High-throughput study of silencer elements in T cells
authors
keywords
- Slicener elements
- REST motifs
- CapSTARR-Seq
- Activator elements
- Transcriptionnal silencer elements
- Lymphocyte T
- Genetic regulation
document type
THESEabstract
The control of gene expression is fundamental to mammalian cell life. Although much of this control occurs at transcriptional and post transcriptional level. Gene expression is controlled by cis regulatory elements along with numbers of transcription factors combinatorial in mammals. The transcription can only be initiated by the assembly of RNAPII machinery around transcription start site of a gene which is also known as core promoter. Other cis regulatory elements like enhancers, silencers and insulators along with several transcription factors are also involved in gene regulation. There has been increasing number of studies relating enhancer and promoters but silencer has not been studied as much. Previous findings have suggested that some gene is regulated by silencers in T cell differentiation. The stagnation of silencer studies raises several questions whether silencers represent a major genomic strategy of gene regulation. There is lack of high throughput screening technique to identify silencers. My project has carried out aiming to answer these above questions. Firstly, I have repurposed the CapSTARR seq technique to identify activity silencer elements; previously this technique has developed in the lab, which used as an approach to exploiting a high-throughput enhancer activity. Performing CapStarr-seq in mouse p5424 cell line, I found a substantial proportion of regions displaying silencer activity. They display silencer activity in dual luciferase assay. The REST motif found to be enriched in several silencer elements. Moreover, by using comprehensive CRISPR/Cas9 genomic deletion approach, I demonstrated that silencer is generally involved in the regulation of nearby genes. Taken together, our results identify a new high throughput reporter assay to identify silencer element. My thesis is structured into 7 chapters. In chapter 1, I summarize the current understanding about gene regulation in mammals and the important factors contributing to transcriptional regulation. In chapter 2, I focus particularly on the regulatory role of silencer elements on gene expression. Here, the classical view of silencer and the classical reporter assays to identify silencer elements are discussed. There is an overview about silencer found in the hematopoietic system specifically during T cell differentiation. In chapter 3, I discussed respectively about the High throughput techniques that can be used to study regulatory elements. Chapter 4 is focused on the T cell differentiation. The results are organized in chapter 5. This chapter tells about how we identify silencer elements using CapSTARR-Seq technique, and functional validation of silencer elements. In Chapter 6, I give general discussion about results. Finally, in chapter 7, perspectives are mentioned. My work continues to identify silencer elements in different cell lines.
