A pinboard by
Sudhir Thakurela

Post Doc, Broad Institute


Understanding the process by which repressed genes become accessible to transcription machinery

Every biological process require involvement of several biological molecules. Among these two most important ones are DNA and proteins. Proteins, that are generated by use of DNA as a template, are produced in a well defined spatiotemporal manner and as a result different body organs/cells only produce those proteins that are required by that cell-type at a particular moment. The DNA template that produces these proteins, called as genes, are usually in a repressed or closed state. This prevents unwarranted access of these genes to specialized proteins, called as transcription factors, to facilitate expression of these genes. However, this tight regulation by closed packaging of DNA also hinders access when these genes are required to be expressed to produce proteins. This process of initiation of gene expression by changing its accessibility is one of the first steps towards protein production. Most transcription factor proteins do not have the ability to bind to DNA when it is in closed conformation, therefore, it required a special of transcription factors that can bind to DNA even when it is in closed conformation. These transcription factors are called as pioneer transcription factors. Over the past decade there has been several advances and great understanding of the process of gene expression, however we still lack complete understanding of the first step that requires efficient opening of closed DNA. My work involves around understanding how a pioneer factor binds to its target locations on DNA and how is it different from other transcription factors. I am also looking it in a novel context by expressing a pioneer factor in cells where it is normally not expressed. This will felicitate to understand if pioneer factors have intrinsic ability to identify its targets in closed DNA even when it is expressed in ectopic environment which lack any partners that might be required in its naturally expressed cell-type. This systems provides us with an opportunity to understand its function on its own and also we aim to identify what are the changes that occurs after expression of pioneer factors. Another important aspect of regulation of expression of a gene is chemical modifications that can either occur on DNA itself or the protein around which DNA is wrapped (histones). These external factor (other than the DNA sequence) are termed as epigenetic factors. My aim is also to understand epigenetic changes in relation to pioneer factors activity.


Genome-wide analysis reveals positional-nucleosome-oriented binding pattern of pioneer factor FOXA1.

Abstract: The compaction of nucleosomal structures creates a barrier for DNA-binding transcription factors (TFs) to access their cognate cis-regulatory elements. Pioneer factors (PFs) such as FOXA1 are able to directly access these cis-targets within compact chromatin. However, how these PFs interplay with nucleosomes remains to be elucidated, and is critical for us to understand the underlying mechanism of gene regulation. Here, we have conducted a computational analysis on a strand-specific paired-end ChIP-exo (termed as ChIP-ePENS) data of FOXA1 in LNCaP cells by our novel algorithm ePEST. We find that FOXA1 chromatin binding occurs via four distinct border modes (or footprint boundary patterns), with a preferential footprint boundary patterns relative to FOXA1 motif orientation. In addition, from this analysis three fundamental nucleotide positions (oG, oS and oH) emerged as major determinants for blocking exo-digestion and forming these four distinct border modes. By integrating histone MNase-seq data, we found an astonishingly consistent, 'well-positioned' configuration occurs between FOXA1 motifs and dyads of nucleosomes genome-wide. We further performed ChIP-seq of eight chromatin remodelers and found an increased occupancy of these remodelers on FOXA1 motifs for all four border modes (or footprint boundary patterns), indicating the full occupancy of FOXA1 complex on the three blocking sites (oG, oS and oH) likely produces an active regulatory status with well-positioned phasing for protein binding events. Together, our results suggest a positional-nucleosome-oriented accessing model for PFs seeking target motifs, in which FOXA1 can examine each underlying DNA nucleotide and is able to sense all potential motifs regardless of whether they face inward or outward from histone octamers along the DNA helix axis.

Pub.: 28 Jul '16, Pinned: 29 Jun '17