Analysis of transcription factor complexes and their associated chromatin interactions during erythropoiesis

Abstract

Cis-regulatory elements can control gene transcription over large distances. Studying the association between cis-regulatory elements and the mechanism of gene regulation is important since the disruption in the transcriptional control can lead to developmental defects and many complex diseases. This thesis describes the identification and characterization of transcription factor complexes and their associated cis-regulatory regions during erythropoiesis. These include the dynamics of protein complexes and their associated binding patterns, the chromatin interactions between them and their target promoter at specific loci, and the involvement of identified cis-regulatory complexes in gene regulation during erythroid cell differentiation. Transcription factor occupancies of multiple key factors were profiled using ChIP-seq during erythroid development, in proerythroblast-like cells and in fully differentiated erythrocyte-like cells. These factors consist of proteins involved in the LDB1 complex (LDB1, GATA1, SCL/TAL1, ETO2, and MTGR1) and several other factors that are critical for gene regulation such as RUNX1, GFI1B, FOG1, LSD1, LMO2, LMO4, P300, TIF1γ, CTCF, and RNAPII. To analyze ChIP-seq data, a set of bioinformatics tools were developed that expanded on the functionality of existing R/Bioconductor packages. These tools provide functions for data processing, manipulation, mining and visualization, thus greatly facilitating hypothesis generation and interpretation of experimental results. Integrative analysis enabled the identification of how protein complexes change during differentiation and how identified complexes affect gene regulation. We show that the dynamics of the LDB1 complex compositions, in particular, the binding of the co-repressor ETO2 and MTGR1 significantly decreases during differentiation. This dynamic LDB1 complex occurs at a very specific subset of genes that are induced late during erythroid differentiation, suggesting the role of LDB1 as an activation complex. Using computational techniques we discovered twelve distinct patterns of P300 binding complexes. These binding patterns showed distinct characteristics and could be classified into enhancer, promoter, and insulator-associated classes. Integrating the identified binding patterns with associated gene expression profiles demonstrated the central roles of P300 TF complexes in both activating and repressing target genes. Finally, an integrative approach using multiple ChIP-seq data sets together with 3C-seq has been used to demonstrate the long-range regulation of regulatory elements and their associated chromatin contacts at the β-globin and Myb loci. To analyze 3C-seq data, I developed a R/Bioconductor package called r3Cseq to facilitate 3C-seq data analyses. Using r3Cseq, we demonstrated the importance of long-range chromatin contacts. We showed that the dynamics of long-range chromatin interactions between TF binding sites and the associated target promoters is involved in the transcriptional control of β-globin and Myb genes. In summary, this thesis work is primarily concerned with the development of computational methods and tools for the analysis of large-scale experimental data, with an emphasis on data generated by ChIP-seq and 3C-seq technologies, and the application of these tools to generating new hypotheses and acquiring new biological knowledge on mammalian gene regulation.