Project description:Use DNase-seq to assess genome-wide chromation remodeling which occurred in CRISPR/Cas9 and TALE genome engineering systems Determining chromatin structural changes in transfected cells vs. the parent HEK293T cells
Project description:Adipogenesis is tightly controlled by a complex network of transcription factors acting at different stages of differentiation. Peroxisome proliferator-activated receptor gamma (PPAR gamma) and CCAAT/enhancer binding protein (C/EBP) family members are key regulators of this process. We have employed DNase I hypersensitive site analysis to investigate the genome-wide changes in chromatin structure that accompany the binding of adipogenic transcription factors. These analyses revealed a dramatic and dynamic modulation of the chromatin landscape during the first hours of adipocyte differentiation that coincides with cooperative binding of multiple early transcription factors (including glucocorticoid receptor, retinoid X receptor, Stat5a, C/EBPbeta and -delta) to transcription factor 'hotspots'. Our results demonstrate that C/EBPbeta marks a large number of these transcription factor 'hotspots' prior to induction of differentiation and chromatin remodeling and is required for their establishment. Furthermore, a subset of early remodeled C/EBP binding sites persists throughout differentiation and is later occupied by PPAR gamma , indicating that early C/EBP family members, in addition to their well established role in activation of PPAR gamma transcription, may act as pioneering factors for PPAR gamma binding. DNase I hypersensitive chromatin regions and transcription factor binding sites were identified at various time points of 3T3-L1 differentiation using DHS-seq and ChIP-seq, respectively.
Project description:Dynamic changes of histone epigenetic modifications and chromatin structure represent an universal mechanism by which cells adapt their transcriptional response to rapidly changing environmental conditions. During neuronal development, extensive chromatin remodeling takes place allowing the transition of pluripotent cells into differentiated neurons. Here we report that the ATP-dependent chromatin remodeling complex NuRD, which couples ATP-dependent nucleosome sliding with histone deacetylase activity, is a major remodeling complex in embryonic brain and plays an instructive role during mouse neuronal development. Importantly, the ATPase subunits of NuRD complex CHD3, CHD4 and CHD5 undergo a functional switch, thereby regulating distinct aspects of neuronal differentiation and migration in a sequential and mostly non-overlapping manner. We conclude that the recruitment of NuRD complexes containing specific CHDs to gene promoters and enhancers plays an instructive role in brain development. Gene expression analysis was performed in the mouse embryonic cortex at three developmental stages: E12.5, E15.5 and E18.5 using total RNA obtained from four embryos for each time point.
Project description:The transcription factor CCCTC-binding factor (CTCF) modulates pleiotropic functions mostly related to gene expression regulation. The role of CTCF in large scale genome organization is also well established. A unifying model to explain relationships between many CTCF-mediated activities involve direct or indirect interactions with numerous protein cofactors recruited to specific binding sites. The co-association of CTCF with other architectural proteins such as cohesin, chromodomain helicases and BRG1 further support the interplay between master regulators of mammalian genome folding. Here we report a comprehensive LC-MS/MS mapping of the components of the SWI/SNF chromatin remodeling complex co-associated with CTCF including subunits belonging to the core, signature and ATPase modules. We further show that the localization patterns of representative SWI/SNF members significantly overlap with CTCF sites on transcriptionally active chromatin regions. Moreover, we provide evidence of a direct binding of the BRK-BRG1 domain to the zinc finger motifs 4-8 of CTCF, thus suggesting that these domains mediate the interaction of CTCF with the SWI/SNF complex. These findings provide an updated view of the cooperative nature between CTCF and the SWI/SNF ATP-dependent chromatin-remodeling complexes, an important step for understanding how these architectural proteins collaborate to shape the genome.
Project description:We carried out multiple functional genomic assays in Capsaspora owczarzaki, the unicellular relative of animals with the largest known gene repertoire for transcriptional regulation. We show that changing chromatin states, differential lincRNA expression and dynamic cis-regulatory sites are associated with life cycle transitions in Capsaspora.