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Cornelia de Lange syndrome (CdLS) is a spectrum disorder due to variants in genes of the cohesin protein complex. The following abstracts are from the Cornelia de Lange Syndrome Scientific and Educational Symposium held virtually in October 2020. Aspects of behavior, including autistic features, impulsivity, adaptive skills, executive function, and anxiety are described. Applied behavioral analysis is a promising approach for autism, and an N-acetylcysteine trial is proposed. Children below 6 years with CdLS have an increased number of and further travel to medical providers, with insurance type comprising a significant barrier. Speech, language, and feeding abilities fall significantly below expectations for age in CdLS. Augmentative alternative communication can yield potential barriers as well as interesting benefits. Developmentally, studies in animal models further elucidate the mechanisms and roles of cohesin: link with mediator transcriptional complex; facilitation of enhancer-promoter communication; regulation of gene expression; allocation of cells to germ layers; and repair of spontaneous DNA damage in placental cells. Genome and RNA sequencing can help identify the molecular cause in the 20% of individuals with suspected CdLS and negative testing. The phenotypes in individuals with variants in the SMC1A gene are distinct, and that with intractable seizures has been further evaluated. AMA CME credits provided by GBMC, Baltimore, MD. All studies approved by an ethics committee.

The ring-like cohesin complex plays an essential role in chromosome segregation, organization, and double-strand break repair through its ability to bring two DNA double helices together. Scc2 (NIPBL in humans) together with Scc4 functions as the loader of cohesin onto chromosomes. Chromatin adapters such as the RSC complex facilitate the localization of the Scc2-Scc4 cohesin loader. Here, we identify a broad range of Scc2-chromatin protein interactions that are evolutionarily conserved and reveal a role for one complex, Mediator, in the recruitment of the cohesin loader. We identified budding yeast Med14, a subunit of the Mediator complex, as a high copy suppressor of poor growth in Scc2 mutant strains. Physical and genetic interactions between Scc2 and Mediator are functionally substantiated in direct recruitment and cohesion assays. Depletion of Med14 results in defective sister chromatid cohesion and the decreased binding of Scc2 at RNA Pol II-transcribed genes. Previous work has suggested that Mediator, Nipbl, and cohesin connect enhancers and promoters of active mammalian genes. Our studies suggest an evolutionarily conserved fundamental role for Mediator in the direct recruitment of Scc2 to RNA Pol II-transcribed genes.

The ring-like cohesin complex plays an essential role in chromosome segregation, organization, and double-strand break repair through its ability to bring two DNA double helices together. Scc2 (NIPBL in humans) together with Scc4 function as the loader of cohesin onto chromosomes. The RSC complex has been shown to act as a chromatin adapter, facilitating localization of the Scc2-Scc4 cohesin loader. Here we identify a broad range of Scc2- chromatin protein interactions that are evolutionarily conserved, consistent with a multifactorial adaptor landscape. We reveal a role for one complex, Mediator, in recruitment of the cohesin loader. Med14, a subunit of the Mediator complex, is a high copy suppressor of Scc2 mutants. Physical and genetic interactions between Scc2 and Mediator are functionally substantiated in direct recruitment and cohesion assays. Mediator can provide recruitment function for Scc2 based on a chromatin tethering experiment. Depletion of Med14 results in defective sister chromatid cohesion and decreased binding of Scc2 at RNA Pol II transcribed genes. Previous work has suggested that Mediator, Nipbl, and cohesin connect enhancers and promoters of active mammalian genes. Our studies in yeast suggest an evolutionarily conserved fundamental role for Mediator in direct recruitment of Scc2 to RNA pol II transcribed genes.

Successful proliferation and function of an organism relies on the equal segregation of its genetic material during cell division. Duplicate sister chromatids need to accurately segregate at mitosis. Precise segregation depends on a multicomplex protein structure called the kinetochore. The kinetochore assembles at centromeres and attaches to microtubules to segregate sister chromatids. Even though the kinetochore structure was first observed nearly a century ago, many aspects of the regulation, function and assembly of this large 100 + protein structure remain to be determined. Improved microscopy and proteomics techniques over the years have helped to reveal the structure, composition and localization of sub-modules of the kinetochore. Recent work suggests that the configuration of the kinetochore is plastic, with extra submodules being added during anaphase to support microtubule tracking and chromosome segregation. We discuss our perspective of how this process might be regulated.

