Researches On The Regulatory Mechanism Of Chromatin Structure And Transcription Factors And Their Roles In Diseases

BackgroundChromatin structure refers to the three-dimensional spatial structure formed by DNA winding histones and folding in the nucleus,including chromatin accessibility,spatial conformation,etc.Transcription factors are proteins that can bind DNA and affect gene expression.Some transcription factors pull chromatin to form spatial structure in the process of regulation,and chromatin structure also affects the binding of transcription factors to DNA.The dynamic regulation of chromatin structure and transcription factors plays an important role in cell replication,repair and transcriptional regulation.These alterations provide a new perspective for revealing the pathogenesis of disease and searching for drug targets.At present,with the maturity of chromatin conformation capture and chromatin immunoprecipitation sequencing technology,the establishment of multi-omics databases and rapid rise of research algorithms based on different data sources have brought new opportunities to study the regulatory mechanisms of chromatin structure and transcription factors under different conditions.Problems and ChallengesIn mammalian cell nuclei,the relationship between chromatin structure and transcription factor regulation is complex,and the regulatory mechanisms are not well defined.Currently,the lack of systematic research algorithms for mechanistic elucidation of chromatin structure and transcription factor regulation with sources of data under diverse conditions represents a key bottleneck limiting the study of chromatin structure and transcription factors.Chromatin structure and transcription factors research requires massive sequencing data with complex analysis and experimental validation,which is characterized by high cost and long cycle.Currently,there is a lack of efficient and accurate research algorithms to provide guidance for data generation,so the impact of a large number of transcription factors on chromatin structure is unknown.At the same time,whether the research algorithm is close to the essence of regulation requires different conditional data sources to verify and improve the algorithm.Due to the lack of research on the chromatin structure of cancer metastasis and SARS-CoV-2 infection tissues,the regulation mechanism of chromatin structure and transcription factors under both conditions is unknown.The two major constraints affect each other and jointly hinder the analysis of chromatin structure and regulatory mechanisms of transcription factors.PurposeTo explore the impact of more transcription factors on chromatin structure,we develop a systematic screening algorithm to identify transcription factors affecting chromatin structure,form a new research paradigm,and promote the discovery and analysis of more transcription factors affecting chromatin structure.Secondly,for the insufficient research on the regulation mechanism of chromatin structure and transcription factors in cancer metastasis and SARS-CoV-2 infected tissues,we study the chromatin structure alteration in the primary and metastatic process of colorectal cancer and regulation mechanism of transcription factors and chromatin accessibility in SARS-CoV-2 infection,and provide new data source for chromatin structure and transcription factor research and analysis algorithms.ContentsFirstly,a method for identifying transcription factors affecting chromatin structure was developed,and the selected transcription factors were knocked out to reveal the regulation of chromatin structure by novel transcription factors;Secondly,we explored the alteration of chromatin structure in normal colon,primary tumor and metastasis of colorectal cancer patients,and revealed the mechanism of chromatin translocation altering three-dimensional structure and regulating gene expression.Thirdly,the dynamic regulation of chromatin accessibility and transcription factor binding in SARS-CoV-2 infection was explored to reveal the regulation mechanism of receptor protein ACE2 expression.Methods1.Based on the high-quality chromatin immunoprecipitation(Ch IP-seq)sequencing data in the Encyclopedia of DNA Elements(ENCODE)project,a chromatin structure-related transcription factor screening algorithm is developed to quantify the co-location of candidate transcription factors and prior chromatin structure-related proteins.Then the candidate gene SIX5 was silenced by small molecule RNA interference(si RNA)experiment,and the chromatin structure alterations after silencing were analyzed by high-throughput chromatin conformation capture technique(Hi-C).The interaction pattern between SIX5 and the known chromatin structure-regulating transcription factor ZNF143 was explored by using the Cleavage Under Targets and Tagmentation(CUT&Tag)experiment,revealing the mechanism by which SIX5 cooperates with ZNF143 to maintain chromatin conformation.2.The normal colon,primary tumor and metastasis samples of 6 patients with metastasis and the normal colon and primary tumor of 7 patients without metastasis obtained by our research group in cooperation with Shougang Hospital were used to study the change laws of chromatin structure in colorectal cancer.Firstly,rigorous quality control was applied to screen high-quality multi-omics sequencing data.Next,the specific expression patterns of colorectal