Research Of WAC And Sc35 Regulating Gene Transcription,R-loop And DNA Damage Repair

Post-translational modification refers to a modifying group adding to one or more amino acid residues of a post-translational protein,which can change the spatial conformation and biological activity of the protein.Post-translational modification is widely involved in regulating a series of life activities such as cell cycle,proliferation,apoptosis,differentiation gene expression,and signal transmission.Abnormal post-translational modification is closely related to the occurrence of diseases and tumors.Histone ubiquitination is one of the covalent post-translational modifications.The ubiquitination modification process usually consists of ubiquitin activating enzyme E1,ubiquitin binding enzyme E2 and ubiquitin ligase E3 to complete the ubiquitin cascade.In eukaryotic cells,chromatin is composed of genomic DNA wrapped around nucleosomes.The core of the nucleosome is composed of histone H2 A,H2B,H3 and H4.The RNF20/RNF40 dimer(RING finger protein)is an E3 ubiquitin ligase complex that catalyzes the monoubiquitination H2 B on lysine 120(H2Bub1).UbH2B1 mediates gene transcription regulation,chromatin remodeling,and plays an important role in homologous repair,which is one of the DNA double-strand break(DSBs)repair pathways.Deletion of the RNF20/RNF40 dimer results in the inability to recruit repair factors to DNA damage sites during homologous repair,leading to the accumulation of DNA double-strand break damage and destroying genome stability.In addition,studies have shown that R-loop accumulation in RNF20/RNF40 dimer depleted cells is one of the causes of replication pressure and genomic instability.R-loop is a DNA:RNA hybrid formed by RNA polymerase II during the transcription process.It is a three-stranded nucleic acid structure composed of a nascent RNA strand and its template DNA.The causes of R-loop include high GC content in DNA sequence,lack of topoisomerase,and impaired RNA synthesis.In RNF20/RNF40 dimer knockout cells,a group of genes related to RNA processing and splicing are up-regulated,which is one of the hallmarks of R-loop accumulation.sc35 is a serine arginine protein splicing factor(also known as SRSF2),which plays an important role in mRNA splicing and RNA metabolism.It has been reported that sc35 exerts effect on maintaining the stability of the genome,because the deletion of sc35 leads to the enrichment of R-loop and DNA DSBs.Many factors such as topoisomerases,chromatin remodelers and RNA processing factors can inhibit R-loop formation.For example,in addition to sc35 inhibiting the R-loop,the R-loop is also degraded by some enzymes.RNases H1 and H2 function to degrade the RNA in the R-loop,and RNA-DNA helicases unwind the DNA:RNA hybrid strand,such assenataxin(SETX)and Aquarius(AQR).Deletion and mutation of RNase or RNA helicase aggravates DNA damage and increase chromatin instability through accumulation of R-loop,and then induce various diseases.From previous studies,we know that the loss of RNF20/RNF40 dimers reduces H2Bub1,which leads to defects in histone mRNA processing and R-loop enrichment.Similarly,the deletion of sc35 also promoted the formation of R-loop.In addition,WAC is an important functional partner of RNF20/RNF40 dimer.The C-terminal coiled coil domain of WAC interacts with RNF20/RNF40 dimer to form the RNF20/RNF40/WAC complex to participate in H2Bub1.Therefore,the main purpose of this project is to investigate whether WAC and sc35 play a synergistic role in gene transcription regulation,R-loop processing and DNA damage repair.First,we demonstrated that WAC interacts with sc35 through endogenous co-immunoprecipitation(Co-IP).Second,we used chromatin-immunorecipitation qPCR(ChIP-qPCR)to detect the protein enrichment of different genes.Our data showed that WAC,sc35,and RNF20 have the similar enrichment on the three genes p21,PUMA,and I?B?,indicating that WAC and sc35 are synergic in the transcription process of p21,PUMA,and I?B?.Next,RNA/DNA Hybrid IP experiments verified that WAC,sc35 participates in the R-loop processing.S9.6(an antibody that recognizes R-Loop)interacts with WAC,sc35,and RNA Pol ?.We know that the collision of R-loop and DNA replication fork causes DNA double-strand break damage.Next,we studied how WAC and sc35-mediated R-loop participates in DNA Damage repair pathways.ATM/ATR kinase is the main regulator of DNA damage and replication stress response.Activation of the ATR pathway stimulates Chk1 phosphorylation,while ATM phosphorylates Chk2.Both of these pathways further activate downstream genes to respond to DNA damage and protect the genome.We used siRNA technology to knock down WAC,sc35,RNF20 and SETX,and found that the expression of Chk1 protein was significantly increased by Western-Blot,indicating that the ATR-Chk1 pathway was activated.In conclusion,WAC and sc35 play a synergistic role in gene transcription,R-loop regulation and DNA damage repair.