NEXMIF encephalopathy:an X-linked disorder with male and female phenotypic patterns

Purpose Pathogenic variants in the X-linked gene NEXMIF (previously KIAA2022) are associated with intellectual disability (ID), autism spectrum disorder, and epilepsy. We aimed to delineate the female and male phenotypic spectrum of NEXMIF encephalopathy. Methods Through an international collaboration, we analyzed the phenotypes and genotypes of 87 patients with NEXMIF encephalopathy. Results Sixty-three females and 24 males (46 new patients) with NEXMIF encephalopathy were studied, with 30 novel variants. Phenotypic features included developmental delay/ID in 86/87 (99%), seizures in 71/86 (83%) and multiple comorbidities. Generalized seizures predominated including myoclonic seizures and absence seizures (both 46/70, 66%), absence with eyelid myoclonia (17/70, 24%), and atonic seizures (30/70, 43%). Males had more severe developmental impairment; females had epilepsy more frequently, and varied from unaffected to severely affected. All NEXMIF pathogenic variants led to a premature stop codon or were deleterious structural variants. Most arose de novo, although X-linked segregation occurred for both sexes. Somatic mosaicism occurred in two males and a family with suspected parental mosaicism. Conclusion NEXMIF encephalopathy is an X-linked, generalized developmental and epileptic encephalopathy characterized by myoclonic-atonic epilepsy overlapping with eyelid myoclonia with absence. Some patients have developmental encephalopathy without epilepsy. Males have more severe developmental impairment. NEXMIF encephalopathy arises due to loss-of-function variants

Stressful situation if CENP-A not front and CENter

The exclusive localization of the histone H3 variant CENP-A to centromeres is essential for accurate chromosome segregation. Ubiquitin-mediated proteolysis helps to ensure that CENP-A does not mislocalize to euchromatin, which can lead to genomic instability. Consistent with this, overexpression of the budding yeast CENP-A(Cse4) is lethal in cells lacking Psh1, the E3 ubiquitin ligase that targets CENP-A(Cse4) for degradation. To identify additional mechanisms that prevent CENP-A(Cse4) misincorporation and lethality, we analyzed the genome-wide mislocalization pattern of overexpressed CENP-A(Cse4) in the presence and absence of Psh1 by chromatin immunoprecipitation followed by high throughput sequencing. We found that ectopic CENP-A(Cse4) is enriched at promoters that contain histone H2A.Z(Htz1) nucleosomes, but that H2A.Z(Htz1) is not required for CENP-A(Cse4) mislocalization. Instead, the INO80 complex, which removes H2A.Z(Htz1) from nucleosomes, promotes the ectopic deposition of CENP-A(Cse4). Transcriptional profiling revealed gene expression changes in the psh1Δ cells overexpressing CENP-A(Cse4). The down-regulated genes are enriched for CENP-A(Cse4) mislocalization to promoters, while the up-regulated genes correlate with those that are also transcriptionally up-regulated in an htz1Δ strain. Together, these data show that regulating centromeric nucleosome localization is not only critical for maintaining centromere function, but also for ensuring accurate promoter function and transcriptional regulation

Magnetic Fields toward Ophiuchus-B Derived from SCUBA-2 Polarization Measurements

We present the results of dust emission polarization measurements of Ophiuchus-B (Oph-B) carried out using the Submillimetre Common-User Bolometer Array 2 (SCUBA-2) camera with its associated polarimeter (POL-2) on the James Clerk Maxwell Telescope in Hawaii. This work is part of the B-fields in Star-forming Region Observations survey initiated to understand the role of magnetic fields in star formation for nearby star-forming molecular clouds. We present a first look at the geometry and strength of magnetic fields in Oph-B. The field geometry is traced over ~0.2 pc, with clear detection of both of the sub-clumps of Oph-B. The field pattern appears significantly disordered in sub-clump Oph-B1. The field geometry in Oph-B2 is more ordered, with a tendency to be along the major axis of the clump, parallel to the filamentary structure within which it lies. The degree of polarization decreases systematically toward the dense core material in the two sub-clumps. The field lines in the lower density material along the periphery are smoothly joined to the large-scale magnetic fields probed by NIR polarization observations. We estimated a magnetic field strength of 630 ± 410 μG in the Oph-B2 sub-clump using a Davis–Chandrasekhar–Fermi analysis. With this magnetic field strength, we find a mass-to-flux ratio λ = 1.6 ± 1.1, which suggests that the Oph-B2 clump is slightly magnetically supercritical

