Hippocampal Neuron Stimulation Promotes Intracellular Tip60 Dynamics with Concomitant Genome Reorganization and Synaptic Gene Activation

Coordinated gene transcription within mammalian nuclei in response to external stimuli is a highly orchestrated process involving the interplay between epigenetics-mediated chromatin remodeling and RNA polymerase-mediated transcription. The emergence of the concept of transcription factories (TFs), characterized as specialized nuclear subcompartments enriched in hyperphosphorylated RNAPII, suggests the potential for an additional mechanism directing coordinated and efficient gene transcription. While these transcriptional ‘hot spots’ have been implicated in the co-regulation of partner genes in other cell types, its presence in hippocampal neurons and its role in activity-dependent transcriptional control within the brain remains relatively unexplored. Furthermore, while previous findings indicate a functional relevance of histone acetylation in activity-dependent gene expression, the full array of histone acetyltransferases (HATs) involved in this process remain to be determined. Our findings reveal altered HAT Tip60 intranuclear dynamics and binding patterns on activity-dependent synaptic plasticity genes following rat hippocampal neuron stimulation. Utilizing immuno-DNA FISH, we show that neuronal stimulation alters the localization of these genes within the nucleus, corresponding to the mobilization of these co-regulated genes to RNAPII-rich TFs. Lastly, we show that Tip60 is found within the same TFs as the co-regulated synaptic plasticity genes following neuronal stimulation. Taken together, these data suggest that specific, directed compartmentalization of target genes, HATs and transcription machinery within hippocampal nuclei occurs following the introduction of external stimuli thereby enabling efficient, coordinated gene transcription. Our findings provide insights into a fundamental physiological gene expression paradigm governing transcriptional activation of co-regulated genes in response to external stimuli in hippocampal neurons.M.S., Neuroscience -- Drexel University, 201

An atlas of lamina-associated chromatin across twelve human cell types reveals an intermediate chromatin subtype

BackgroundAssociation of chromatin with lamin proteins at the nuclear periphery has emerged as a potential mechanism to coordinate cell type-specific gene expression and maintain cellular identity via gene silencing. Unlike many histone modifications and chromatin-associated proteins, lamina-associated domains (LADs) are mapped genome-wide in relatively few genetically normal human cell types, which limits our understanding of the role peripheral chromatin plays in development and disease.ResultsTo address this gap, we map LAMIN B1 occupancy across twelve human cell types encompassing pluripotent stem cells, intermediate progenitors, and differentiated cells from all three germ layers. Integrative analyses of this atlas with gene expression and repressive histone modification maps reveal that lamina-associated chromatin in all twelve cell types is organized into at least two subtypes defined by differences in LAMIN B1 occupancy, gene expression, chromatin accessibility, transposable elements, replication timing, and radial positioning. Imaging of fluorescently labeled DNA in single cells validates these subtypes and shows radial positioning of LADs with higher LAMIN B1 occupancy and heterochromatic histone modifications primarily embedded within the lamina. In contrast, the second subtype of lamina-associated chromatin is relatively gene dense, accessible, dynamic across development, and positioned adjacent to the lamina. Most genes gain or lose LAMIN B1 occupancy consistent with cell types along developmental trajectories; however, we also identify examples where the enhancer, but not the gene body and promoter, changes LAD state.ConclusionsAltogether, this atlas represents the largest resource to date for peripheral chromatin organization studies and reveals an intermediate chromatin subtype

BRD4 orchestrates genome folding to promote neural crest differentiation

Higher-order chromatin structure regulates gene expression, and mutations in proteins mediating genome folding underlie developmental disorders known as cohesinopathies. However, the relationship between three-dimensional genome organization and embryonic development remains unclear. Here we define a role for bromodomain-containing protein 4 (BRD4) in genome folding, and leverage it to understand the importance of genome folding in neural crest progenitor differentiation. Brd4 deletion in neural crest results in cohesinopathy-like phenotypes. BRD4 interacts with NIPBL, a cohesin agonist, and BRD4 depletion or loss of the BRD4-NIPBL interaction reduces NIPBL occupancy, suggesting that BRD4 stabilizes NIPBL on chromatin. Chromatin interaction mapping and imaging experiments demonstrate that BRD4 depletion results in compromised genome folding and loop extrusion. Finally, mutation of individual BRD4 amino acids that mediate an interaction with NIPBL impedes neural crest differentiation into smooth muscle. Remarkably, loss of WAPL, a cohesin antagonist, rescues attenuated smooth muscle differentiation resulting from BRD4 loss. Collectively, our data reveal that BRD4 choreographs genome folding and illustrates the relevance of balancing cohesin activity for progenitor differentiation