Kristin D. Kernohan

ATRX Partners with Cohesin and MeCP2 and Contributes to Developmental Silencing of Imprinted Genes in the Brain

Human developmental disorders caused by chromatin dysfunction often display overlapping clinical manifestations, such as cognitive deficits, but the underlying molecular links are poorly defined. Here, we show that ATRX, MeCP2, and cohesin, chromatin regulators implicated in ATR-X, RTT, and CdLS syndromes, respectively, interact in the brain and colocalize at the H19 imprinting control region (ICR) with preferential binding on the maternal allele. Importantly, we show that ATRX loss of function alters enrichment of cohesin, CTCF, and histone modifications at the H19 ICR, without affecting DNA methylation on the paternal allele. ATRX also affects cohesin, CTCF, and MeCP2 occupancy within the Gtl2/Dlk1 imprinted domain. Finally, we show that loss of ATRX interferes with the postnatal silencing of the maternal H19 gene along with a larger network of imprinted genes. We propose that ATRX, cohesin, and MeCP2 cooperate to silence a subset of imprinted genes in the postnatal mouse brain.

Autosomal-Recessive Intellectual Disability with Cerebellar Atrophy Syndrome Caused by Mutation of the Manganese and Zinc Transporter Gene SLC39A8

Manganese (Mn) and zinc (Zn) are essential divalent cations used by cells as protein cofactors; various human studies and animal models have demonstrated the importance of Mn and Zn for development. Here we describe an autosomal-recessive disorder in six individuals from the Hutterite community and in an unrelated Egyptian sibpair; the disorder is characterized by intellectual disability, developmental delay, hypotonia, strabismus, cerebellar atrophy, and variable short stature. Exome sequencing in one affected Hutterite individual and the Egyptian family identified the same homozygous variant, c.112G>C (p.Gly38Arg), affecting a conserved residue of SLC39A8. The affected Hutterite and Egyptian individuals did not share an extended common haplotype, suggesting that the mutation arose independently. SLC39A8 is a member of the solute carrier gene family known to import Mn, Zn, and other divalent cations across the plasma membrane. Evaluation of these two metal ions in the affected individuals revealed variably low levels of Mn and Zn in blood and elevated levels in urine, indicating renal wasting. Our findings identify a human Mn and Zn transporter deficiency syndrome linked to SLC39A8, providing insight into the roles of Mn and Zn homeostasis in human health and development.

Analysis of neonatal brain lacking ATRX or MeCP2 reveals changes in nucleosome density, CTCF binding and chromatin looping

ATRX and MeCP2 belong to an expanding group of chromatin-associated proteins implicated in human neurodevelopmental disorders, although their gene-regulatory activities are not fully resolved. Loss of ATRX prevents full repression of an imprinted gene network in the postnatal brain and in this study we address the mechanistic aspects of this regulation. We show that ATRX binds many imprinted domains individually but that transient co-localization between imprinted domains in the nuclei of neurons does not require ATRX. We demonstrate that MeCP2 is required for ATRX recruitment and that deficiency of either ATRX or MeCP2 causes decreased frequency of long-range chromatin interactions associated with altered nucleosome density at CTCF-binding sites and reduced CTCF occupancy. These findings indicate that MeCP2 and ATRX regulate gene expression at a subset of imprinted domains by maintaining a nucleosome configuration conducive to CTCF binding and to the maintenance of higher order chromatin structure.

ATRX promotes gene expression by facilitating transcriptional elongation through guanine-rich coding regions

ATRX is a chromatin remodeling protein involved in deposition of the histone variant H3.3 at telomeres and pericentromeric heterochromatin. It also influences the expression level of specific genes; however, deposition of H3.3 at transcribed genes is currently thought to occur independently of ATRX. We focused on a set of genes, including the autism susceptibility gene Neuroligin 4 (Nlgn4), that exhibit decreased expression in ATRX-null cells to investigate the mechanisms used by ATRX to promote gene transcription. Overall TERRA levels, as well as DNA methylation and histone modifications at ATRX target genes are not altered and thus cannot explain transcriptional dysregulation. We found that ATRX does not associate with the promoter of these genes, but rather binds within regions of the gene body corresponding to high H3.3 occupancy. These intragenic regions consist of guanine-rich DNA sequences predicted to form non-B DNA structures called G-quadruplexes during transcriptional elongation. We demonstrate that ATRX deficiency corresponds to reduced H3.3 incorporation and stalling of RNA polymerase II at these G-rich intragenic sites. These findings suggest that ATRX promotes the incorporation of histone H3.3 at particular transcribed genes and facilitates transcriptional elongation through G-rich sequences. The inability to transcribe genes such as Nlgn4 could cause deficits in neuronal connectivity and cognition associated with ATRX mutations in humans.

