Genes regulated by SATB2 during neurodevelopment contribute to schizophrenia and educational attainment.

SATB2 is associated with schizophrenia and is an important transcription factor regulating neocortical organization and circuitry. Rare mutations in SATB2 cause a syndrome that includes developmental delay, and mouse studies identify an important role for SATB2 in learning and memory. Interacting partners BCL11B and GATAD2A are also schizophrenia risk genes indicating that other genes interacting with or are regulated by SATB2 are making a contribution to schizophrenia and cognition. We used data from Satb2 mouse models to generate three gene-sets that contain genes either functionally related to SATB2 or targeted by SATB2 at different stages of development. Each was tested for enrichment using the largest available genome-wide association studies (GWAS) datasets for schizophrenia and educational attainment (EA) and enrichment analysis was also performed for schizophrenia and other neurodevelopmental disorders using data from rare variant sequencing studies. These SATB2 gene-sets were enriched for genes containing common variants associated with schizophrenia and EA, and were enriched for genes containing rare variants reported in studies of schizophrenia, autism and intellectual disability. In the developing cortex, genes targeted by SATB2 based on ChIP-seq data, and functionally affected when SATB2 is not expressed based on differential expression analysis using RNA-seq data, show strong enrichment for genes associated with EA. For genes expressed in the hippocampus or at the synapse, those targeted by SATB2 are more strongly enriched for genes associated EA than gene-sets not targeted by SATB2. This study demonstrates that single gene findings from GWAS can provide important insights to pathobiological processes. In this case we find evidence that genes influenced by SATB2 and involved in synaptic transmission, axon guidance and formation of the corpus callosum are contributing to schizophrenia and cognition.

Cognitive analysis of schizophrenia risk genes that function as epigenetic regulators of gene expression.

Epigenetic mechanisms are an important heritable and dynamic means of regulating various genomic functions, including gene expression, to orchestrate brain development, adult neurogenesis, and synaptic plasticity. These processes when perturbed are thought to contribute to schizophrenia pathophysiology. A core feature of schizophrenia is cognitive dysfunction. For genetic disorders where cognitive impairment is more severe such as intellectual disability, there are a disproportionally high number of genes involved in the epigenetic regulation of gene transcription. Evidence now supports some shared genetic aetiology between schizophrenia and intellectual disability. GWAS have identified 108 chromosomal regions associated with schizophrenia risk that span 350 genes. This study identified genes mapping to those loci that have epigenetic functions, and tested the risk alleles defining those loci for association with cognitive deficits. We developed a list of 350 genes with epigenetic functions and cross-referenced this with the GWAS loci. This identified eight candidate genes: BCL11B, CHD7, EP300, EPC2, GATAD2A, KDM3B, RERE, SATB2. Using a dataset of Irish psychosis cases and controls (n = 1235), the schizophrenia risk SNPs at these loci were tested for effects on IQ, working memory, episodic memory, and attention. Strongest associations were for rs6984242 with both measures of IQ (P = 0.001) and episodic memory (P = 0.007). We link rs6984242 to CHD7 via a long range eQTL. These associations were not replicated in independent samples. Our study highlights that a number of genes mapping to risk loci for schizophrenia may function as epigenetic regulators of gene expression but further studies are required to establish a role for these genes in cognition. © 2016 Wiley Periodicals, Inc.

Msl2 is a novel component of the vertebrate DNA damage response.

hMSL2 (male-specific lethal 2, human) is a RING finger protein with ubiquitin ligase activity. Although it has been shown to target histone H2B at lysine 34 and p53 at lysine 351, suggesting roles in transcription regulation and apoptosis, its function in these and other processes remains poorly defined. To further characterize this protein, we have disrupted the Msl2 gene in chicken DT40 cells. Msl2(-/-) cells are viable, with minor growth defects. Biochemical analysis of the chromatin in these cells revealed aberrations in the levels of several histone modifications involved in DNA damage response pathways. DNA repair assays show that both Msl2(-/-) chicken cells and hMSL2-depleted human cells have defects in non-homologous end joining (NHEJ) repair. DNA damage assays also demonstrate that both Msl2 and hMSL2 proteins are modified and stabilized shortly after induction of DNA damage. Moreover, hMSL2 mediates modification, presumably ubiquitylation, of a key DNA repair mediator 53BP1 at lysine 1690. Similarly, hMSL1 and hMOF (males absent on the first) are modified in the presence of hMSL2 shortly after DNA damage. These data identify a novel role for Msl2/hMSL2 in the cellular response to DNA damage. The kinetics of its stabilization suggests a function early in the NHEJ repair pathway. Moreover, Msl2 plays a role in maintaining normal histone modification profiles, which may also contribute to the DNA damage response.

Core and linker histone modifications involved in the DNA damage response.

The stability of the genome is constantly under attack from both endogenous and exogenous DNA damaging agents. These agents, as well as naturally occurring processes such as DNA replication and recombination can result in DNA double-strand breaks (DSBs). DSBs are potentially lethal and so eukaryotic cells have evolved an elaborate pathway, the DNA damage response, which detects the damage, recruits proteins to the DSBs, activates checkpoints to stall cell cycle progression and ultimately mediates repair of the damaged DNA. As the DSBs occur in the context of chromatin, execution of this response is partly orchestrated through the modification of the DNA-bound histone proteins. These histone modifications include the addition or removal of various chemical groups or small peptides and function to change the chromatin structure or to attract factors involved in the DNA damage response, and as such, are particularly important in the early stages of the DNA damage response. This review will focus on such modifications, the enzymes responsible and also highlights their importance by reporting known roles for these modifications in genome stability and disease.