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Mechanisms and Functions of Chromosome Compartmentalization

Active and inactive chromatin are spatially separated in the nucleus. In Hi-C data, this is reflected by the formation of compartments, whose interactions form a characteristic checkerboard pattern in chromatin interaction maps. Only recently have the mechanisms that drive this separation come into view. Here, we discuss new insights into these mechanisms and possible functions in genome regulation. Compartmentalization can be understood as a microphase-segregated block co-polymer. Microphase separation can be facilitated by chromatin factors that associate with compartment domains, and that can engage in liquid-liquid phase separation to form subnuclear bodies, as well as by acting as bridging factors between polymer sections. We then discuss how a spatially segregated state of the genome can contribute to gene regulation, and highlight experimental challenges for testing these structure-function relationships

CENP-A^Cse4 peak information.

<p>CENP-A^Cse4 peak information.</p

INO80-C contributes to CENP-A^Cse4 misincorporation.

<p>(A) Mean chromatin CENP-A^Cse4:H2B fold change vs. <i>pGAL-3Flag-CSE4 HA-HTZ1</i> strain +/- 1 SEM from quantitative immunoblots of the chromatin fraction. Strains used: <i>pGAL-3Flag-CSE4 HA-HTZ1</i> (SBY12832), <i>nhp10Δ pGAL-3Flag-CSE4 3HA-HTZ1</i> (SBY12930), <i>psh1Δ pGAL-3Flag-CSE4 3HA-HTZ1</i> (SBY12833), and <i>nhp10Δ psh1Δ pGAL-3Flag-CSE4 3HA-HTZ1</i> (SBY12959). (n = 3). p = 0.0593 (paired t-test comparing the Cse4:H2B relative ratio in <i>psh1Δ pGAL-3Flag-CSE4</i> to <i>nhp10Δ psh1Δ pGAL-3Flag-CSE4</i>). (B) Co-Immunoprecipitation (co-IP) of overexpressed 3Flag-Cse4 with either Psh1-13Myc or Ino80-13Myc from the following strains: <i>PSH1-13Myc pGAL-3Flag-CSE4</i> (SBY14482), <i>pGAL-3Flag-CSE4</i> (SBY9540), <i>INO80-13Myc</i> (SBY14527), <i>INO80-13Myc pGAL-3Flag-CSE4</i> (SBY14526), <i>INO80-13Myc psh1Δ pGAL-3Flag-CSE4</i> (SBY14515). (C) Five-fold serial dilutions of strains <i>3Flag-CSE4</i> (SBY10419), <i>nhp10Δ 3Flag-CSE4</i> (SBY12958), <i>psh1Δ 3Flag-CSE4</i> (SBY10484), <i>nhp10Δ psh1Δ 3Flag-CSE4</i> (SBY12928), <i>pGAL-3Flag-CSE4</i> (SBY10425), <i>nhp10Δ pGAL-3Flag-CSE4</i> (SBY12930), <i>psh1Δ pGAL-3Flag-CSE4</i> (SBY10484), and <i>nhp10Δ psh1Δ pGAL-3Flag-CSE4</i> (SBY12959) were plated on indicated media.</p

Intergenic regions are the major sites of overexpressed CENP-A^Cse4 mislocalization.

