The Roles Of Transcription Factor ?A And Its Recognition Element In Gene Transcription Directed By Rna Polymerase ? And Their Regulatory Mechanisms

How eukaryotic gene transcription occurs is one of the most fundamental questions that need to be addressed in the field of life science research.Transcription initiation directed by RNA polymerase ??Pol ??requires general transcription factors,cofactors,mediators and Pol ? to be assembled at the core promoter of a gene and to form pre-initiation complexes.Previous studies revealed that TF?B recognition sites are present in both upstream and downstream of the TATA box at core promoters,and mutations of TF?B recognition elements(BRE^ud)significantly reduce the formation of the TF?A-TBP-DNA complex,suggesting that TF?A DNA-binding region could overlap with BRE^ud.The primary objectives of this project are:1)Determine whether there is a TF?A recognition element in the core promoter;2)Define a TF?A recognition element??ARE?and determine the role of ?ARE in gene transcription directed by Pol ?;3)Examine how the interaction between TF?A and ?ARE regulates Pol ? gene transcription.Conventional in vitro transcription assay based on radioactive isotope-labeled primer and primer extension is one of the most widely used approaches applied to the study of gene transcription.However,this method has various drawbacks due to using the radioactive isotope-labeled primer that restricts its application broadly.Thus,in this project,we have initially established a novel non-radioactive method for in vitro transcription assay based on quantitative PCR and primer extension.We show that the DNA templates in in vitro transcription system can be effectively eliminated to very low level by the approach developed in this study,and that the products synthesized by primer-extension can be recognized and amplified by the primers uniquely designed for primer extension and qPCR.Using this novel method,the effects of TF?B recognition element and TF?B mutations on the activities of the AdE4 and AdML promoters were investigated.The results are consistent with those obtained using conventional in vitro transcription assay.These results indicate that the novel method can be used to detect the effect of core promoter element on the promoter activity and to investigate transcription factor-mediated transcription activity.This novel method is simple and green,and can be widely applied to the research of gene transcription in the laboratories worldwide,and provides a convenient approach that can be used in this project.As described above,mutations of BRE^ud severely affect the formation of the TF?A-TBP-DNA complex,which suggests that the TF?A DNA-binding region might overlap with the sequence of BRE^ud.To determine the specific region of TF?A DNA-binding,EMSA and DNA methylation interference assays were performed using purified natural human TF?A protein and AdML promoter DNA.The results showed that TF?A could bind the region upstream of the TATA box at the AdML promoter.Using this information,we generated a core promoter DNA library that contains randomized bases at the TF?A-binding sites,and random selection was performed using the DNA library and SELEX technique.A TF?A recognition element??ARE?upstream of the TATA box is defined by analyzing the selection result.This element displays strong preferences for G and T,and against A and C at its two ends.We found that the element is widely present in human natural promoter by analyzing human promoter database.To confirm whether the ?ARE consensus affects the affinity of TF?A to DNA and transcription activity of promoter,protein-DNA binding assays,in vitro transcription assays and luciferase assays were performed using the promoters containing an optimal or defective ?ARE.The results from these assays demonstrated that the ?ARE enhanced TF?A binding to the promoter and the activity of TATA-containing promoters,but it repressed the activity of the promoters that lack a TATA box.Chromatin immunoprecipitation?ChIP?assays were performed using Flp-In293integrated with AdML promoters containing an optimal or defective ?ARE,the results showed that the ?ARE enhanced transcription by increasing the recruitments of Pol ?,TF?A,TAF4 and p300 at the promoters containing the TATA box.To understand the effect of the interaction between TF?A and ?ARE on transcription initiation assembly and transcription activity,the ?ARE and TF?A?/?sites that potentially bind to the ?ARE element were mutated and protein-DNA binding assays were performed.The results showed that mutations of the ?ARE reduced TF?A binding to the AdML promoter as did mutations of TF?A?/?.The results from in vitro transcription assays and luciferase assays revealed that mutations of TF?A?/?residues,K348 and K350,inhibited the activities of the AdML and natural promoters,suggesting that TF?A?/?involves the regulation of Pol ?-directed gene transcription by the bindings of its K348 and K350 to the promoter DNA containing the ?ARE element.ChIP assays using HEK293T cell lines with silence of endogenous TF?A and expression of HA-TF?A?/?or the 2SM mutant showed that expression of the 2SM mutant repressed the recruitments of TF?A,TBP,p300 and Pol ? at the ?ARE-containing promoters,suggesting that TF?A?/?mutations can inhibit transcriptional activities of the promoters by reducing the bindings of these factors to the promoter.These results indicate that disruption of the interaction between TF?A?/?and ?ARE can inhibit the formation of TF?A-TBP-DNA complex and Pol ?-directed gene transcription.Taken together,in this project,we have established a novel non-radioactive method for in vitro transcription assay.Using this method and other molecular and biological approaches,the roles of TF?A and its recognition element in Pol ?-directed gene transcription have been investigated;and the role and regulatory mechanism of the interaction between TF?A and ?ARE in Pol ?-directed gene transcription has been explored.The findings from this study extend the roles of TF?A and core promoter elements in basal transcription,and provide a novel insight into the regulatory mechanism of gene transcription directed by RNA Pol ?.