Chromatin proteins play critical roles in gene regulation, yet frequently we do not fully understand how weak DNA binding affinity of such proteins contributes to their locus-specific actions. Here, we studied SMCHD1, a non-canonical SMC-family protein involved in three-dimensional genome organization and gene repression of the inactive X chromosome and its autosomal targets. We replaced endogenous SMCHD1 with GFP-tagged wild-type or hinge-domain DNA-binding mutant SMCHD1 to define the cellular role of DNA binding. The mutant showed reduced enrichment at the inactive X chromosome in female cells, while retaining stable binding at most autosomal binding sites. Impaired DNA binding weakens SMCHD1-mediated gene repression and chromatin-state regulation, producing hypomorphic effect. Multiple live-cell imaging methods reveal that DNA binding constrains SMCHD1 mobility and supports maintenance, rather than initial recruitment, of chromatin-bound SMCHD1 both during interphase and mitosis. Thus, SMCHD1’s weak and sequence-independent DNA binding is a key determinant of its chromatin residence, localization and function. Our findings provide a framework for understanding SMCHD1 and other chromatin proteins with sequence-independent DNA binding activity.
Rui-Feng Hu, Julissa J. Sánchez-Velásquez, Jieqiong Lou et al.· bioRxiv· 0 citations
Nuclear export of mRNA is extensively coupled to transcription and processing of mRNA. How mRNA export is mechanistically regulated remains poorly understood. Here, we uncover a physical checkpoint at nuclear speckles that regulates mRNA export during DNA replication in S-phase of the cell cycle. Unbiased screening approaches identify WEE1 and CHK1 as novel mRNA export regulators. mRNA export complexes are recruited to sites of replication stress in S-phase. WEE1 inhibition prematurely activates CDK1 and PLK1, leading to accumulation of R-loop associated mRNA in large nuclear speckles, with late markers of replication stress present around their periphery. This recruitment is dependent on CDK1 activity. Phosphorylation of ALYREF by CDK1 and PLK1 regulates nuclear speckle accumulation of mRNA following replication stress. mRNA export factors including ALYREF are subsequently mis-localised from these speckles, preventing nuclear export of R-loop associated mRNA. Thus, WEE1, CDK1 and PLK1 enforce a cell cycle regulated checkpoint at nuclear speckles that serves to protect the cell from major sources of genome instability by ensuring that R-loop associated mature mRNA is not exported to the cytoplasm.
Tobias D. Williams, Kirstyn T. Carey, W. Hamilton et al.· bioRxiv· 0 citations
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