Study On The Regulatory Mechanism Of Murine Padi4 Gene Expression

Peptidyl arginine deiminases type4(PAD4)is a calcium-dependent enzyme that catalyzes histone citrullination.Recent studies have shown that histone citrullination promotes the formation of neutrophil extracellular traps(NETs)and cancer extracellular chromatin networks(CECNs),thereby contributing to inflammation injury,tumor metastasis,and other disease development.As the relationship between PAD4 and diseases has been studied,it has been found that PAD4 protein is highly expressed in various tumor tissues such as rectal cancer,breast cancer,esophageal cancer and gastric cancer,and the inhibition of endogenous PAD4 expression or its activity is beneficial for the treatment of these diseases.A large amount of scientific work has been focused on the function of PAD4 protein and its role in disease development,but there is a lack of research on the mechanisms of upstream gene expression regulation.The regulation of eukaryotic gene expression includes transcriptional,post-transcriptional and translational regulation.For the analysis of gene transcriptional regulation,the key point is to identify cisregulatory element sequences such as promoters,enhancers and silencers,etc.Prediction of gene regulatory elements with the help of databases and validation with dual luciferase reporter gene technology allows analysis of promoter regions as well as transcription factor binding sites.Based on the CRISPR/Cas9 technology,Feng Zhang’s team has designed and developed CRISPR/Cas9 g RNA library screening technology,which can be used for genome-scale functional gene screening.Based on published research results,different targeting designs can be applied to g RNAs in CRISPR/Cas9 libraries,for example,g RNA libraries targeting coding regions of genes were used to screen drug resistance genes,and g RNA libraries targeting non-coding regions of genes were used to screen DNA sequences with specific functions,including cis-regulatory elements such as promoters and enhancers.g RNA library technology is a powerful technique for large-scale analysis,and attempts are made to apply it in more research directions.In this study,the mechanism of mouse Padi4 gene expression regulation is explored.The first part of the experiment is to analyze the upstream cis-regulatory sequences of the mouse Padi4 gene,using the following experimental methods:(1)bioinformatics analysis tools to predict the possible cis-regulatory elements and transcription factor binding sites contained in the 4 kb upstream sequence of the mouse Padi4gene;(2)generate fluorescent reporter constructs with different lengths of cis-regulatory sequence deletion and transcription factor mutations and perform reporter assays;(3)quantitative PCR experiments to analyze the effect of overexpression of transcription factors on Padi4 gene expression.The second part focuses on the screening of genes that can affect PAD4 expression using CRISPR/Cas9 g RNA libraries targeting the CDS region of mouse genes,using the following experimental methods:(1)introduction of g RNA libraries into 4T1 recipient cells by lentiviral technology;(2)candidate gene screening by flow sorting technology,high-throughput sequencing technology and bioinformatics analysis;(3)introduction of candidate gene g RNA into 4T1 cells for validation.In this thesis,I successfully constructed luciferase expression vectors with 12 different length deletions in the upstream-3867/+62 region of the mouse Padi4 gene and expression vectors containing transcription factor mutation sites in the-1464/+62 region,and the results of dual luciferase experiments show that:(1)the mouse Padi4 gene-1464/+62 is the promoter region,-125/+62 is the core promoter region;(2)the TATAbox position is at-31/-25;(3)there are two Sp1 binding sites in the upstream-1464/+62 region of mouse Padi4 gene located at-154/-144 and-946/-936,respectively.Two regulatory factors affecting Padi4 expression,Ppp3ca and Calu,were successfully screened and validated by CRISPR/Cas9 g RNA library and high-throughput sequencing technology,with Ppp3 ca acting as a positive regulator and Calu acting as a negative regulator.