Project description:In order to assess Tet1 binding, we first generated a Flag tagged Tet1 ES cells and then knocked out Dnmt3a in the [WT, Tet1-Flag] cells. By Tet1 ChIP and Flag ChIP, we showed that Tet1 binding was complementary to Dnmt3a. And Tet1 binding was not affected or slightly increased at majority of its targets.
Project description:We compared the genome occupancy for FLAG-tagged versions of the ETS factors ERG and EHF in the normal prostate epithelial cell line RWPE1. Our in vitro binding studies support a model whereby oncogenic ETS factors like ERG bind cooperativly with AP1 factors at closly spaced ETS-AP1 sites, while certain non-oncogenic factors like EHF bind anti-cooperatively with AP1 at the same sites. ETS and AP1 binding motifs were enriched in both ChIP datasets, but the ERG-FLAG bound reginos contained a much higher percentage of ETS-AP1 sites spaced in close proximity, consistent with our in vitro binding data.
Project description:DNA alkylation at the N2 position of guanine (N2-dG) is a prevalent type of DNA minor-groove lesions arising from various exogenous environmental contaminants and endogenous cellular processes. These N2-alkyl-dG lesions can induce G → T mutations during transcription if left unrepaired. However, the repair pathways of N2-alkyl-dG lesions remain incompletely elucidated. In this study, we identified a series of potential N2-alkyl-dG-binding proteins utilizing an affinity pulldown coupled with quantitative proteomic approach. We investigated their roles in DNA damage response and repair of these lesions. High-mobility group protein B3 (HMGB3) and activated RNA polymerase II transcriptional coactivator P15 (SUB1) exhibited preferential binding toward N2-nBudG-containing duplex DNA in quantitative proteomics analysis and in vitro binding assay using recombinant proteins. The presence of HMGB3 and SUB1 protected cells against alkylating agents (e.g., BPDE). Both HMGB3 and SUB1 modulated the repair of N2-nBudG and trans-N2-BPDE-dG in genomic DNA, while HMGB3 wasn’t involved in the repair of cis-N2-BPDE-dG. Together, our findings provided new knowledge about the cellular sensing and repair of minor-groove N2-alkyl-dG lesions.