<p>(A) Quantification of CENP-A^Cse4 levels in MNase-digested chromatin in untagged (SBY3, black), <i>3Flag-CSE4</i> (SBY10419, blue), <i>psh1Δ 3Flag-CSE4</i> (SBY10484, pink), <i>pGAL-3Flag-CSE4</i> (SBY10425, green) and <i>psh1Δ pGAL-3Flag-Cse4</i> (SBY10483, orange) strains. The ratio of CENP-A^Cse4:H2B in the chromatin in each strain was quantified relative to the CENP-A^Cse4:H2B ratio from the <i>3Flag-CSE4</i> (SBY10419) strain. Quantification is based on two biological replicates. Error bars are +/- 1 standard error of the mean (SEM) of the two biological replicates. (B) The total number of CENP-A^Cse4 peaks called in the indicated strains: <i>3Flag-CSE4</i> (SBY10419, blue), <i>psh1Δ 3Flag-CSE4</i> (SBY10484, pink), <i>pGAL-3Flag-CSE4</i> (SBY10425, green) and <i>psh1Δ pGAL-3Flag-CSE4</i> (SBY10483, orange). (C) A representative region of the CENP-A^Cse4 ChIP-seq coverage on Chromosome 4 between 429,000 base pairs (bp) and 470,000 bp is shown. The CENP-A^Cse4 ChIP-seq coverage for the strains in (B) is normalized to the coverage at the centromeres after subtracting the input. Peaks are shown as lines below each coverage signal (the cutoff is the average minimum coverage at the centromere in the <i>3Flag-CSE4</i> strain). The scale of the normalized coverage is from 0–20,000 for all strains. (D) The percentage of CENP-A^Cse4 peak centers in each type of genomic region is graphed for each strain, as in (B). The percentage of each feature in the genome is: genes (68.23%), intergenic (27.04%), pericentromeres (2.62%), telomeres (1.16%), origins (0.92%) and centromeres (0.02%).</p

CENP-A^Cse4 mislocalization does not depend on H2A.Z^Htz1 incorporation.

<p>(A) ChIP was performed with anti-Cse4 antibody on negative control cells (SBY15924), as well as <i>pTET-CSE4</i> (SBY15903), <i>psh1Δ pTET-CSE4</i> (SBY15904) and <i>psh1Δ htz1Δ pTET-CSE4</i> (SBY15906) cells overexpressing CENP-A^Cse4 for six hours. As a control, we also performed a ChIP experiment with no antibody in <i>psh1Δ pTET-CSE4</i> cells. Input dilutions are 1:100, 1:300, 1:100 and ChIP dilutions are 1x, 1:3, 1:9. (B) ChIP-PCR of 3HA-Htz1 at the <i>RDS1</i> promoter. Strains used: negative (neg.) control (SBY3), <i>psh1Δ pGAL-3Flag-CSE4 3HA-HTZ1</i> (SBY12833), <i>psh1Δ pGAL-3Flag-CSE4</i>, <i>3HA-HTZ1 swr1Δ</i> (SBY12924). Input dilutions: 1:100, 1:300, 1:900. ChIP dilutions: 1:3, 1:9, 1:21. (C) Relative CENP-A^Cse4 levels were measured by quantifying the mean chromatin CENP-A^Cse4:H2B fold change vs. <i>pGAL-3Flag-CSE4 HA-HTZ1</i> +/- 1 SEM using quantitative immunoblots of chromatin fraction (n = 3), p = 0.0204 (paired t-test comparing the Cse4:H2B relative ratio in <i>psh1Δ pGAL-3Flag-CSE4</i> to <i>swr1Δ psh1Δ pGAL-3Flag-CSE4</i>). Strains used were <i>pGAL-3Flag-CSE4 3HA-HTZ1</i> (SBY12832), <i>swr1Δ pGAL-3Flag-CSE4 3HA-HTZ1</i> (SBY12956), <i>psh1Δ pGAL-3Flag-CSE4 3HA-HTZ1</i> (SBY12833) and <i>swr1Δ psh1Δ pGAL-3Flag-CSE4 3HA-HTZ1</i> (SBY12924).</p