Dual Effect of CTCF Loss on Neuroprogenitor Differentiation and Survival

An increasing number of proteins involved in genome organization have been implicated in neurodevelopmental disorders, highlighting the importance of chromatin architecture in the developing CNS. The CCCTC-binding factor (CTCF) is a zinc finger DNA binding protein involved in higher-order chromatin organization, and mutations in the human CTCF gene cause an intellectual disability syndrome associated with microcephaly. However, information on CTCF function in vivo in the developing brain is lacking. To address this gap, we conditionally inactivated the Ctcf gene at early stages of mouse brain development. Cre-mediated Ctcf deletion in the telencephalon and anterior retina at embryonic day 8.5 triggered upregulation of the p53 effector PUMA (p53 upregulated modulator of apoptosis), resulting in massive apoptosis and profound ablation of telencephalic structures. Inactivation of Ctcf several days later at E11 also resulted in PUMA upregulation and increased apoptotic cell death, and the Ctcf-null forebrain was hypocellular and disorganized at birth. Although deletion of both Ctcf and Puma in the embryonic brain efficiently rescued Ctcf-null progenitor cell apoptosis, it failed to improve neonatal hypocellularity due to decreased proliferative capacity of rescued apical and outer radial glia progenitor cells. This was exacerbated by an independent effect of CTCF loss that resulted in depletion of the progenitor pool due to premature neurogenesis earlier in development. Our findings demonstrate that CTCF activities are required for two distinct events in early cortex formation: first, to correctly regulate the balance between neuroprogenitor cell proliferation and differentiation, and second, for the survival of neuroprogenitor cells, providing new clues regarding the contributions of CTCF in microcephaly/intellectual disability syndrome pathologies.

Genetic and epigenetic dysregulation of imprinted genes in the brain

Imprinted genes are an epigenetically regulated class of genes that are preferentially expressed from one parental allele. A number of these genes are crucial for placental function and embryonic growth in mice and humans. Disruption of imprinted genes is also associated with several neurodevelopmental disorders, although the role of genomic imprinting in the brain remains largely unresolved. In this article, we describe current knowledge on the various epigenetic mechanisms that can drive monoallelic expression, provide examples of imprinted genes with relevant function in the brain and discuss imprinted gene deregulation in various neurodevelopmental disorders. Continued efforts in this field will be necessary in order to fully appreciate how the modulation of imprinted gene expression is essential to achieve normal development, and therefore function, of the mammalian nervous system.

Stanniocalcin 2 alters PERK signalling and reduces cellular injury during cerulein induced pancreatitis in mice

BackgroundStanniocalcin 2 (STC2) is a secreted protein activated by (PKR)-like Endoplasmic Reticulum Kinase (PERK) signalling under conditions of ER stress in vitro. Over-expression of STC2 in mice leads to a growth-restricted phenotype; however, the physiological function for STC2 has remained elusive. Given the relationship of STC2 to PERK signalling, the objective of this study was to examine the role of STC2 in PERK signalling in vivo.ResultsSince PERK signalling has both physiological and pathological roles in the pancreas, STC2 expression was assessed in mouse pancreata before and after induction of injury using a cerulein-induced pancreatitis (CIP) model. Increased Stc2 expression was identified within four hours of initiating pancreatic injury and correlated to increased activation of PERK signalling. To determine the effect of STC2 over-expression on PERK, mice systemically expressing human STC2 (STC2Tg) were examined. STC2Tgpancreatic tissue exhibited normal pancreatic morphology, but altered activation of PERK signalling, including increases in Activating Transcription Factor (ATF) 4 accumulation and autophagy. Upon induction of pancreatic injury, STC2Tgmice exhibited limited increases in circulating amylase levels and increased maintenance of cellular junctions.ConclusionsThis study links STC2 to the pathological activation of PERK in vivo, and suggests involvement of STC2 in responding to pancreatic acinar cell injury.

