the only altered protein (Figure S3) suggesting that there could still be some differences between human and yeast about the roles of RAD6 in regulating the expression of cell cycle related genes. Here, our work indicates that RAD6 is a novel regulator of G1-S transition and cell proliferation in human cells by targeting CCND1 expression. cells were subjected to cell cycle assay. The quantification of the cell cycle assay is shown. (C) Soft agar colony formation assays were performed using control, or RAD6 overexpressing, or RAD6 overexpressing together with CCND1 knocking down HeLa cells.(TIF) pone.0113727.s002.tif (2.9M) GUID:?410B165F-8F3E-4BFB-90B5-2E94C47CFE56 Figure S3: RAD6 overexpression does not affect the protein levels of cyclin A, cyclin E, cyclin B1 and Chk2. HL-7702 cells transfected with an empty control or Myc-RAD6 expressing plasmids were lysed and subjected to western blot assays with antibodies as indicated.(TIF) pone.0113727.s003.tif (790K) GUID:?8C5DD9FB-E854-47E0-95EB-97A909CA033F Data Availability StatementThe authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Abstract Protein ubiquitinylation regulates protein stability and activity. RAD6, an E2 ubiquitin-conjugating enzyme, which that has been substantially biochemically characterized, functions in a number of biologically relevant pathways, including cell cycle progression. In this study, we show that RAD6 promotes the G1-S transition and cell proliferation by regulating the expression of cyclin D1 (CCND1) IITZ-01 in human cells. Furthermore, our IITZ-01 data IITZ-01 indicate that RAD6 influences the transcription of CCND1 by increasing monoubiquitinylation of histone H2B and trimethylation of H3K4 in the CCND1 promoter region. Our study presents, for the first time, an evidence for the function of RAD6 in cell cycle progression and cell proliferation in human cells, raising the possibility that RAD6 could be a new target for molecular diagnosis and prognosis in cancer therapeutics. Introduction Protein ubiquitination plays multiple roles in different life processes. It is well known that protein ubiquitination is crucial for protein degradation . Increasing evidence indicates that protein ubiquitination also have other biochemical functions in addition to protein degradation C. For example, ubiquitination of histones (e.g. H2A and H2B) always associates with gene transcriptional regulation. Multiple DNA damage repair related proteins (e.g. PCNA, FANCD2-FANCI complex etc.) can be mono- or polyubiquitinated by different E2 or E3 ubiquitin ligases. This kind of modification usually affects their abilities in the regulation of DNA damage repair and genome stability . RAD6 is an E2 ubiquitin-conjugating enzyme, which exhibits its biological functions mainly through targeting different substrates for ubiquitination . For instance, in promoter region ( Figure 4A , upper left), and the levels of H2B monoubiquitination and H3K4me3 are increased in RAD6 overexpressing cells compared with the control cells ( Figure 4A , upper middle and right). We also examined the effect of RAD6 RNAi on the enrichments of H2B monoubiquitination and H3K4me3 on promoter, and an opposite result to RAD6 overexpression were observed supporting the same conclusion of RAD6 on expression ( Figure 4A , lower). Open in a separate window Figure 4 RAD6 enhances the enrichment of H2B monoubiquitination and H3K4me3 level at CCND1 promoter region.(A) HL-7702 cells transfected with an empty control or Myc-RAD6 expressing plasmids, or a control siRNA or RAD6 specific siRNAs were lysed and sonicated, then subjected to chromatin immunoprecipitation (ChIP) assays with antibodies against Myc-tag (indicating exogenous RAD6), H2B monoubiquitination and H3K4me3. Nonspecific antibody, normal rabbit IgG (NIgG), was used as a negative control. The precipitated DNA fragments were supplied for PCR assay with primers specific for CCND1 promoter. (B) Working model. RAD6 regulates the transcription of CCND1 likely through Rabbit polyclonal to ADAM17 affecting the H2B monoubiquitination and H3K4me3 levels at the CCND1 promoter region. Taken together, these results suggest that RAD6 might regulate the transcription of CCND1 through modulating the H2B monoubiquitination and H3K4me3 levels at the promoter region ( Figure 4B ). Discussion G1-S transition is critical for cell proliferation and tumor growth . Therefore, understanding the regulation mechanism and finding novel regulator of G1-S transition is essential for the development of cancer molecular diagnosis and anticancer therapeutics. In this study, we demonstrated for the first time that RAD6 is a novel regulator of G1-S transition and cell proliferation in human cells. Our results.