Correct localization of the centromeric histone variant CenH3/CENP-A/Cse4 is an important part of faithful chromosome segregation. Mislocalization of CenH3 could affect chromosome segregation, DNA replication and transcription. CENP-A is often overexpressed and mislocalized in cancer genomes, but the underlying mechanisms are not understood. One major regulator of Cse4 deposition is Psh1, an E3 ubiquitin ligase that controls levels of Cse4 to prevent deposition into non-centromeric regions. We present evidence that Chromatin assembly factor-1 (CAF-1), an evolutionarily conserved histone H3/H4 chaperone with subunits shown previously to interact with CenH3 in flies and human cells, regulates Cse4 deposition in budding yeast. yCAF-1 interacts with Cse4 and can assemble Cse4 nucleosomes in vitro. Loss of yCAF-1 dramatically reduces the amount of Cse4 deposited into chromatin genome-wide when Cse4 is overexpressed. The incorporation of Cse4 genome-wide may have multifactorial effects on growth and gene expression. Loss of yCAF-1 can rescue growth defects and some changes in gene expression associated with Cse4 deposition that occur in the absence of Psh1-mediated proteolysis. Incorporation of Cse4 into promoter nucleosomes at transcriptionally active genes depends on yCAF-1. Overall our findings suggest CAF-1 can act as a CenH3 chaperone, regulating levels and incorporation of CenH3 in chromatin.

Background: Psh1 is an E3 ubiquitin ligase that controls CenH3/Cse4 levels through proteolysis in budding yeast. Results: Psh1 is phosphorylated in vivo by CK2, and its E3 ligase activity is promoted. Conclusion: Phosphorylation is crucial in Psh1-assisted control of Cse4 levels, and the Psh1-Cse4 association itself functions to prevent Cse4 misincorporation. Significance: This work reports a previously unknown function of CK2 in CenH3/Cse4 regulation., Cse4 is the centromeric histone H3 variant in budding yeast. Psh1 is an E3 ubiquitin ligase that controls Cse4 levels through proteolysis. Here we report that Psh1 is phosphorylated by the Cka2 subunit of casein kinase 2 (CK2) to promote its E3 activity for Cse4. Deletion of CKA2 significantly stabilized Cse4. Consistent with phosphorylation promoting the activity of Psh1, Cse4 was stabilized in a Psh1 phosphodepleted mutant strain in which the major phosphorylation sites were changed to alanines. Phosphorylation of Psh1 did not control Psh1-Cse4 or Psh1-Ubc3(E2) interactions. Although Cse4 was highly stabilized in a cka2Δ strain, mislocalization of Cse4 was mild, suggesting that Cse4 misincorporation was prevented by the intact Psh1-Cse4 association. Supporting this idea, Psh1 was also stabilized in a cka2Δ strain. Collectively our data suggest that phosphorylation is crucial in Psh1-assisted control of Cse4 levels and that the Psh1-Cse4 association itself functions to prevent Cse4 misincorporation.

The Cse4 nucleosome at each budding yeast centromere must be faithfully assembled each cell cycle to specify the site of kinetochore assembly and microtubule attachment for chromosome segregation. Although Scm3 is required for the localization of the centromeric H3 histone variant Cse4 to centromeres, its role in nucleosome assembly has not been tested. We demonstrate that Scm3 is able to mediate the assembly of Cse4 nucleosomes in vitro, but not H3 nucleosomes, as measured by a supercoiling assay. Localization of Cse4 to centromeres and the assembly activity depend on an evolutionarily conserved core motif in Scm3, but localization of the CBF3 subunit Ndc10 to centromeres does not depend on this motif. The centromere targeting domain of Cse4 is sufficient for Scm3 nucleosome assembly activity. Assembly does not depend on centromeric sequence. We propose that Scm3 plays an active role in centromeric nucleosome assembly.

SummaryThe centromere is a specialized chromosomal structure that regulates chromosome segregation. Centromeres are marked by a histone H3 variant. In budding yeast, the histone H3 variant Cse4 is present in a single centromeric nucleosome. Experimental evidence supports several different models for the structure of centromeric nucleosomes. To investigate Cse4 copy number in live yeast, we developed a method coupling fluorescence correlation spectroscopy and calibrated imaging. We find that centromeric nucleosomes have one copy of Cse4 during most of the cell cycle, whereas two copies are detected at anaphase. The proposal of an anaphase-coupled structural change is supported by Cse4-Cse4 interactions, incorporation of Cse4, and the absence of Scm3 in anaphase. Nucleosome reconstitution and ChIP suggests both Cse4 structures contain H2A/H2B. The increase in Cse4 intensity and deposition at anaphase are also observed in Candida albicans. Our experimental evidence supports a cell-cycle-coupled oscillation of centromeric nucleosome structure in yeast.

