Project description:The generation of a diverse antibody repertoire is essential for humoral immunity and requires the participation of all V genes in V(D)J recombination, which depends on the Pax5-regulated contraction of the 2.8-Mb long immunoglobulin heavy-chain (Igh) locus. How Pax5 controls Igh contraction in pro-B cells is, however, not known. Here, we demonstrate that locus contraction is caused by cohesin-mediated chromatin loop extrusion across the entire Igh locus. Notably, expression of the cohesin-release factor Wapl is repressed by Pax5 specifically in pro-B and pre-B cells, which facilitates extended loop extrusion by increasing the residence time of cohesin on chromatin. Pax5 mediates the transcriptional repression of Wapl through a single Pax5-binding site by recruiting the Polycomb repressive complex 2 to induce bivalent chromatin at the Wapl promoter. Reduced Wapl expression causes global alterations of the three-dimensional chromatin architecture, indicating that the potential to recombine all V genes entails structural changes of the entire genome in pro-B cells.
Project description:Nuclear speckles are prominent nuclear bodies that contain a myriad of factors involved in gene expression. The role of nuclear speckles as activating transcriptional compartments is emerging. However, the extent that the association between speckles and DNA is regulatable, and the mechanisms that govern regulated speckle association are currently unclear. Using DNA- and RNA-FISH, we show that speckle association can be mediated by the p53 transcription factor, finding that p53 activation drives speckle association of specific p53 transcriptional targets. Analysis of a key p53 target, p21, revealed an increase in nascent transcripts at speckle-adjacent transcription sites, supporting a role for speckles in amplifying transcriptional output. Importantly, p53-regulated speckle association of p21 did not depend on transcriptional activation, demonstrating that speckle association is not merely a consequence of gene expression. In contrast, speckle association of p21 did require DNA binding functions of p53, providing a mechanism for the specificity by which speckle association is regulated. Beyond p21, a substantial subset of p53 targets have p53-regulated speckle association, while other p53 targets do not, and we find that genomic context is highly deterministic of which target genes have regulated speckle association. These findings reveal a novel means by which transcription factors may control gene expression and provide a mechanism for the specificity of regulated speckle association.
Project description:Recent findings indicate that nuclear speckles, a distinct type of nuclear body, interact with certain chromatin regions in a ground state. Here, we report that the chromatin structural factors CTCF and cohesin are required for full ground-state association between DNA and nuclear speckles. We identified a putative speckle targeting motif (STM) within cohesin subunit RAD21 and demonstrated that the STM is required for chromatin-nuclear speckle association, disruption of which impaired induction of speckle-associated genes. Depletion of the cohesin-releasing factor WAPL, which stabilizes cohesin on chromatin and reinforces DNA–speckle contacts, results in enhanced inducibility of speckle-associated genes. Additionally, we observed disruption of chromatin–nuclear speckle association in patient-derived cells with Cornelia de Lange syndrome, a congenital neurodevelopmental disorder involving defective cohesin pathways. In summary, our findings reveal a mechanism for establishing the ground state of chromatin–speckle association and promoting gene inducibility, with relevance to human disease.
Project description:Emerging evidence reveals the critical role of the nuclear RNA-protein meshwork complex, termed nuclear matrix, in stabilizing chromatin architecture. While chromatin associates with transcriptional machinery within nuclear bodies to regulate gene expression through hierarchical architecture formation, how the nuclear matrix mediates chromatin-nuclear body associations and their spatial organization remains unclear. Here, we demonstrate that depletion of nuclear matrix factor SAF-A leads to genome-wide chromatin compaction and enhanced chromatin-speckle associations. Using super-resolution imaging and genomic analyses, we show that these chromatin organizational changes alter the inducibility of speckle-associated genes. Mechanistically, we reveal that nuclear matrix exhibits a layered spatial distribution, forming distinct compartments with RNA polymerase II clusters and chromatin in the perispeckle region. Our findings demonstrate that nuclear matrix maintains chromatin architecture and regulates gene expression through spatial coupling with nuclear bodies, providing new insights into the hierarchical organization of nuclear structure-function relationships.
Project description:Emerging evidence reveals the critical role of the nuclear RNA-protein meshwork complex, termed nuclear matrix, in stabilizing chromatin architecture. While chromatin associates with transcriptional machinery within nuclear bodies to regulate gene expression through hierarchical architecture formation, how the nuclear matrix mediates chromatin-nuclear body associations and their spatial organization remains unclear. Here, we demonstrate that depletion of nuclear matrix factor SAF-A leads to genome-wide chromatin compaction and enhanced chromatin-speckle associations. Using super-resolution imaging and genomic analyses, we show that these chromatin organizational changes alter the inducibility of speckle-associated genes. Mechanistically, we reveal that nuclear matrix exhibits a layered spatial distribution, forming distinct compartments with RNA polymerase II clusters and chromatin in the perispeckle region. Our findings demonstrate that nuclear matrix maintains chromatin architecture and regulates gene expression through spatial coupling with nuclear bodies, providing new insights into the hierarchical organization of nuclear structure-function relationships.
