The SET Domain Protein, Set3p, Promotes the Reliable Execution of Cytokinesis in Schizosaccharomyces pombe

In response to perturbation of the cell division machinery fission yeast cells activate regulatory networks that ensure the faithful completion of cytokinesis. For instance, when cells are treated with drugs that impede constriction of the actomyosin ring (low doses of Latrunculin A, for example) these networks ensure that cytokinesis is complete before progression into the subsequent mitosis. Here, we identify three previously uncharacterized genes, hif2, set3, and snt1, whose deletion results in hyper-sensitivity to LatA treatment and in increased rates of cytokinesis failure. Interestingly, these genes are orthologous to TBL1X, MLL5, and NCOR2, human genes that encode components of a histone deacetylase complex with a known role in cytokinesis. Through co-immunoprecipitation experiments, localization studies, and phenotypic analysis of gene deletion mutants, we provide evidence for an orthologous complex in fission yeast. Furthermore, in light of the putative role of the complex in chromatin modification, together with our results demonstrating an increase in Set3p levels upon Latrunculin A treatment, global gene expression profiles were generated. While this analysis demonstrated that the expression of cytokinesis genes was not significantly affected in set3Δ backgrounds, it did reveal defects in the ability of the mutant to regulate genes with roles in the cellular response to stress. Taken together, these findings support the existence of a conserved, multi-protein complex with a role in promoting the successful completion of cytokinesis

Genomic analyses in Cornelia de Lange Syndrome and related diagnoses: Novel candidate genes, <scp>genotype–phenotype</scp> correlations and common mechanisms

Cornelia de Lange Syndrome (CdLS) is a rare, dominantly inherited multisystem developmental disorder characterized by highly variable manifestations of growth and developmental delays, upper limb involvement, hypertrichosis, cardiac, gastrointestinal, craniofacial, and other systemic features. Pathogenic variants in genes encoding cohesin complex structural subunits and regulatory proteins (NIPBL, SMC1A, SMC3, HDAC8, and RAD21) are the major pathogenic contributors to CdLS. Heterozygous or hemizygous variants in the genes encoding these five proteins have been found to be contributory to CdLS, with variants in NIPBL accounting for the majority (&gt;60%) of cases, and the only gene identified to date that results in the severe or classic form of CdLS when mutated. Pathogenic variants in cohesin genes other than NIPBL tend to result in a less severe phenotype. Causative variants in additional genes, such as ANKRD11, EP300, AFF4, TAF1, and BRD4, can cause a CdLS‐like phenotype. The common role that these genes, and others, play as critical regulators of developmental transcriptional control has led to the conditions they cause being referred to as disorders of transcriptional regulation (or “DTRs”). Here, we report the results of a comprehensive molecular analysis in a cohort of 716 probands with typical and atypical CdLS in order to delineate the genetic contribution of causative variants in cohesin complex genes as well as novel candidate genes, genotype–phenotype correlations, and the utility of genome sequencing in understanding the mutational landscape in this population

PLAG1 and USF2 Co-regulate Expression of Musashi-2 in Human Hematopoietic Stem and Progenitor Cells

Summary: MSI2, which is expressed predominantly in hematopoietic stem and progenitor cells (HSPCs), enforces HSPC expansion when overexpressed and is upregulated in myeloid leukemias, indicating its regulated transcription is critical to balanced self-renewal and leukemia restraint. Despite this, little is understood of the factors that enforce appropriate physiological levels of MSI2 in the blood system. Here, we define a promoter region that reports on endogenous expression of MSI2 and identify USF2 and PLAG1 as transcription factors whose promoter binding drives reporter activity. We show that these factors co-regulate, and are required for, efficient transactivation of endogenous MSI2. Coincident overexpression of USF2 and PLAG1 in primitive cord blood cells enhanced MSI2 transcription and yielded cellular phenotypes, including expansion of CD34+ cells in vitro, consistent with that achieved by direct MSI2 overexpression. Global chromatin immunoprecipitation sequencing analyses confirm a preferential co-binding of PLAG1 and USF2 at the promoter of MSI2, as well as regulatory regions corresponding to genes with roles in HSPC homeostasis. PLAG1 and USF2 cooperation is thus an important contributor to stem cell-specific expression of MSI2 and HSPC-specific transcriptional circuitry. : MSI2 is an essential human hematopoietic stem and progenitor cell (HSPC) regulator, but knowledge of the mechanisms ensuring its appropriate expression in this context are lacking. Here, Hope and colleagues map the MSI2 promoter functional in hematopoietic cells and identify USF2 and PLAG1 as essential, cooperative enforcers of endogenous MSI2 expression and stemness traits in human HSPCs. Keywords: human hematopoietic stem cells, self-renewal, promoter, transcriptional regulation, transcription factors, Musashi-2, genome-wide DNA binding site mapping, PLAG1, USF

