This proposal aims at providing mechanistic insights into MYOD regulation of the high-order chromatin interactions that define the 3D nuclear architecture of skeletal muscle cells, by capitalizing on the unprecedented availability of high-resolution matrices of the genomic interactions induced by MYOD during skeletal myogenesis from our preliminary studies. This study will fill a critical gap of basic knowledge in the control of transcription and muscle stem cell biology, by exploring a higher level of complexity in the regulation of gene expression in muscle satellite cells introduced by the spatial dimension.
The Specific Aims are:
Aim 1 Molecular analysis of MYOD-directed regulation of 3D chromatin structure in muscle progenitors. 1a To determine the contribution of specific MYOD domains to structural and functional alterations of high-order chromatin interactions that regulate gene expression. We will monitor the effect of MYOD mutants on 3D chromatin structure and transcriptional output at specific loci. 1b To determine the role of co-factors in MYOD- directed rewiring of chromatin interactions that regulate gene expression. We will investigate the role of architectural proteins (CTCF and cohesin) as well as specific components of the chromatin-remodeling complex and the transcriptional machinery - including BAF60C, Polymerase II (PolII), TAF3, Bromodomain and Extra-Terminal Domain (BET) proteins - in MYOD-directed reconfiguration of chromatin interactions within specific nuclear topological domains. 1c To investigate MYOD-directed control of high-order chromatin interactions in satellite cells. We will determine the role of MYOD and co-factors on regulation of 3D chromatin interactome and transcriptional output in satellite cells isolated from a mouse model of conditional genetic ablation of MyoD.
Aim 2 MYOD-directed regulation of 3D chromatin structure for signal-dependent control of gene expression 2a. To functionally challenge MYOD-regulated cis-regulatory elements for signal- regulated gene expression. We will exploit Cas9-based genome editing, RNAi-based knockdown and pharmacological targeting of key epigenetic events to interrogate the dynamics of enhancer activation and chromatin interactions in response to extrinsic signals in MYOD-converted IMR90 fibroblasts. 2b To investigate MYOD-regulated cis-regulatory elements for satellite cell response to regeneration cues. We will use mouse models of satellite cell conditional genetic ablation of MyoD or the signal-responsive component of the SWI/SNF chromatin-remodeling complex BAF60C, to investigate the functional relationship between MYOD, chromatin remodeling and changes in chromatin interactions for signal-regulated gene expression. Data from this study will provide fundamental insights into the spatial control of eukaryotic transcription and satellite cell biology that will favor the transition in regenerative medicine from current therapeutic strategies affecting global gene expression to tailored approaches, based on genome editing techniques, toward a selective targeting of individual genes in satellite cells exposed to pathogenic cues from diseased muscles.
This research will advance the knowledge on the regulation of gene expression during skeletal myogenesis, by adding the spatial dimension of transcriptional control. We have generated high-resolution maps of the genome interactome during nuclear reprogramming toward the myogenic lineage induced by MYOD expression, whereby formation of higher-order chromatin domains constrains interactions between regulatory elements of the genome to spatially regulate gene expression. This resource provides an unprecedented opportunity to interrogate the three-dimensional organization of the genome in skeletal muscle progenitors, and to determine how this enables proper response to regeneration cues.