Epigenomic programming in early fetal brain development.

Aim: To provide a comprehensive understanding of gene regulatory networks in the developing human brain and a foundation for interpreting pathogenic deregulation. Materials & methods: We generated reference epigenomes and transcriptomes of dissected brain regions and primary neural progenitor cells (NPCs) derived from cortical and ganglionic eminence tissues of four normal human fetuses. Results: Integration of these data across developmental stages revealed a directional increase in active regulatory states, transcription factor activities and gene transcription with developmental stage. Consistent with differences in their biology, NPCs derived from cortical and ganglionic eminence regions contained common, region specific, and gestational week specific regulatory states. Conclusion: We provide a high-resolution regulatory network for NPCs from different brain regions as a comprehensive reference for future studies.

Rapid and accurate species identification for ecological studies and monitoring using CRISPR-based SHERLOCK.

One of the most fundamental aspects of ecological research and monitoring is accurate species identification, but cryptic speciation and observer error can confound phenotype-based identification. The CRISPR-Cas toolkit has facilitated remarkable advances in many scientific disciplines, but the fields of ecology and conservation biology have yet to fully embrace this powerful technology. The recently developed CRISPR-Cas13a platform SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) enables highly accurate taxonomic identification and has all the characteristics needed to transition to ecological and environmental disciplines. Here we conducted a series of "proof of principle" experiments to characterize SHERLOCK's ability to accurately, sensitively and rapidly distinguish three fish species of management interest co-occurring in the San Francisco Estuary that are easily misidentified in the field. We improved SHERLOCK's ease of field deployment by combining the previously demonstrated rapid isothermal amplification and CRISPR genetic identification with a minimally invasive and extraction-free DNA collection protocol, as well as the option of instrument-free lateral flow detection. This approach opens the door for redefining how, where and by whom genetic identifications occur in the future.

Discovery of Isoxazole Amides as Potent and Selective SMYD3 Inhibitors.

We report herein the discovery of isoxazole amides as potent and selective SET and MYND Domain-Containing Protein 3 (SMYD3) inhibitors. Elucidation of the structure-activity relationship of the high-throughput screening (HTS) lead compound 1 provided potent and selective SMYD3 inhibitors. The SAR optimization, cocrystal structures of small molecules with SMYD3, and mode of inhibition (MOI) characterization of compounds are described. The synthesis and biological and pharmacokinetic profiles of compounds are also presented.

Non-invasive genetic monitoring for the threatened valley elderberry longhorn beetle.

The valley elderberry longhorn beetle (VELB), Desmocerus californicus dimorphus (Coleoptera: Cerambycidae), is a federally threatened subspecies endemic to the Central Valley of California. The VELB range partially overlaps with that of its morphologically similar sister taxon, the California elderberry longhorn beetle (CELB), Desmocerus californicus californicus (Coleoptera: Cerambycidae). Current surveying methods are limited to visual identification of larval exit holes in the VELB/CELB host plant, elderberry (Sambucus spp.), into which larvae bore and excavate feeding galleries. Unbiased genetic approaches could provide a much-needed complementary approach that has more precision than relying on visual inspection of exit holes. In this study we developed a DNA sequencing-based method for indirect detection of VELB/CELB from frass (insect fecal matter), which can be easily and non-invasively collected from exit holes. Frass samples were collected from 37 locations and the 12S and 16S mitochondrial genes were partially sequenced using nested PCR amplification. Three frass-derived sequences showed 100% sequence identity to VELB/CELB barcode references from museum specimens sequenced for this study. Database queries of frass-derived sequences also revealed high similarity to common occupants of old VELB feeding galleries, including earwigs, flies, and other beetles. Overall, this non-invasive approach is a first step towards a genetic assay that could augment existing VELB monitoring and accurately discriminate between VELB, CELB, and other insects. Furthermore, a phylogenetic analysis of 12S and 16S data from museum specimens revealed evidence for the existence of a previously unrecognized, genetically distinct CELB subpopulation in southern California.

Anti-tumor Activity of the Type I PRMT Inhibitor, GSK3368715, Synergizes with PRMT5 Inhibition through MTAP Loss.

