CHD8 Regulates Neurodevelopmental Pathways Associated with Autism Spectrum Disorder in Neural Progenitors

Truncating mutations of chromodomain helicase DNA-binding protein 8 (CHD8), and of many other genes with diverse functions, are strong-effect risk factors for autism spectrum disorder (ASD), suggesting multiple mechanisms of pathogenesis. We explored the transcriptional networks that CHD8 regulates in neural progenitor cells (NPCs) by reducing its expression and then integrating transcriptome sequencing (RNA sequencing) with genome-wide CHD8 binding (ChIP sequencing). Suppressing CHD8 to levels comparable with the loss of a single allele caused altered expression of 1,756 genes, 64.9% of which were up-regulated. CHD8 showed widespread binding to chromatin, with 7,324 replicated sites that marked 5,658 genes. Integration of these data suggests that a limited array of direct regulatory effects of CHD8 produced a much larger network of secondary expression changes. Genes indirectly down-regulated (i.e., without CHD8-binding sites) reflect pathways involved in brain development, including synapse formation, neuron differentiation, cell adhesion, and axon guidance, whereas CHD8-bound genes are strongly associated with chromatin modification and transcriptional regulation. Genes associated with ASD were strongly enriched among indirectly down-regulated loci (P < 10[superscript −8]) and CHD8-bound genes (P = 0.0043), which align with previously identified coexpression modules during fetal development. We also find an intriguing enrichment of cancer-related gene sets among CHD8-bound genes (P < 10[superscript −10]). In vivo suppression of chd8 in zebrafish produced macrocephaly comparable to that of humans with inactivating mutations. These data indicate that heterozygous disruption of CHD8 precipitates a network of gene-expression changes involved in neurodevelopmental pathways in which many ASD-associated genes may converge on shared mechanisms of pathogenesis.Simons FoundationNancy Lurie Marks Family FoundationNational Institutes of Health (U.S.) (Grant MH095867)National Institutes of Health (U.S.) (Grant MH095088)National Institutes of Health (U.S.) (Grant GM061354)March of Dimes Birth Defects FoundationCharles H. Hood FoundationBrain & Behavior Research FoundationAutism Genetic Resource ExchangeAutism Speaks (Organization)Pitt–Hopkins Research Foundatio

Common location of determinants in initiator transfer RNAs for initiator-elongator discrimination in bacteria and in eukaryotes

Initiator tRNAs are used exclusively for initiation of protein synthesis and not for elongation. We show that both Escherichia coli and eukaryotic initiator tRNAs have negative determinants, at the same positions, that block their activity in elongation. The primary negative determinant in E. coliinitiator tRNA is the C1xA72 mismatch at the end of the acceptor stem. The primary negative determinant in eukaryotic initiator tRNAs is located in the Tψ C stem, whereas a secondary negative determinant is the A1:U72 base pair at the end of the acceptor stem. Here we show thatE. coli initiator tRNA also has a secondary negative determinant for elongation and that it is the U50·G64 wobble base pair, located at the same position in the Tψ C stem as the primary negative determinant in eukaryotic initiator tRNAs. Mutation of the U50·G64 wobble base pair to C50:G64 or U50:A64 base pairs increases the in vivo amber suppressor activity of initiator tRNA mutants that have changes in the acceptor stem and in the anticodon sequence necessary for amber suppressor activity. Binding assays of the mutant aminoacyl-tRNAs carrying the C50 and A64 changes to the elongation factor EF-Tu·GTP show marginally higher affinity of the C50 and A64 mutant tRNAs and increased stability of the EF-Tu·GTP· aminoacyl-tRNA ternary complexes. Other results show a large effect of the amino acid attached to a tRNA, glutamineversus methionine, on the binding affinity toward EF-Tu·GTP and on the stability of the EF-Tu·GTP·aminoacyl-tRNA ternary complex

Human RECQ1 Interacts with Ku70/80 and Modulates DNA End-Joining of Double-Strand Breaks

<div><p></p><p>Genomic instability is a known precursor to cancer and aging. The RecQ helicases are a highly conserved family of DNA-unwinding enzymes that play key roles in maintaining genome stability in all living organisms. Human RecQ homologs include RECQ1, BLM, WRN, RECQ4, and RECQ5β, three of which have been linked to diseases with elevated risk of cancer and growth defects (Bloom Syndrome and Rothmund-Thomson Syndrome) or premature aging (Werner Syndrome). RECQ1, the first RecQ helicase discovered and the most abundant in human cells, is the least well understood of the five human RecQ homologs. We have previously described that knockout of RECQ1 in mice or knockdown of its expression in human cells results in elevated frequency of spontaneous sister chromatid exchanges, chromosomal instability, increased load of DNA damage and heightened sensitivity to ionizing radiation. We have now obtained evidence implicating RECQ1 in the nonhomologous end-joining pathway of DNA double-strand break repair. We show that RECQ1 interacts directly with the Ku70/80 subunit of the DNA-PK complex, and depletion of RECQ1 results in reduced end-joining in cell free extracts. In vitro, RECQ1 binds and unwinds the Ku70/80-bound partial duplex DNA substrate efficiently. Linear DNA is co-bound by RECQ1 and Ku70/80, and DNA binding by Ku70/80 is modulated by RECQ1. Collectively, these results provide the first evidence for an interaction of RECQ1 with Ku70/80 and a role of the human RecQ helicase in double-strand break repair through nonhomologous end-joining.</p></div

RECQ1-null cells promote end-joining but show reduced Ku-DNA binding activity.

