Chromatin Domain Structure and Function

Transcriptional regulation is fundamental to most basic molecular and cell biological processes. Our long-term goals are to determine how 10 and 30 nm chromatin fibers fold into large-scale chromatin domains, how these chromatin domains are moved and positioned within nuclei relative to specific nuclear bodies and compartments, and what this means for DNA functions such as transcription and replication. Our first Aim is to dissect both transcription independent and transcription dependent mechanisms for genome positioning relative to nuclear speckles. Our rationale is that gene positioning relative to nuclear speckles modulates levels of gene expression possibly for thousands of genes, and therefore dissection of these mechanisms will reveal novel aspects of gene regulation. This Aim builds on strong preliminary work. During the last grant cycle, we discovered gene expression amplification after nuclear speckle contact. We also discovered a surprising conservation of genome distance to nuclear speckles, with small distance shifts relative to speckles highly correlated with large changes in gene expression and with many inducible genes “pre-positioned” near nuclear speckles even before transcriptional activation. Our second Aim is to map, visualize, reconstitute, and then dissect cis and trans determinants for large- scale chromatin domains with distinct levels of compaction; these domains can be visualized by live-cell microscopy and electron microscopy but are not yet measured by current genomic methods such as Hi-C and are not well-preserved by conventional FISH methods. Our rationale is that this level of large-scale compaction modulates both transcriptional initiation and elongation rates and therefore our analysis of chromatin domains with different levels of large-scale chromatin compaction will reveal novel aspects of gene regulation. This Aim also builds on strong preliminary work. During the last grant cycle, we used high slopes of TSA-seq scores to identify unusually decondensed large-scale chromatin domains (DLCDs). DLCDs mapped predominately to Hi-C compartment, subcompartment, and TAD boundaries separating active and repressed chromatin domains. Acidic activators and chromatin factors recruited by acidic activators showed the highest enrichment over DLCDS among hundreds of chromatin-modifying factors. Strikingly, this observed enrichment connects with results from the early years of this grant showing that among four classes of transcriptional activators, only acidic transcriptional activator domains showed the common activity of inducing large-scale decondensation of an engineered heterochromatic chromosome region. During this last grant cycle, we also made technological advances in developing TSA-MS to identify proteins localizing to immunostained nuclear bodies and in the manipulation and transgenesis of large DNA constructs.