CTCF/cohesin organize the ground state of chromatin-nuclear speckle association [CUT&RUN, CUT&Tag and RNA-seq]

The interchromatin space in the cell nucleus contains various membrane-less nuclear bodies. Recent findings indicate that nuclear speckles, comprising a distinct nuclear body, exhibit interactions with certain chromatin regions in a ground state. Key questions are how this ground state of chromatin-nuclear speckle association is established and what are the gene regulatory roles of this layer of nuclear organization. We report here that chromatin structural factors CTCF and cohesin are required for full ground state association between DNA and nuclear speckles. Disruption of ground state DNA-speckle contacts via either CTCF depletion or cohesin depletion had minor effects on basal level expression of speckle-associated genes, however we show strong negative effects on stimulus-dependent induction of speckle-associated genes. We identified a putative speckle targeting motif (STM) within cohesin subunit RAD21 and demonstrated that the STM is required for chromatin-nuclear speckle association. In contrast to reduction of CTCF or RAD21, depletion of the cohesin releasing factor WAPL stabilized cohesin on chromatin and DNA-speckle contacts, resulting in enhanced inducibility of speckle-associated genes. In addition, we observed disruption of chromatin-nuclear speckle association in patient derived cells with Cornelia de Lange syndrome (CdLS), a congenital neurodevelopmental diagnosis involving defective cohesin pathways, thus revealing nuclear speckles as an avenue for therapeutic inquiry. In summary, our findings reveal a mechanism to establish the ground organizational state of chromatin-speckle association, to promote gene inducibility, and with relevance to human disease.