Homozygous mutation in the eukaryotic translation initiation factor 2alpha phosphatase gene, PPP1R15B, is associated with severe microcephaly, short stature and intellectual disability

Protein translation is an essential cellular process initiated by the association of a methionyl–tRNA with the translation initiation factor eIF2. The Met-tRNA/eIF2 complex then associates with the small ribosomal subunit, other translation factors and mRNA, which together comprise the translational initiation complex. This process is regulated by the phosphorylation status of the α subunit of eIF2 (eIF2α); phosphorylated eIF2α attenuates protein translation. Here, we report a consanguineous family with severe microcephaly, short stature, hypoplastic brainstem and cord, delayed myelination and intellectual disability in two siblings. Whole-exome sequencing identified a homozygous missense mutation, c.1972G>A; p.Arg658Cys, in protein phosphatase 1, regulatory subunit 15b (PPP1R15B), a protein which functions with the PPP1C phosphatase to maintain dephosphorylated eIF2α in unstressed cells. The p.R658C PPP1R15B mutation is located within the PPP1C binding site. We show that patient cells have greatly diminished levels of PPP1R15B–PPP1C interaction, which results in increased eIF2α phosphorylation and resistance to cellular stress. Finally, we find that patient cells have elevated levels of PPP1R15B mRNA and protein, suggesting activation of a compensatory program aimed at restoring cellular homeostasis which is ineffective due to PPP1R15B alteration. PPP1R15B now joins the expanding list of translation-associated proteins which when mutated cause rare genetic diseases.

Receptor tyrosine kinase mutations in developmental syndromes and cancer: two sides of the same coin

Receptor tyrosine kinases (RTKs) are a family of ligand-binding cell surface receptors that regulate a wide range of essential cellular activities, including proliferation, differentiation, cell-cycle progression, survival and apoptosis. As such, these proteins play an important role during development and throughout life; germline mutations in genes encoding RTKs cause several developmental syndromes, while somatic alterations contribute to the pathogenesis of many aggressive cancers. This creates an interesting paradigm in which mutation timing, type and location in a gene leads to different cell signaling and biological responses, and ultimately phenotypic outcomes. In this review, we highlight the roles of RTKs in developmental disorders and cancer. The multifaceted roles of these receptors, their genetic signatures and their signaling during developmental morphogenesis and oncogenesis are discussed. Additionally, we propose that comparative analysis of RTK mutations responsible for developmental syndromes may shed light on those driving tumorigenesis.

Concordance between whole‐exome sequencing and clinical Sanger sequencing: implications for patient care

The clinical translation of next‐generation sequencing has created a paradigm shift in the diagnostic assessment of individuals with suspected rare genetic diseases. Whole‐exome sequencing (WES) simultaneously examines the majority of the coding portion of the genome and is rapidly becoming accepted as an efficient alternative to clinical Sanger sequencing for diagnosing genetically heterogeneous disorders. Among reports of the clinical and diagnostic utility of WES, few studies to date have directly compared its concordance to Sanger sequencing, which is considered the clinical “gold standard”. We performed a direct comparison of 391 coding and noncoding polymorphisms and variants of unknown significance identified by clinical Sanger sequencing to the WES results of 26 patients. Of the 150 well‐covered coding variants identified by Sanger sequencing, 146 (97.3%) were also reported by WES. Nine genes were excluded from the comparison due to consistently low coverage in WES, which might be attributed to the use of older exome capture kits. We performed confirmatory Sanger sequencing of discordant variants; including five variants with discordant bases and four with discordant zygosity. Confirmatory Sanger sequencing supported the original Sanger report for three of the five discordant bases, one was shown to be a false positive supporting the WES data, and one result differed from both the Sanger and WES data. Two of the discordant zygosity results supported Sanger and the other two supported WES data. We report high concordance for well‐covered coding variants, supporting the use of WES as a screening tool for heterogeneous disorders, and recommend the use of supplementary Sanger sequencing for poorly‐covered genes when the clinical suspicion is high. Importantly, despite remaining difficulties with achieving complete coverage of the whole exome, 10 (38.5%) of the 26 compared patients were diagnosed through WES.