Type I protein arginine methyltransferases (PRMTs) catalyze asymmetric dimethylation of arginines on proteins. Type I PRMTs and their substrates have been implicated in human cancers, suggesting inhibition of type I PRMTs may offer a therapeutic approach for oncology. The current report describes GSK3368715 (EPZ019997), a potent, reversible type I PRMT inhibitor with anti-tumor effects in human cancer models. Inhibition of PRMT5, the predominant type II PRMT, produces synergistic cancer cell growth inhibition when combined with GSK3368715. Interestingly, deletion of the methylthioadenosine phosphorylase gene (MTAP) results in accumulation of the metabolite 2-methylthioadenosine, an endogenous inhibitor of PRMT5, and correlates with sensitivity to GSK3368715 in cell lines. These data provide rationale to explore MTAP status as a biomarker strategy for patient selection.

A High-Throughput Dose-Response Cellular Thermal Shift Assay for Rapid Screening of Drug Target Engagement in Living Cells, Exemplified Using SMYD3 and IDO1.

A persistent problem in early small-molecule drug discovery is the frequent lack of rank-order correlation between biochemical potencies derived from initial screens using purified proteins and the diminished potency and efficacy observed in subsequent disease-relevant cellular phenotypic assays. The introduction of the cellular thermal shift assay (CETSA) has bridged this gap by enabling assessment of drug target engagement directly in live cells based on ligand-induced changes in protein thermal stability. Initial success in applying CETSA across multiple drug target classes motivated our investigation into replacing the low-throughput, manually intensive Western blot readout with a quantitative, automated higher-throughput assay that would provide sufficient capacity to use CETSA as a primary hit qualification strategy. We introduce a high-throughput dose-response cellular thermal shift assay (HTDR-CETSA), a single-pot homogenous assay adapted for high-density microtiter plate format. The assay features titratable BacMam expression of full-length target proteins fused to the DiscoverX 42 amino acid ePL tag in HeLa suspension cells, facilitating enzyme fragment complementation-based chemiluminescent quantification of ligand-stabilized soluble protein. This simplified format can accommodate determination of full-dose CETSA curves for hundreds of individual compounds/analyst/day in replicates. HTDR-CETSA data generated for substrate site and alternate binding mode inhibitors of the histone-lysine N-methyltransferase SMYD3 in HeLa suspension cells demonstrate excellent correlation with rank-order potencies observed in cellular mechanistic assays and direct translation to target engagement of endogenous Smyd3 in cancer-relevant cell lines. We envision this workflow to be generically applicable to HTDR-CETSA screening spanning a wide variety of soluble intracellular protein target classes.

Migration-related phenotypic divergence is associated with epigenetic modifications in rainbow trout.

Migration is essential for the reproduction and survival of many animals, yet little is understood about its underlying molecular mechanisms. We used the salmonid Oncorhynchus mykiss to gain mechanistic insight into smoltification, which is a morphological, physiological and behavioural transition undertaken by juveniles in preparation for seaward migration. O. mykiss is experimentally tractable and displays intra- and interpopulation variation in migration propensity. Migratory individuals can produce nonmigratory progeny and vice versa, indicating a high degree of phenotypic plasticity. One potential way that phenotypic plasticity might be linked to variation in migration-related life history tactics is through epigenetic regulation of gene expression. To explore this, we quantitatively measured genome-scale DNA methylation in fin tissue using reduced representation bisulphite sequencing of F2 siblings produced from a cross between steelhead (migratory) and rainbow trout (nonmigratory) lines. We identified 57 differentially methylated regions (DMRs) between smolt and resident O. mykiss juveniles. DMRs were high in magnitude, with up to 62% differential methylation between life history types, and over half of the gene-associated DMRs were in transcriptional regulatory regions. Many of the DMRs encode proteins with activity relevant to migration-related transitions (e.g. circadian rhythm pathway, nervous system development, protein kinase activity). This study provides the first evidence of a relationship between epigenetic variation and life history divergence associated with migration-related traits in any species.

Cancer. The transcription factor GABP selectively binds and activates the mutant TERT promoter in cancer.