<p><i>A.</i> Level of <i>in vitro</i> end-joining activity is comparable in WT and RECQ1 KO MEFs. Cell free extracts prepared from non-transformed RECQ1 WT or KO MEFs were used in end-joining reaction containing EcoRI-linearized pUC19 DNA as substrate. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer, trimer and tetramer are indicated (right panel). Western blot showed no detectable difference in Ku protein level in WT and KO cell extracts (left panel). GAPDH serves as loading control. <i>B.</i> Presence of PARP-1 antibody interferes with RECQ1 KO cell free extract mediated end-joining. In addition to standard reactions, <i>in vitro</i> end-joining reactions were performed with WT or KO cell extracts in the presence of a DNA-PKcs inhibitor Nu7026 (1.2 µM) or a specific anti-PARP-1 antibody (3 µg). IgG (3 µg) was included as unrelated antibody in a control reaction. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. Western blot showed no detectable difference in PARP-1 protein level in WT and KO cell extracts (left panel). GAPDH serves as loading control. <i>C.</i> Ku70/80 DNA binding assay performed by using Active Motif kit shows diminished DNA binding in RECQ1 KO extract as compared to WT extract (p<0.05). The results shown are the average of at least three independent experiments, with SD indicated by error bars. <i>D.</i> DNA binding analysis for Ku70/80 in extracts prepared from control or RECQ1-depleted HeLa cells. Nuclear extracts prepared from control or RECQ1 siRNA transfected cells either untreated or treated with NCS (0.01 µM, 3 h) were used to perform Ku70/80 DNA binding assay (Active Motif). Following NCS treatment, RECQ1 siRNA transfected cells showed significantly reduced Ku70/80 DNA binding activity as compared to control siRNA transfected cells (p<0.05). The results shown are average of at least three independent experiments, with SD indicated by error bars. <i>E.</i> RECQ1-depleted HeLa cells show reduced chromatin bound Ku following NCS treatment. Ku80 and PARP-1 was detected by Western blot in the soluble and insoluble fractions prepared from control or RECQ1 siRNA transfected cells that were either untreated or treated with indicated dose of NCS for 3 h. GAPDH serves as cytoplasmic marker and H3 serves as marker for chromatin enriched fractions. <i>F.</i> RECQ1 protein in the chromatin enriched fraction of control or Ku80-depleted cells. Cells were untreated or treated with indicated dose of NCS for 3 h, followed by biochemical fractionation and the chromatin containing insoluble fractions were examined by Western blotting. H3 serves as marker for chromatin enriched fractions.</p

RECQ1 interacts with Ku70/80 <i>in vivo</i>.

<p><i>A</i>. Co-immunoprecipitation analysis of RECQ1 interaction with Ku70/80 using HeLa nuclear extracts. Immunoprecipitations (IP) with antibodies specific for RECQ1, Ku70/80 and preimmune IgG are indicated. Eluted proteins in immunoprecipitate were analyzed by Western blotting and are indicated. RECQ1 IP contained Ku70 and Ku80 subunits but DNA-PKcs was not detected. Reciprocal co-IPs of Ku70/80 also contained RECQ1. <i>B.</i> Association of RECQ1 and Ku70/80 is not mediated via DNA. RECQ1 antibody co-precipitated RECQ1 and Ku70/80 using benzonase-treated extract in IP reaction. <i>C.</i> RECQ1 interacts with Ku in DNA-PKcs deficient and proficient cells. Lysates of MO59J (DNA-PKcs deficient) or MO59K (DNA-PKcs proficient) cells were used for IP using RECQ1 antibody or IgG and analyzed by Western blotting as indicated. <i>D</i>. Immunofluorescence staining of endogenous RECQ1 and Ku70/80. HeLa cells grown on coverslips were either mock-treated or treated with NCS (100 ng/ml, 3 h). Cells were fixed and immunostained using a mouse monoclonal Ku70/80 antibody (1∶200) and a rabbit polyclonal RECQ1 antibody (1∶500). RECQ1 and Ku70/80 were visualized with Alexa Fluor 488- or Alexa Fluor 568-conjugated secondary antibodies, respectively, followed by confocal microscopy. Inset shows enlarged portion of the nucleus after NCS treatment; colocalization of RECQ1 (green) and Ku70/80 (red) in cells appears yellow in merged images. In all experiments, input corresponds to 5% of total protein used in IP reactions.</p

RECQ1 modulates DNA end-joining.