Palmoplantar keratoderma with deafness phenotypic variability in a patient with an inherited GJB2 frameshift variant and novel missense variant

Abstract Background Variants in the GJB2 gene encoding the gap junction protein connexin‐26 (Cx26) can cause autosomal recessive nonsyndromic hearing loss or a variety of phenotypically variable autosomal dominant disorders that effect skin and hearing, such as palmoplantar keratoderma (PPK) with deafness and keratitis–ichthyosis–deafness (KID) syndrome. Here, we report a patient with chronic mucocutaneous candidiasis, hyperkeratosis with resorption of the finger tips, profound bilateral sensorineural hearing loss, and normal hair and ocular examination. Exome analysis identified a novel missense variant in GJB2 (NM_004004.5:c.101T>A, p.Met34Lys) that was inherited from a mosaic unaffected parent in the setting of a well‐reported GJB2 loss of function variant (NM_004004.5:c.35delG, p.Gly12Valfs*2) on the other allele. Method Rat epidermal keratinocytes were transfected with cDNA encoding wildtype Cx26 and/or the Met34Lys mutant of Cx26. Fixed cells were immunolabeled in order to assess the subcellular location of the Cx26 mutant and cell images were captured. Results Expression in rat epidermal keratinocytes revealed that the Met34Lys mutant was retained in the endoplasmic reticulum, unlike wildtype Cx26, and failed to reach the plasma membrane to form gap junctions. Additionally, the Met34Lys mutant acted dominantly to wildtype Cx26, restricting its delivery to the cell surface. Conclusion Overall, we show the p.Met34Lys variant is a novel dominant acting variant causing PPK with deafness. The presence of a loss a function variant on the other allele creates a more severe clinical phenotype, with some features reminiscent of KID syndrome

Double-strand breaks are not the main cause of spontaneous sister chromatid exchange in wild-type yeast cells

Homologous recombination involving sister chromatids is the most accurate, and thus most frequently used, form of recombination-mediated DNA repair. Despite its importance, sister chromatid recombination is not easily studied because it does not result in a change in DNA sequence, making recombination between sister chromatids difficult to detect. We have previously developed a novel DNA template strand sequencing technique, called Strand-seq, that can be used to map sister chromatid exchange (SCE) events genome-wide in single cells. An increase in the rate of SCE is an indicator of elevated recombination activity and of genome instability, which is a hallmark of cancer. In this study, we have adapted Strand-seq to detect SCE in the yeast Saccharomyces cerevisiae. Contrary to what is commonly thought, we find that most spontaneous SCE events are not due to the repair of DNA double-strand breaks

Set3p, Snt1p and Hif2p levels increase two- to three-fold upon treatment with low doses of LatA.

<p>(<b>A</b>) Strains expressing the indicated fusion proteins were grown to early log phase in YES at 30°C and treated with 0.5 µM LatA (left panel) or 1 µM LatA (right panel). Extracts were subjected to SDS-PAGE, transferred to PVDF membranes, and immunoblotted with anti-HA antibody. Tubulin was used as a loading control. (<b>B and C</b>) Strains expressing the indicated fusion proteins were grown to early log phase in YES at 30°C and treated with 0.5 µM LatA. Extracts were subjected to SDS-PAGE, transferred to PVDF membranes, and immunoblotted with anti-HA antibody. Tubulin or the RNA pol II carboxy-terminal domain (CTD) were used as loading controls. Signal intensities relative to the loading controls of three independent trials were quantified using ImageJ 1.41 Gel Analyzer software and are plotted below representative blots. Error bars indicate sd.</p