The histone acetyltransferase hMOF is frequently downregulated in primary breast carcinoma and medulloblastoma and constitutes a biomarker for clinical outcome in medulloblastoma.

Loss of H4 lysine 16 (H4K16) acetylation was shown to be a common feature in human cancer. However, it remained unclear which enzyme is responsible for the loss of this modification. Having recently identified the histone acetyltransferase human MOF (hMOF) to be required for bulk H4K16 acetylation, here we examined the involvement of hMOF expression and H4K16 acetylation in breast cancer and medulloblastoma. Analysis of a recent mRNA expression profiling study in breast cancer (n = 100 cases) and an array-CGH screen in medulloblastomas (n = 102 cases), revealed downregulation in 40% and genomic loss in 11% of cases, respectively. We investigated hMOF protein expression as well as H4K16 acetylation in large series of primary breast carcinomas (n = 298) and primary medulloblastomas (n = 180) by immunohistochemistry. In contrast to nontransformed control tissues, significant fractions of both primary breast carcinomas and medulloblastomas showed markedly reduced hMOF mRNA and protein expression. In addition, hMOF protein expression tightly correlated with acetylation of H4K16 in all tested samples. For medulloblastoma, downregulation of hMOF protein expression was associated with lower survival rates identifying hMOF as an independent prognostic marker for clinical outcome in univariate as well as multivariate analyses.

Reversal of H3K9me2 by a small-molecule inhibitor for the G9a histone methyltransferase.

Histone lysine methylation has important roles in the organization of chromatin domains and the regulation of gene expression. To analyze its function and modulate its activity, we screened for specific inhibitors against histone lysine methyltransferases (HMTases) using recombinant G9a as the target enzyme. From a chemical library comprising 125,000 preselected compounds, seven hits were identified. Of those, one inhibitor, BIX-01294 (diazepin-quinazolin-amine derivative), does not compete with the cofactor S-adenosyl-methionine, and selectively impairs the G9a HMTase and the generation of H3K9me2 in vitro. In cellular assays, transient incubation of several cell lines with BIX-01294 lowers bulk H3K9me2 levels that are restored upon removal of the inhibitor. Importantly, chromatin immunoprecipitation at several G9a target genes demonstrates reversible reduction of promoter-proximal H3K9me2 in inhibitor-treated mouse ES cells and fibroblasts. Our data identify a biologically active HMTase inhibitor that allows for the transient modulation of H3K9me2 marks in mammalian chromatin.

hMOF histone acetyltransferase is required for histone H4 lysine 16 acetylation in mammalian cells.

Reversible histone acetylation plays an important role in regulation of chromatin structure and function. Here, we report that the human orthologue of Drosophila melanogaster MOF, hMOF, is a histone H4 lysine K16-specific acetyltransferase. hMOF is also required for this modification in mammalian cells. Knockdown of hMOF in HeLa and HepG2 cells causes a dramatic reduction of histone H4K16 acetylation as detected by Western blot analysis and mass spectrometric analysis of endogenous histones. We also provide evidence that, similar to the Drosophila dosage compensation system, hMOF and hMSL3 form a complex in mammalian cells. hMOF and hMSL3 small interfering RNA-treated cells also show dramatic nuclear morphological deformations, depicted by a polylobulated nuclear phenotype. Reduction of hMOF protein levels by RNA interference in HeLa cells also leads to accumulation of cells in the G(2) and M phases of the cell cycle. Treatment with specific inhibitors of the DNA damage response pathway reverts the cell cycle arrest caused by a reduction in hMOF protein levels. Furthermore, hMOF-depleted cells show an increased number of phospho-ATM and gammaH2AX foci and have an impaired repair response to ionizing radiation. Taken together, our data show that hMOF is required for histone H4 lysine 16 acetylation in mammalian cells and suggest that hMOF has a role in DNA damage response during cell cycle progression.

Central role of Drosophila SU(VAR)3-9 in histone H3-K9 methylation and heterochromatic gene silencing.

Su(var)3-9 is a dominant modifier of heterochromatin-induced gene silencing. Like its mammalian and Schizosaccharomyces pombe homologues, Su(var) 3-9 encodes a histone methyltransferase (HMTase), which selectively methylates histone H3 at lysine 9 (H3-K9). In Su(var)3-9 null mutants, H3-K9 methylation at chromocentre heterochromatin is strongly reduced, indicating that SU(VAR)3-9 is the major heterochromatin-specific HMTase in Drosophila. SU (VAR)3-9 interacts with the heterochromatin-associated HP1 protein and with another silencing factor, SU(VAR)3-7. Notably, SU(VAR)3-9-HP1 interaction is interdependent and governs distinct localization patterns of both proteins. In Su(var)3-9 null mutants, concentration of HP1 at the chromocentre is nearly lost without affecting HP1 accumulation at the fourth chromosome. By contrast, in HP1 null mutants SU(VAR)3-9 is no longer restricted at heterochromatin but broadly dispersed across the chromosomes. Despite this interdependence, Su(var)3-9 dominates the PEV modifier effects of HP1 and Su(var)3-7 and is also epistatic to the Y chromosome effect on PEV. Finally, the human SUV39H1 gene is able to partially rescue Su(var)3-9 silencing defects. Together, these data indicate a central role for the SU(VAR)3-9 HMTase in heterochromatin-induced gene silencing in Drosophila.