<p><i>A.</i> RECQ1 affects DNA end-joining by T4 ligase. Comparison of ligation products of 5′-cohesive (left panel) or blunt (right panel) ended linear DNA after incubation with T4 ligase alone, or after pre-incubation with the increasing amount of RECQ1 (0–260 ng) or Ku70/80 (0–400 ng) as described in materials and methods. IgG (1µg) was included as unrelated protein in a control reaction. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. O.C., open circular. DNA size marker is shown in leftmost lane. <i>B.</i> RECQ1 antibody interferes with cell free extract mediated end-joining <i>in vitro</i>. The effect of antibodies against human RECQ1 (1.5 and 3 µg) or Ku70/80 (1 and 2 µg) was tested in DNA end-joining reactions assembled with 5 µg HeLa cell free extract. Antibodies at the indicated amounts were incubated with cell free extract for 10 min at room temperature in NHEJ buffer without DNA and ATP. Subsequently, EcoRI-linearized pUC19 DNA and ATP were added to start the reaction followed by 2 h incubation at room temperature. Reaction products were purified and analyzed by SYBR Gold staining after resolution on agarose gel. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. <i>C.</i> Immunodepletion of RECQ1 or Ku70/80 from cell extracts results in similarly reduced end-joining. DNA end joining reactions were performed using HeLa extract depleted either with anti-RECQ1 polyclonal antibody (upper panel) or with the anti-Ku70/80 monoclonal antibody (lower panel) as described. Mock-depleted extracts used preimmune rabbit or mouse IgG as control for RECQ1 or Ku70/80 depletion, respectively. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. Inverted image of a typical gel is shown. ImageJ was used to quantitate dimer product in each case, and the average from at least three independent experiments are indicated including SD. Western blot of control un-depleted, mock-depleted and RECQ1 or Ku70/80 depleted extracts used in the end-joining reaction is shown along with a loading control (actin) (right).</p

RECQ1 and Ku70/80 co-bind a linear blunt duplex DNA.

<p><i>A.</i> RECQ1 binds a 322 bp blunt duplex fragment derived from pUC19 plasmid DNA. An electrophoretic mobility shift assay (EMSA) was performed to examine the ability of increasing concentration of purified RECQ1 to bind linearized plasmid DNA (30 ng) under DNA binding conditions as described in materials and methods. Protein-DNA complexes were resolved by native 6% polyacrylamide gels and detected by staining with SYBR Gold and a typical inverted image is shown. <i>B.</i> RECQ1 facilitates the formation of higher order DNA complex with Ku70/80. EMSA was performed to examine the ability of increasing concentration of Ku70/80 (0–100 nM) to interact with RECQ1 (12.5 nM) when bound to linear plasmid DNA (30 ng). Both RECQ1 and Ku70/80 bind to blunt duplex resulting in a series of progressively retarded bands, but the mobility of RECQ1-DNA and Ku70/80-DNA complex is distinct. Additional slow migrating bands were observed in Ku70/80 (25, 50 and 100 nM) in the presence of RECQ1 (12.5 nM) (lanes 4–6 and 7–9). <i>C.</i> Molar excess of RECQ1 may compete with Ku70/80 for DNA binding. EMSA was performed to examine the ability of increasing concentration of RECQ1 (0–100 nM) to interact with Ku70/80 (12.5 nM) when bound to linearized plasmid DNA (30 ng) (lane 7 and 8). Arrow indicates the change in band shift pattern of DNA-protein complexes at 50 nM RECQ1. <i>D.</i> RECQ1 and Ku70/80 are co-bound to linear blunt duplex. EMSA reactions were performed using biotinylated DNA probe and the indicated concentration of RECQ1, Ku70/80 or both. The DNA probe was either exposed to a mixture of RECQ1 and Ku70/80, or pre-incubated with one protein followed by addition of the second protein as indicated by parentheses. The DNA-protein complexes were pulled-down on streptavidin magnetic beads and DNA-bound RECQ1 and Ku70/80 were analyzed by Western blotting. Comparable amount of DNA was pull-down in all reactions as shown by agarose gel analyses (bottom). Lane 1 represents biotinylated DNA bound to the streptavidin beads in the absence of RECQ1 or Ku70/80. DNA size marker is shown in leftmost lane of each gel.</p

Efficient Ablation of Genes in Human Hematopoietic Stem and Effector Cells using CRISPR/Cas9.

Genome editing via CRISPR/Cas9 has rapidly become the tool of choice by virtue of its efficacy and ease of use. However, CRISPR/Cas9-mediated genome editing in clinically relevant human somatic cells remains untested. Here, we report CRISPR/Cas9 targeting of two clinically relevant genes, B2M and CCR5, in primary human CD4(+) T cells and CD34(+) hematopoietic stem and progenitor cells (HSPCs). Use of single RNA guides led to highly efficient mutagenesis in HSPCs but not in T cells. A dual guide approach improved gene deletion efficacy in both cell types. HSPCs that had undergone genome editing with CRISPR/Cas9 retained multilineage potential. We examined predicted on- and off-target mutations via target capture sequencing in HSPCs and observed low levels of off-target mutagenesis at only one site. These results demonstrate that CRISPR/Cas9 can efficiently ablate genes in HSPCs with minimal off-target mutagenesis, which could have broad applicability for hematopoietic cell-based therapy