Reactivation of telomerase reverse transcriptase (TERT) expression enables cells to overcome replicative senescence and escape apoptosis, which are fundamental steps in the initiation of human cancer. Multiple cancer types, including up to 83% of glioblastomas (GBMs), harbor highly recurrent TERT promoter mutations of unknown function but specific to two nucleotide positions. We identified the functional consequence of these mutations in GBMs to be recruitment of the multimeric GA-binding protein (GABP) transcription factor specifically to the mutant promoter. Allelic recruitment of GABP is consistently observed across four cancer types, highlighting a shared mechanism underlying TERT reactivation. Tandem flanking native E26 transformation-specific motifs critically cooperate with these mutations to activate TERT, probably by facilitating GABP heterotetramer binding. GABP thus directly links TERT promoter mutations to aberrant expression in multiple cancers.

Epigenetic and transcriptional determinants of the human breast.

While significant effort has been dedicated to the characterization of epigenetic changes associated with prenatal differentiation, relatively little is known about the epigenetic changes that accompany post-natal differentiation where fully functional differentiated cell types with limited lifespans arise. Here we sought to address this gap by generating epigenomic and transcriptional profiles from primary human breast cell types isolated from disease-free human subjects. From these data we define a comprehensive human breast transcriptional network, including a set of myoepithelial- and luminal epithelial-specific intronic retention events. Intersection of epigenetic states with RNA expression from distinct breast epithelium lineages demonstrates that mCpG provides a stable record of exonic and intronic usage, whereas H3K36me3 is dynamic. We find a striking asymmetry in epigenomic reprogramming between luminal and myoepithelial cell types, with the genomes of luminal cells harbouring more than twice the number of hypomethylated enhancer elements compared with myoepithelial cells.

Recurrent epimutations activate gene body promoters in primary glioblastoma.

Aberrant DNA hypomethylation may play an important role in the growth rate of glioblastoma (GBM), but the functional impact on transcription remains poorly understood. We assayed the GBM methylome with MeDIP-seq and MRE-seq, adjusting for copy number differences, in a small set of non-glioma CpG island methylator phenotype (non-G-CIMP) primary tumors. Recurrent hypomethylated loci were enriched within a region of chromosome 5p15 that is specified as a cancer amplicon and also encompasses TERT, encoding telomerase reverse transcriptase, which plays a critical role in tumorigenesis. Overall, 76 gene body promoters were recurrently hypomethylated, including TERT and the oncogenes GLI3 and TP73. Recurring hypomethylation also affected previously unannotated alternative promoters, and luciferase reporter assays for three of four of these promoters confirmed strong promoter activity in GBM cells. Histone H3 lysine 4 trimethylation (H3K4me3) ChIP-seq on tissue from the GBMs uncovered peaks that coincide precisely with tumor-specific decrease of DNA methylation at 200 loci, 133 of which are in gene bodies. Detailed investigation of TP73 and TERT gene body hypomethylation demonstrated increased expression of corresponding alternate transcripts, which in TP73 encodes a truncated p73 protein with oncogenic function and in TERT encodes a putative reverse transcriptase-null protein. Our findings suggest that recurring gene body promoter hypomethylation events, along with histone H3K4 trimethylation, alter the transcriptional landscape of GBM through the activation of a limited number of normally silenced promoters within gene bodies, in at least one case leading to expression of an oncogenic protein.

Functional DNA methylation differences between tissues, cell types, and across individuals discovered using the M&M algorithm.

DNA methylation plays key roles in diverse biological processes such as X chromosome inactivation, transposable element repression, genomic imprinting, and tissue-specific gene expression. Sequencing-based DNA methylation profiling provides an unprecedented opportunity to map and compare complete DNA methylomes. This includes one of the most widely applied technologies for measuring DNA methylation: methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq), coupled with a complementary method, methylation-sensitive restriction enzyme sequencing (MRE-seq). A computational approach that integrates data from these two different but complementary assays and predicts methylation differences between samples has been unavailable. Here, we present a novel integrative statistical framework M&M (for integration of MeDIP-seq and MRE-seq) that dynamically scales, normalizes, and combines MeDIP-seq and MRE-seq data to detect differentially methylated regions. Using sample-matched whole-genome bisulfite sequencing (WGBS) as a gold standard, we demonstrate superior accuracy and reproducibility of M&M compared to existing analytical methods for MeDIP-seq data alone. M&M leverages the complementary nature of MeDIP-seq and MRE-seq data to allow rapid comparative analysis between whole methylomes at a fraction of the cost of WGBS. Comprehensive analysis of nineteen human DNA methylomes with M&M reveals distinct DNA methylation patterns among different tissue types, cell types, and individuals, potentially underscoring divergent epigenetic regulation at different scales of phenotypic diversity. We find that differential DNA methylation at enhancer elements, with concurrent changes in histone modifications and transcription factor binding, is common at the cell, tissue, and individual levels, whereas promoter methylation is more prominent in reinforcing fundamental tissue identities.

