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.
Abstract: Distinct combinations of transcription factors are necessary to elicit cell fate changes in embryonic development. Yet within each group of fate-changing transcription factors, a subset called 'pioneer factors' are dominant in their ability to engage silent, unmarked chromatin and initiate the recruitment of other factors, thereby imparting new function to regulatory DNA sequences. Recent studies have shown that pioneer factors are also crucial for cellular reprogramming and that they are implicated in the marked changes in gene regulatory networks that occur in various cancers. Here, we provide an overview of the contexts in which pioneer factors function, how they can target silent genes, and their limitations at regions of heterochromatin. Understanding how pioneer factors regulate gene expression greatly enhances our understanding of how specific developmental lineages are established as well as how cell fates can be manipulated.
Pub.: 02 Jun '16, Pinned: 29 Jun '17
Abstract: Pioneer factors such as FoxA target nucleosomal DNA and initiate cooperative interactions at silent genes during development, cellular reprogramming, and steroid hormone induction. Biophysical studies previously showed that the nuclear mobility of FoxA1 is slower than for many other transcription factors, whereas a new single molecule study (Swinstead et al., 2016, Cell) shows comparable chromatin residence times for FoxA1 and steroid receptors. Despite that steroid receptors engage nucleosome-remodeling complexes, the vast majority of co-bound sites with FoxA are dependent upon FoxA, not vice versa. Taken together, the distinguishing feature of pioneer factors remains nucleosomal access rather than an exceptional residence time in chromatin.
Pub.: 04 Jun '16, Pinned: 29 Jun '17
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
Abstract: Although many approaches have been employed to generate defined fate in vitro, the resultant cells often appear developmentally immature or incompletely specified, limiting their utility. Growing evidence suggests that current methods of direct lineage conversion may rely on the transition through a developmental intermediate. Here, I hypothesize that complete conversion between cell fates is more probable and feasible via reversion to a developmentally immature state. I posit that this is due to the role of pioneer transcription factors in engaging silent, unmarked chromatin and activating hierarchical gene regulatory networks responsible for embryonic patterning. Understanding these developmental contexts will be essential for the precise engineering of cell identity.
Pub.: 04 Aug '16, Pinned: 29 Jun '17
Abstract: Pioneer transcription factors recognise and bind their target sequences in inaccessible chromatin to establish new transcriptional networks during development and cellular reprogramming. During this process, pioneer factors establish an accessible chromatin state to facilitate additional transcription factor binding, yet how different pioneer factors achieve this remains unclear. Here, we discover that the pluripotency-associated pioneer factor OCT4 binds chromatin to shape accessibility, transcription factor co-binding, and regulatory element function in mouse embryonic stem cells. Chromatin accessibility at OCT4-bound sites requires the chromatin remodeller BRG1, which is recruited to these sites by OCT4. BRG1 occupancy supports transcription factor binding and expression of the pluripotency-associated transcriptome. Furthermore, the requirement for BRG1 in shaping OCT4 binding reflects how these target sites are used during cellular reprogramming and early mouse development. Together this reveals a distinct requirement for a chromatin remodeller in shaping the activity of the pioneer factor OCT4 and regulating the pluripotency network.
Pub.: 14 Mar '17, Pinned: 29 Jun '17
Abstract: Recruitment of transcription factors (TFs) to repressed genes in euchromatin is essential to activate new transcriptional programs during cell differentiation. However, recruitment of all TFs, including pioneer factors, is impeded by condensed H3K27me3-containing chromatin. Single-cell and gene-specific analyses revealed that, during the first hours of induction of differentiation of mammalian embryonic stem cells (ESCs), accumulation of the repressive histone mark H3K27me3 is delayed after DNA replication, indicative of a decondensed chromatin structure in all regions of the replicating genome. This delay provides a critical "window of opportunity" for recruitment of lineage-specific TFs to DNA. Increasing the levels of post-replicative H3K27me3 or preventing S phase entry inhibited recruitment of new TFs to DNA and significantly blocked cell differentiation. These findings suggest that recruitment of lineage-specifying TFs occurs soon after replication and is facilitated by a decondensed chromatin structure. This insight may explain the developmental plasticity of stem cells and facilitate their exploitation for therapeutic purposes.
Pub.: 16 Apr '17, Pinned: 29 Jun '17