tumorigenesis and metastasis were analyzed based on transcriptome data(RNA-seq).Further,the correlation between Hi-C data and gene expression were analyzed to reveal the effect chromatin structure changes at the level of A/B compartment and topological association domain(TAD)on gene expression regulation during the occurrence and metastasis of colorectal cancer.Finally,the recognition algorithm of chromatin translocation based on Hi-C data was applied to reveal the influence of chromatin translocation on chromatin structure.3.Our group validated two cell lines were used as SARS-CoV-2 infection model,including susceptible Caco-2 cell line with accessible ACE2 promoter and insusceptible A549 cell line with inaccessible ACE2 promoter.To explore the effect of chromatin accessibility on the binding of transcription factors,type I interferon(IFN-α)treatment was conducted on both two cell lines to induce ACE2 overexpression.The expression and translation alteration of ACE2 after IFN-α treatment were compared by RNA-seq and Western blot.Next,ATAC-seq and q PCR experiments were used to confirm that the accessibility of ACE2 promoter remained unchanged after induction.Further,the transcription factor HNF1 A enriched in ACE2 promoter region was selected and CUT&Tag and q PCR experiment were conducted to observe the binding of HNF1 A.Results1.The recognition method of transcription factors affecting chromatin structure was developed,and the regulatory mechanism of new chromatin structure-related transcription factor SIX5 on chromatin structure was explored.Firstly,SIX5 was recognized by chromatin structure-related transcription factor recognition algorithm in different cell lines.Next,the selected transcription factor SIX5 was significantly enriched at high-level TAD boundaries,regulating the tumor suppressor and DNA repair gene RMI2.Silencing one of SIX5 and ZNF143 affected the binding of the other transcription factor to DNA,and altered chromatin structure.2.The changes of chromatin structure and gene expression during the occurrence and metastasis of colorectal cancer were explored.Firstly,the continuously changes of transcriptome in the order of normal colon,colorectal tumor,lymph node metastasis,liver metastasis and normal liver were observed.Secondly,the differential genes between metastatic tumor and primary tumor were significantly correlated with the survival of colorectal patients.During the tumorigenesis and metastasis of colorectal cancer,the chromatin structure changed at the scale of A/B compartment and TAD,which led to alterations of gene expression.Finally,chromatin translocation accumulated during the development of the colon and affected the chromatin structure.3.The regulation mechanism of chromatin accessibility on transcription factors during SARS-CoV-2 infection was explored.In susceptible Caco-2 cell line with accessible ACE2 promoter,the binding of transcription factor HNF1 A in ACE2 promoter and the expression of ACE2 increased after IFN-α treatment.However,in insusceptible A549 cell line with inaccessible ACE2 promoter,the binding of transcription factor HNF1 A in ACE2 promoter and the expression of ACE2 remained unchanged after IFN-α treatment.Pathway analysis based on transcriptional factor regulatory network and protein interaction network showed that genes interacting with receptor proteins of SRAS-CoV-2 were enriched in virus infection related pathways.ConclusionsIn this paper,we confirmed that chromatin structure and transcription factors play important regulatory roles under various conditions.The algorithm we developed to identify transcription factors that affect chromatin structure is robust and effective.The research on SIX5 revealed its unique preference for chromatin structure binding.Further research revealed that SIX5 and ZNF143 jointly regulated and maintained chromatin structure homeostasis.During the tumorigenesis and metastasis of colorectal cancer,the chromatin structure changes dynamically and regulates the gene expression patterns of different samples.The translocation between chromatin accumulates in this process and affected the chromatin structure.Finally,in SARS-CoV-2 infection,the accessible of ACE2 promoter enabled the increasing of transcription binding and ACE2 expression.Innovation and significance of workThis study provides a new research algorithm and data source for the analysis of chromatin structure and transcription factor mechanism.Firstly,a recognition algorithm for systematically screening transcription factors affecting chromatin structure was proposed,which had potential application value in the research of three-dimensional genome based on molecular level.The mechanism of the new transcription factor SIX5 regulating the chromatin structure was revealed,providing potential drug targets for altering chromatin structure.Next,TAD hierarchy was used to explore the relationship between structural changes and gene expression in colorectal cancer,and a new regulatory unit named square domain was proposed,providing a new paradigm for the research of three-dimensional genome of tumors at the TAD level.Finally,based on previous research of our group on SARS-CoV-2 susceptibility and chromatin accessibility,the association mechanism between was revealed from perspective of transcription factor,enhancing the value of chromatin accessibility in SARS-CoV-2susceptibility research.