Methods for cancer epigenome analysis.

Accurate detection of epimutations in tumor cells is crucial for -understanding the molecular pathogenesis of cancer. Alterations in DNA methylation in cancer are functionally important and clinically relevant, but even this well-studied area is continually re-evaluated in light of unanticipated results, such as the strong association between aberrant DNA methylation in adult tumors and polycomb group profiles in embryonic stem cells, cancer-associated genetic mutations in epigenetic regulators such as DNMT3A and TET family genes, and the discovery of altered 5-hydroxymethylcytosine, a product of TET proteins acting on 5-methylcytosine, in human tumors with TET mutations. The abundance and distribution of covalent histone modifications in primary cancer tissues relative to normal cells is an important but largely uncharted area, although there is good evidence for a mechanistic role of cancer-specific alterations in histone modifications in tumor etiology, drug response, and tumor progression. Meanwhile, the discovery of new epigenetic marks continues, and there are many useful methods for epigenome analysis applicable to primary tumor samples, in addition to cancer cell lines. For DNA methylation and hydroxymethylation, next-generation sequencing allows increasingly inexpensive and quantitative whole-genome profiling. Similarly, the refinement and maturation of chromatin immunoprecipitation with next-generation sequencing (ChIP-seq) has made possible genome-wide mapping of histone modifications, open chromatin, and transcription factor binding sites. Computational tools have been developed apace with these epigenome methods to better enable accurate interpretation of the profiling data.

Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications.

Analysis of DNA methylation patterns relies increasingly on sequencing-based profiling methods. The four most frequently used sequencing-based technologies are the bisulfite-based methods MethylC-seq and reduced representation bisulfite sequencing (RRBS), and the enrichment-based techniques methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylated DNA binding domain sequencing (MBD-seq). We applied all four methods to biological replicates of human embryonic stem cells to assess their genome-wide CpG coverage, resolution, cost, concordance and the influence of CpG density and genomic context. The methylation levels assessed by the two bisulfite methods were concordant (their difference did not exceed a given threshold) for 82% for CpGs and 99% of the non-CpG cytosines. Using binary methylation calls, the two enrichment methods were 99% concordant and regions assessed by all four methods were 97% concordant. We combined MeDIP-seq with methylation-sensitive restriction enzyme (MRE-seq) sequencing for comprehensive methylome coverage at lower cost. This, along with RNA-seq and ChIP-seq of the ES cells enabled us to detect regions with allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression.

Genome-scale DNA methylation analysis.

The haploid human genome contains approximately 29 million CpGs that exist in a methylated, hydroxymethylated or unmethylated state, collectively referred to as the DNA methylome. The methylation status of cytosines in CpGs and occasionally in non-CpG cytosines influences protein­DNA interactions, gene expression, and chromatin structure and stability. The degree of DNA methylation at particular loci may be heritable transgenerationally and may be altered by environmental exposures and diet, potentially contributing to the development of human diseases. For the vast majority of normal and disease methylomes however, less than 1% of the CpGs have been assessed, revealing the formative stage of methylation mapping techniques. Thus, there is significant discovery potential in new genome-scale platforms applied to methylome mapping, particularly oligonucleotide arrays and the transformative technology of next-generation sequencing. Here, we outline the currently used methylation detection reagents and their application to microarray and sequencing platforms. A comparison of the emerging methods is presented, highlighting their degrees of technical complexity, methylome coverage and precision in resolving methylation. Because there are hundreds of unique methylomes to map within one individual and interindividual variation is likely to be significant, international coordination is essential to standardize methylome platforms and to create a full repository of methylome maps from tissues and unique cell types.

