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Structural maintenance of chromosomes flexible hinge domain-containing 1 (SMCHD1) is an epigenetic regulator that mediates gene expression silencing at targeted sites across the genome. Our current understanding of SMCHD1's molecular mechanism, and how substitutions within SMCHD1 lead to the diseases, facioscapulohumeral muscular dystrophy (FSHD) and Bosma arhinia microphthalmia syndrome (BAMS), are only emerging. Recent structural studies of its two component domains — the N-terminal ATPase and C-terminal SMC hinge — suggest that dimerization of each domain plays a central role in SMCHD1 function. Here, using biophysical techniques, we demonstrate that the SMCHD1 ATPase undergoes dimerization in a process that is dependent on both the N-terminal UBL (Ubiquitin-like) domain and ATP binding. We show that neither the dimerization event, nor the presence of a C-terminal extension past the transducer domain, affect SMCHD1's in vitro catalytic activity as the rate of ATP turnover remains comparable to the monomeric protein. We further examined the functional importance of the N-terminal UBL domain in cells, revealing that its targeted deletion disrupts the localization of full-length SMCHD1 to chromatin. These findings implicate UBL-mediated SMCHD1 dimerization as a crucial step for chromatin interaction, and thereby for promoting SMCHD1-mediated gene silencing.

Summary: Neural tube defects (NTDs) are common birth defects in humans and show an unexplained female bias. Female mice lacking the tumor suppressor p53 display NTDs with incomplete penetrance. We found that the combined loss of pro-apoptotic BIM and p53 caused 100% penetrant, female-exclusive NTDs, which allowed us to investigate the female-specific functions of p53. We report that female p53−/− embryonic neural tube samples show fewer cells with inactive X chromosome markers Xist and H3K27me3 and a concomitant increase in biallelic expression of the X-linked genes, Huwe1 and Usp9x. Decreased Xist and increased X-linked gene expression was confirmed by RNA sequencing. Moreover, we found that p53 directly bound response elements in the X chromosome inactivation center (XIC). Together, these findings suggest p53 directly activates XIC genes, without which there is stochastic failure in X chromosome inactivation, and that X chromosome inactivation failure may underlie the female bias in neural tube closure defects. : The molecular mechanisms underlying the female bias of neural tube defects are currently unclear. Delbridge et al. present evidence that p53 is required for normal Xist expression and X chromosome inactivation and show in two models that partial failure of X chromosome inactivation is associated with female-biased neural tube defects. Keywords: female-specific neural tube defects, X-chromosome inactivation, p53, Xist, Ftx, Tsix, biallelic expression, BIM, BCL2L11, SMCHD1

Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) is an epigenetic regulator in which polymorphisms cause the human developmental disorder, Bosma arhinia micropthalmia syndrome, and the degenerative disease, facioscapulohumeral muscular dystrophy. SMCHD1 is considered a noncanonical SMC family member because its hinge domain is C-terminal, because it homodimerizes rather than heterodimerizes, and because SMCHD1 contains a GHKL-type, rather than an ABC-type ATPase domain at its N terminus. The hinge domain has been previously implicated in chromatin association; however, the underlying mechanism involved and the basis for SMCHD1 homodimerization are unclear. Here, we used x-ray crystallography to solve the three-dimensional structure of the Smchd1 hinge domain. Together with structure-guided mutagenesis, we defined structural features of the hinge domain that participated in homodimerization and nucleic acid binding, and we identified a functional hotspot required for chromatin localization in cells. This structure provides a template for interpreting the mechanism by which patient polymorphisms within the SMCHD1 hinge domain could compromise function and lead to facioscapulohumeral muscular dystrophy.

Human adipose-derived mesenchymal stromal cells (ASC) are showing clinical promise for the treatment of a range of inflammatory and degenerative conditions. These lipoaspirate-derived cells are part of the abundant and accessible source of heterogeneous stromal vascular fraction (SVF). They are typically isolated and expanded from the SVF via adherent cell culture for at least 2 weeks and as such represent a relatively undefined population of cells. We isolated ex vivo ASC directly from lipoaspirate using a cocktail of antibodies combined with immunomagnetic bead sorting. This method allowed for the rapid enrichment of a defined and untouched ex vivo ASC population (referred to as MACS-derived ASC) that were then compared to culture-derived ASC. This comparison found that MACS-derived ASC contain a greater proportion of cells with activity in in vitro differentiation assays. There were also significant differences in the secretion levels of some key paracrine molecules. Moreover, when the MACS-derived ASC were subjected to adherent tissue culture, rapid changes in gene expression were observed. This indicates that culturing cells may alter the clinical utility of these cells. Although MACS-derived ASC are more defined compared to culture-derived ASC, further investigations using a comprehensive multicolor flow cytometry panel revealed that this cell population is more heterogeneous than previously appreciated. Additional studies are therefore required to more precisely delineate phenotypically distinct ASC subsets with the most therapeutic potential. This research highlights the disparity between ex vivo MACS-derived and culture-derived ASC and the need for further characterization.

Summary: We and others have recently reported that the SMC protein Smchd1 is a regulator of chromosome conformation. Smchd1 is critical for the structure of the inactive X chromosome and at autosomal targets such as the Hox genes. However, it is unknown how Smchd1 is recruited to these sites. Here, we report that Smchd1 localizes to the inactive X via the Xist-HnrnpK-PRC1 (polycomb repressive complex 1) pathway. Contrary to previous reports, Smchd1 does not bind Xist or other RNA molecules with any specificity. Rather, the localization of Smchd1 to the inactive X is H2AK119ub dependent. Following perturbation of this interaction, Smchd1 is destabilized, which has consequences for gene silencing genome-wide. Our work adds Smchd1 to the PRC1 silencing pathway for X chromosome inactivation. : Jansz et al. report that the chromatin protein Smchd1 depends on polycomb repressive complex 1-mediated ubiquitylation of histone H2A for its recruitment to the inactive X chromosome and for its protein stability. These data have implications for Smchd1 targeting genome-wide. Keywords: Smchd1, X inactivation, Xist, PRC1, Hnrnpk, Ring1B

The regulation of higher order chromatin structure is complex and dynamic; however we do not yet understand the full suite of mechanisms governing architecture. Here we reveal the non-canonical SMC protein Smchd1 as a novel regulator of long-range chromatin interactions, and add it to the canon of epigenetic proteins required forHoxgene regulation. The effect of losing Smchd1-dependent chromatin interactions has varying outcomes dependent on chromatin context. At autosomal targets transcriptionally sensitive to Smchd1 deletion, we find increased short-range interactions and ectopic enhancer activation. By contrast, the inactive X chromosome is transcriptionally refractive to Smchd1 ablation, despite chromosome-wide increases in short-range interactions. There we observe spreading of H3K27me3 domains into regions not normally decorated by this mark. Together these data suggest Smchd1 has the capacity to insulate the chromatin, thereby limiting access to other chromatin modifying proteins.