46 research outputs found
Regulation of Imprinted Genes in the brain by the ATRX Chromatin Remodeling Protein
Alpha-thalassemia mental retardation, X-linked (ATRX) is a SWI/SNF-like chromatin remodeling protein, enriched at heterochromatic regions of the genome. Disruption of ATRX in humans causes a neurodevelopmental disorder known as ATRâX Syndrome, and has been linked to paediatric neuronal cancers, suggesting an important role for ATRX in the regulation of chromatin structure in the developing brain. At the outset of this study direct ATRX target genes had not yet been identified. This thesis identifies imprinted genes as targets of ATRX in the developing brain, and explores the mechanism of ATRX regulation at these sites, using the H19/Igf2 imprinted domain as a model. The findings indicate that in the forebrain ATRX localizes to the maternal allele of the H19 imprinting control region (H19 ICR) with methyl CpG binding protein 2 (MeCP2), CCCTCâbinding factor (CTCF) and Cohesin, three important regulators of chromatin structure. ATRX is recruited by MeCP2 to the H19 ICR, where it then governs the profile of postâtranslational histone modifications and nucleosome occupancy to maintain CTCF and Cohesin binding. CTCF and Cohesin are essential constituents of the cis and trans chromatin interactions that regulate the expression of imprinted genes. Loss of either ATRX or MeCP2 disrupts cis chromosomal interactions across H19/Igf2. A role for ATRX in cis at several imprinted genes is supported by its ability to bind directly to many imprinted domains. Taken together, these findings indicate that ATRX can regulate the expression of target genes in the brain by altering nucleosome positioning to control local chromatin interactions
Three Dimensional Dual Labelled DNA Fluorescent In Situ Hybridization Analysis in Fixed Tissue Sections
Emerging studies demonstrate that three-dimensional organization of chromatin in the nucleus plays a vital role in regulating the genome. DNA fluorescent in situ hybridization (FISH) is a common molecular technique used to visualize the location of DNA sequences. The vast majority of DNA FISH studies are conducted on cultured cells due to the technical difficulties encountered using fixed tissue sections. However, the use of cultured cells poses important limitations that could yield misleading results, making in vivo analysis a far superior approach. Here we present a protocol for multiplexed three dimensional DNA FISH in mouse brain sections, which is also applicable to other tissues. Paraffin-embedded tissues could be used but the embedding and preparation of the samples is time-consuming and often associated with poor antigenicity. To overcome this problem we:âądeveloped a FISH technique using fixed, frozen cryosections;âąprovide specific instructions for tissue processing for proper fixation and freezing, including equilibration in sucrose gradients to maintain proper cellular structure;âąinclude optimized permeabilization and washing steps to achieve specific signal and to limit background fluorescence in tissue sections
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 thatATRX 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 thatATRX deficiency corresponds to reduced H3.3 incorporation and stalling ofRNApolymerase 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
Stanniocalcin 2 alters PERK signalling and reduces cellular injury during cerulein induced pancreatitis in mice
BACKGROUND: Stanniocalcin 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.
RESULTS: Since 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. STC2Tg pancreatic 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, STC2Tg mice exhibited limited increases in circulating amylase levels and increased maintenance of cellular junctions.
CONCLUSIONS: This study links STC2 to the pathological activation of PERK in vivo, and suggests involvement of STC2 in responding to pancreatic acinar cell injury
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. © 2014 the authors
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
A novel <i>RLIM/RNF12</i> variant disrupts protein stability and function to cause severe Tonne-Kalscheuer syndrome
Identification of a methylation profile for DNMT1-associated autosomal dominant cerebellar ataxia, deafness, and narcolepsy
Background: DNA methylation is an essential epigenetic mark, controlled by DNA methyltransferase (DNMT) proteins, which regulates chromatin structure and gene expression throughout the genome. In this study, we describe a family with adult-onset autosomal dominant cerebellar ataxia with deafness and narcolepsy (ADCA-DN) caused by mutations in the maintenance methyltransferase DNMT1 and assess the DNA methylation profile of these individuals. Results: We report a family with six individuals affected with ADCA-DN; specifically, patients first developed hearing loss and ataxia, followed by narcolepsy, and cognitive decline. We identified a heterozygous DNMT1 variant, c.1709C\u3eT [p.Ala570Val] by Sanger sequencing, which had been previously reported as pathogenic for ADCA-DN and segregated with disease in the family. DNA methylation analysis by high-resolution genome-wide DNA methylation array identified a decrease in CpGs with 0â10 % methylation and 80â95 % methylation and a concomitant increase in sites with 10â30 % methylation and \u3e95 % methylation. This pattern suggests an increase in methylation of normally unmethylated regions, such as promoters and CpG islands, as well as further methylation of highly methylated gene bodies and intergenic regions. Furthermore, a regional analysis identified 82 hypermethylated loci with consistent robust differences across â„5 consecutive probes compared to our large reference cohort. Conclusions: This report identifies robust changes in the DNA methylation patterns in ADCA-DN patients, which is an important step towards elucidating disease pathogenesis
Identification of Epigenetic Signature Associated With Alpha Thalassemia/Mental Retardation X-linked Syndrome
BACKGROUND: Alpha thalassemia/mental retardation X-linked syndrome (ATR-X) is caused by a mutation at the chromatin regulator gene
RESULTS: We performed genome-wide DNA methylation assessment of the peripheral blood samples from 18 patients with ATR-X and compared it to 210 controls. We demonstrated the evidence of a unique and highly specific DNA methylation epi-signature in the peripheral blood of ATRX patients, which was corroborated by targeted bisulfite sequencing experiments. Although genomically represented, differentially methylated regions showed evidence of preferential clustering in pericentromeric and telometric chromosomal regions, areas where ATRX has multiple functions related to maintenance of heterochromatin and genomic integrity.
CONCLUSION: Most significant methylation changes in the 14 genomic loci provide a unique epigenetic signature for this syndrome that may be used as a highly sensitive and specific diagnostic biomarker to support the diagnosis of ATR-X, particularly in patients with phenotypic complexity and in patients wit
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Identification of rare-disease genes using blood transcriptome sequencing and large control cohorts.
It is estimated that 350 million individuals worldwide suffer from rare diseases, which are predominantly caused by mutation in a single gene1. The current molecular diagnostic rate is estimated at 50%, with whole-exome sequencing (WES) among the most successful approaches2-5. For patients in whom WES is uninformative, RNA sequencing (RNA-seq) has shown diagnostic utility in specific tissues and diseases6-8. This includes muscle biopsies from patients with undiagnosed rare muscle disorders6,9, and cultured fibroblasts from patients with mitochondrial disorders7. However, for many individuals, biopsies are not performed for clinical care, and tissues are difficult to access. We sought to assess the utility of RNA-seq from blood as a diagnostic tool for rare diseases of different pathophysiologies. We generated whole-blood RNA-seq from 94 individuals with undiagnosed rare diseases spanning 16 diverse disease categories. We developed a robust approach to compare data from these individuals with large sets of RNA-seq data for controls (nâ=â1,594 unrelated controls and nâ=â49 family members) and demonstrated the impacts of expression, splicing, gene and variant filtering strategies on disease gene identification. Across our cohort, we observed that RNA-seq yields a 7.5% diagnostic rate, and an additional 16.7% with improved candidate gene resolution