Conserved role of intragenic DNA methylation in regulating alternative promoters.

Although it is known that the methylation of DNA in 5' promoters suppresses gene expression, the role of DNA methylation in gene bodies is unclear. In mammals, tissue- and cell type-specific methylation is present in a small percentage of 5' CpG island (CGI) promoters, whereas a far greater proportion occurs across gene bodies, coinciding with highly conserved sequences. Tissue-specific intragenic methylation might reduce, or, paradoxically, enhance transcription elongation efficiency. Capped analysis of gene expression (CAGE) experiments also indicate that transcription commonly initiates within and between genes. To investigate the role of intragenic methylation, we generated a map of DNA methylation from the human brain encompassing 24.7 million of the 28 million CpG sites. From the dense, high-resolution coverage of CpG islands, the majority of methylated CpG islands were shown to be in intragenic and intergenic regions, whereas less than 3% of CpG islands in 5' promoters were methylated. The CpG islands in all three locations overlapped with RNA markers of transcription initiation, and unmethylated CpG islands also overlapped significantly with trimethylation of H3K4, a histone modification enriched at promoters. The general and CpG-island-specific patterns of methylation are conserved in mouse tissues. An in-depth investigation of the human SHANK3 locus and its mouse homologue demonstrated that this tissue-specific DNA methylation regulates intragenic promoter activity in vitro and in vivo. These methylation-regulated, alternative transcripts are expressed in a tissue- and cell type-specific manner, and are expressed differentially within a single cell type from distinct brain regions. These results support a major role for intragenic methylation in regulating cell context-specific alternative promoters in gene bodies.

Molecular epigenetics and genetics in neuro-oncology.

Gliomas arise through genetic and epigenetic alterations of normal brain cells, although the exact cell of origin for each glioma subtype is unknown. The alteration-induced changes in gene expression and protein function allow uncontrolled cell division, tumor expansion, and infiltration into surrounding normal brain parenchyma. The genetic and epigenetic alterations are tumor subtype and tumor-grade specific. Particular alterations predict tumor aggressiveness, tumor response to therapy, and patient survival. Genetic alterations include deletion, gain, amplification, mutation, and translocation, which result in oncogene activation and tumor suppressor gene inactivation, or in some instances the alterations may simply be a consequence of tumorigenesis. Epigenetic alterations in brain tumors include CpG island hypermethylation associated with tumor suppressor gene silencing, gene-specific hypomethylation associated with aberrant gene activation, and genome-wide hypomethylation potentially leading to loss of imprinting, chromosomal instability, and cellular hyperproliferation. Other epigenetic alterations, such as changes in the position of histone variants and changes in histone modifications are also likely to be important in the molecular pathology of brain tumors. Given that histone deacetylases are targets for drugs that are already in clinical trial, surprisingly little is known about histone acetylation in primary brain tumors. Although a majority of epigenetic alterations are independent of genetic alterations, there is interaction on specific genes, signaling pathways and within chromosomal domains. Next-generation sequencing technology is now the method of choice for genomic and epigenome profiling, allowing more comprehensive understanding of genetic and epigenetic contributions to tumorigenesis in the brain.

Epigenetic mechanisms in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is an aggressive and lethal cancer, accounting for the majority of primary brain tumors in adults. GBMs are characterized by genetic alterations large and small, affecting genes that control cell growth, apoptosis, angiogenesis, and invasion. Epigenetic alterations also affect the expression of cancer genes alone, or in combination with genetic mechanisms. For example, in each GBM, hundreds of genes are subject to DNA hypermethylation at their CpG island promoters. A subset of GBMs is also characterized by locus-specific and genome-wide decrease in DNA methylation, or DNA hypomethylation. Other epigenetic alterations, such as changes in the position of histone variants and changes in histone modifications are also likely important in the molecular pathology of GBM, but somewhat surprisingly there are very limited data about these in GBM. Alterations in histone modifications are especially important to understand, given that histone deacetylases are targets for drugs that are in clinical trial for GBMs. The technological wave of next-generation sequencing will accelerate GBM epigenome profiling, allowing the direct integration of DNA methylation, histone modification and gene expression profiles. Ultimately, genomic and epigenomic data should provide new predictive markers of response and lead to more effective therapies for GBM.