Cse4 is a variant of histone H3 that is incorporated into a single nucleosome at each centromere in budding yeast. We have discovered an E3 ubiquitin ligase, called Psh1, which controls the cellular level of Cse4 via ubiquitylation and proteolysis. The activity of Psh1 is dependent on both its RING and zinc finger domains. We demonstrate the specificity of the ubiquitylation activity of Psh1 toward Cse4 in vitro and map the sites of ubiquitylation. Mutation of key lysines prevents ubiquitylation of Cse4 by Psh1 in vitro and stabilizes Cse4 in vivo. While deletion of Psh1 stabilizes Cse4, elimination of the Cse4-specific chaperone Scm3 destabilizes Cse4, and the addition of Scm3 to the Psh1-Cse4 ubiquitylation reaction prevents Cse4 ubiquitylation, together suggesting Scm3 may protect Cse4 from ubiquitylation. Without Psh1, Cse4 overexpression is toxic and Cse4 is found at ectopic locations. Our results suggest Psh1 functions to prevent the mislocalization of Cse4.

The budding yeast CenH3 histone variant Cse4 localizes to centromeric nucleosomes and is required for kinetochore assembly and chromosome segregation. The exact composition of centromeric Cse4–containing nucleosomes is a subject of debate. ChIP-chip experiments and high resolution quantitative PCR confirm that there is a single Cse4 nucleosome at each centromere, and additional regions of the genome contain Cse4 nucleosomes at low levels. Using unbiased biochemical, cell biological, and genetic approaches we have tested the composition of Cse4-containing nucleosomes. Using micrococcal nuclease-treated chromatin, we find that Cse4 is associated with the histones H2A, H2B, and H4, but not H3 or the non-histone protein Scm3. Overexpression of Cse4 rescues the lethality of a scm3 deletion, indicating Scm3 is not essential for the formation of functional centromeric chromatin. Additionally, octameric Cse4 nucleosomes can be reconstituted in vitro. The Cse4-Cse4 interaction domain appears to be essential and interaction occurs in vivo in the centromeric nucleosome. Taken together, our experimental evidence supports the model that the Cse4-nucleosome is an octamer, containing two copies each of Cse4, H2A, H2B, and H4.

PURPOSE: Fecal diversion is frequently required in critically ill patients who may not be able to tolerate a laparotomy. Laparoscopic-assisted and trephine colostomies are alternative methods for colostomy without laparotomy, but require general anesthetic. The objective of this study was to evaluate the possibility of performing fecal diversion with the assistance of a colonoscope and without the additional morbidity of abdominal exploration or general anesthesia. METHODS: Patients were diverted using a colonoscope to identify a site of the sigmoid colon that could easily be approximated to the anterior abdominal wall as confirmed by transillumination of the abdominal wall. A small skin disc was then removed at this location and a loop colostomy was made. The colonoscope was also used as a guide to identify the proximal and distal limbs of the loop colostomy. Four patients were considered to be critically ill and local or regional anesthetic with sedation was used in these patients. RESULTS: A total of 15 patients were reviewed during the past five years. All 15 patients were successfully diverted using minimally invasive techniques with the aid of the colonoscope. Four of these patients were diverted using local or regional anesthetic without complication, thus avoiding the morbidity associated with a general anesthetic in critically ill patients. CONCLUSION: No complications related to this technique were noted in this five-year review. Endoscopically assisted colostomy is an acceptable method for fecal diversion without the need for laparotomy and can be accomplished using a local or regional anesthetic with sedation.

We demonstrate the utility of variable-temperature (VT) magic-angle-spinning (MAS) Fourier transform nuclear magnetic resonance (FT-NMR) spectroscopy for the study of mobile species in solids, especially when quadrupolar nuclei are being observed. A new VT-MAS probe is described for use in the temperature range 150 - 400 K, using spectrometers with high-field superconducting magnets. It has been utilized in studies of the bonding and dynamics of alkali cations in hydrated zeolites at an applied field of 7.05 T. Results are presented for the cases of Cs/Na-A and Cs/Li-A zeolites, in which the Cs/sup +/ ions exchange rapidly, at 293 K, between six-ring and eight-ring sites in the large cage, so that on the time scale of the NMR measurements the ions are indistinguishable and a single /sup 133/Cs resonance is observed. Below 250 K, two well-resolved signals are observed in the Cs/Na-zeolite, 107 ppM apart. These are easily assigned to the Cs/sup +/ ions in the two sites, the large difference in chemical shifts being attributed to the stronger bonding of Cs/sup +/ ions at the six-ring sites. Three sites are distinguished in the Cs/Li-A zeolite, the third site being assigned to a position near to a four-ring in the large more » cage. 26 refs., 3 figs., 1 tab. « less