Periodic Reporting for period 2 - DNAccess (Overcoming chromatin restricted DNA access)
Okres sprawozdawczy: 2022-09-01 do 2024-02-29
The variety of cell types in our body all contain the same genome yet their large differences in form and function results from a differential usage of this blueprint. They express different set of genes, a process controlled by intricate networks of transcription factors (TFs) that bind DNA in a sequence specific fashion. Our genome however is chromatinized and packaged in nucleosomes that can restrict access to DNA. Chromatin is itself an essential component of gene regulation, as this packaging accounts for the fact that most TFs only bind to a small subset of their motif occurrences, in a way we currently do not fully understand. Our inability to predict binding of a TF based on its cognate motif and location in the genome is a serious obstacle towards predictive models of gene regulation such as the impact of sequence variations within control regions.
The ERC funded project DNAccess aims to define in vivo the sensitivities of TFs to nucleosomes and their reliance on chromatin remodeling enzymes for binding. DNA access uses novel genomics and genome editing tools to systematically vary the sequences that are bound by TFs, the presence of nucleosomes and their modifications at a defined chromosomal locus and quantify resulting TF binding. We will further explore genome-wide the ability of TFs to engage with a chromatinized genome in the absence of host cofactor engagement and study proteins capable of moving nucleosomes, so called chromatin remodelers. More specifically we aim to dissect the interplay between chromatin remodelers and TF binding so to learn the temporal order that leads to stable binding and the function of carious remodeler complexes in this process.
DNAccess is building a highly integrated setup to comprehensively characterize how nucleosomes, their modifications and mobility restricts genome access. We will characterize existing chromatin barriers and identify how TFs overcome them. This represents a crucial step towards a comprehensive understanding of the role of chromatin in gene regulation, and will advance our understanding of how specificity is generated in large eukaryotic genomes
The ERC funded project DNAccess aims to define in vivo the sensitivities of TFs to nucleosomes and their reliance on chromatin remodeling enzymes for binding. DNA access uses novel genomics and genome editing tools to systematically vary the sequences that are bound by TFs, the presence of nucleosomes and their modifications at a defined chromosomal locus and quantify resulting TF binding. We will further explore genome-wide the ability of TFs to engage with a chromatinized genome in the absence of host cofactor engagement and study proteins capable of moving nucleosomes, so called chromatin remodelers. More specifically we aim to dissect the interplay between chromatin remodelers and TF binding so to learn the temporal order that leads to stable binding and the function of carious remodeler complexes in this process.
DNAccess is building a highly integrated setup to comprehensively characterize how nucleosomes, their modifications and mobility restricts genome access. We will characterize existing chromatin barriers and identify how TFs overcome them. This represents a crucial step towards a comprehensive understanding of the role of chromatin in gene regulation, and will advance our understanding of how specificity is generated in large eukaryotic genomes
In the first and second period of DNA access we have successfully established a system that enables us to interrogate a large number of sequence motifs for their ability to recruit transcription factors to a defined genomic locus. Our experimental setup enables us to monitor different motifs in parallel and since we use single molecule footprinting a s readout we obtain a quantitative overview of the TF engagements. We were furthermore able to monitor TF binding as a function of nucleosomes presence and positioning. The latter is a critical addition as it allows to ask, in the context of the cell, how motif engagement changes depending on position relative to the complex and context dependent interface of the 140 bp that are covered by the nucleosome (Grand, Pregnaloto et al., in revision for Molecular Cell). We furthermore identified how the important TF and tumor suppressor p53 engages with chromatin and how its cofactor TRIM24 reads out local histone modifications to modulated p53 function in a chromatin dependent manner (Isbel et al. NSMB 2023). By generating genetic deletions of a family of Methyl DNA Binding Domain proteins (MBDs) and contrasting the epigenome and transcriptome changes with those when we remove DNA methylation we were able to demonstrate that MBDs do nod function in repressing methylated promoters in stem and differentiated cells (Kaluscha, Domcke et al. Nature Genetics, 2022). We have in addition summarized the research field and challenges that we try to address within DNAaccess in a review article (Isbel et al. Nature Review Genetics 2022). We furthermore identified and reported the contribution of different ISWI subunit to guiding binding of CTCF revealing a novel function for DNA accessibility in nuclear organization (Iurlaro, Masoni et al., BioRX and Nature Genetics, in press).
Using novel genomic tools DNAccess has established an analytical platform to study nucleosome sensitivities of transcription factors in the cellular context. This is an important for a better understanding how DNA sequence gets translated into gene regulation and to decipher the dependencies and hierarchies of transcription factors. Our hope is that this improves our ability to predict the impact of sequence variation on the regulation of our genome.