Reciprocal co-regulation of EGR2 and MECP2 is disrupted in Rett syndrome and autism.

Mutations in MECP2, encoding methyl-CpG-binding protein 2 (MeCP2), cause the neurodevelopmental disorder Rett syndrome (RTT). Although MECP2 mutations are rare in idiopathic autism, reduced MeCP2 levels are common in autism cortex. MeCP2 is critical for postnatal neuronal maturation and a modulator of activity-dependent genes such as Bdnf (brain-derived neurotropic factor) and JUNB. The activity-dependent early growth response gene 2 (EGR2), required for both early hindbrain development and mature neuronal function, has predicted binding sites in the promoters of several neurologically relevant genes including MECP2. Conversely, MeCP2 family members MBD1, MBD2 and MBD4 bind a methylated CpG island in an enhancer region located in EGR2 intron 1. This study was designed to test the hypothesis that MECP2 and EGR2 regulate each other's expression during neuronal maturation in postnatal brain development. Chromatin immunoprecipitation analysis showed EGR2 binding to the MECP2 promoter and MeCP2 binding to the enhancer region in EGR2 intron 1. Reduction in EGR2 and MeCP2 levels in cultured human neuroblastoma cells by RNA interference reciprocally reduced expression of both EGR2 and MECP2 and their protein products. Consistent with a role of MeCP2 in enhancing EGR2, Mecp2-deficient mouse cortex samples showed significantly reduced EGR2 by quantitative immunofluorescence. Furthermore, MeCP2 and EGR2 show coordinately increased levels during postnatal development of both mouse and human cortex. In contrast to age-matched Controls, RTT and autism postmortem cortex samples showed significant reduction in EGR2. Together, these data support a role of dysregulation of an activity-dependent EGR2/MeCP2 pathway in RTT and autism.

MECP2 promoter methylation and X chromosome inactivation in autism.

Epigenetic mechanisms have been proposed to play a role in the etiology of autism. This hypothesis is supported by the discovery of increased MECP2 promoter methylation associated with decreased MeCP2 protein expression in autism male brain. To further understand the influence of female X chromosome inactivation (XCI) and neighboring methylation patterns on aberrant MECP2 promoter methylation in autism, multiple methylation analyses were peformed on brain and blood samples from individuals with autism. Bisulfite sequencing analyses of a region 0.6 kb upstream of MECP2 in brain DNA samples revealed an abrupt transition from a highly methylated region in both sexes to a region unmethylated in males and subject to XCI in females. Chromatin immunoprecipitation analysis demonstrated that the CCTC-binding factor (CTCF) bound to this transition region in neuronal cells, consistent with a chromatin boundary at the methylation transition. Male autism brain DNA samples displayed a slight increase in methylation in this transition region, suggesting a possible aberrant spreading of methylation into the MECP2 promoter in autism males across this boundary element. In addition, autistic female brain DNA samples showed evidence for aberrant MECP2 promoter methylation as an increase in the number of bisulfite sequenced clones with undefined XCI status for MECP2 but not androgen receptor (AR). To further investigate the specificity of MECP2 methylation alterations in autism, blood DNA samples from females and mothers of males with autism were also examined for XCI skewing at AR, but no significant increase in XCI skewing was observed compared to controls. These results suggest that the aberrant MECP2 methylation in autism brain DNA samples is due to locus-specific rather than global X chromosome methylation changes.

Integrated epigenomic analyses of neuronal MeCP2 reveal a role for long-range interaction with active genes.

Mutations in MECP2 cause the autism-spectrum disorder Rett syndrome. MeCP2 is predicted to bind to methylated promoters and silence transcription. However, the first large-scale mapping of neuronal MeCP2-binding sites on 26.3 Mb of imprinted and nonimprinted loci revealed that 59% of MeCP2-binding sites are outside of genes and that only 6% are in CpG islands. Integrated genome-wide promoter analysis of MeCP2 binding, CpG methylation, and gene expression revealed that 63% of MeCP2-bound promoters are actively expressed and that only 6% are highly methylated. These results indicate that the primary function of MeCP2 is not the silencing of methylated promoters.