Human-specific histone methylation signatures at transcription start sites in prefrontal neurons

Cognitive abilities and disorders unique to humans are thought to result from adaptively driven changes in brain transcriptomes, but little is known about the role of cis-regulatory changes affecting transcription start sites (TSS). Here, we mapped in human, chimpanzee, and macaque prefrontal cortex the genome-wide distribution of histone H3 trimethylated at lysine 4 (H3K4me3), an epigenetic mark sharply regulated at TSS, and identified 471 sequences with human-specific enrichment or depletion. Among these were 33 loci selectively methylated in neuronal but not non-neuronal chromatin from children and adults, including TSS at DPP10 (2q14.1), CNTN4 and CHL1 (3p26.3), and other neuropsychiatric susceptibility genes. Regulatory sequences at DPP10 and additional loci carried a strong footprint of hominid adaptation, including elevated nucleotide substitution rates and regulatory motifs absent in other primates (including archaic hominins), with evidence for selective pressures during more recent evolution and adaptive fixations in modern populations. Chromosome conformation capture at two neurodevelopmental disease loci, 2q14.1 and 16p11.2, revealed higher order chromatin structures resulting in physical contact of multiple human-specific H3K4me3 peaks spaced 0.5-1 Mb apart, in conjunction with a novel cis-bound antisense RNA linked to Polycomb repressor proteins and downregulated DPP10 expression. Therefore, coordinated epigenetic regulation via newly derived TSS chromatin could play an important role in the emergence of human-specific gene expression networks in brain that contribute to cognitive functions and neurological disease susceptibility in modern day humans

Comparative Analyses of SUV420H1 Isoforms and SUV420H2 Reveal Differences in Their Cellular Localization and Effects on Myogenic Differentiation

Methylation of histone H4 on lysine 20 plays critical roles in chromatin structure and function via mono- (H4K20me1), di- (H4K20me2), and trimethyl (H4K20me3) derivatives. In previous analyses of histone methylation dynamics in mid-gestation mouse embryos, we documented marked changes in H4K20 methylation during cell differentiation. These changes were particularly robust during myogenesis, both in vivo and in cell culture, where we observed a transition from H4K20me1 to H4K20me3. To assess the significance of this change, we used a gain-of-function strategy involving the lysine methyltransferases SUV420H1 and SUV420H2, which catalyze H4K20me2 and H4K20me3. At the same time, we characterized a second isoform of SUV420H1 (designated SUV420H1_i2) and compared the activity of all three SUV420H proteins with regard to localization and H4K20 methylation.Immunofluorescence revealed that exogenous SUV420H1_i2 was distributed throughout the cell, while a substantial portion of SUV420H1_i1 and SUV420H2 displayed the expected association with constitutive heterochromatin. Moreover, SUV420H1_i2 distribution was unaffected by co-expression of heterochromatin protein-1α, which increased the targeting of SUV420H1_i1 and SUV420H2 to regions of pericentromeric heterochromatin. Consistent with their distributions, SUV420H1_i2 caused an increase in H4K20me3 levels throughout the nucleus, whereas SUV420H1_i1 and SUV420H2 facilitated an increase in pericentric H4K20me3. Striking differences continued when the SUV420H proteins were tested in the C2C12 myogenic model system. Specifically, although SUV420H1_i2 induced precocious appearance of the differentiation marker Myogenin in the presence of mitogens, only SUV420H2 maintained a Myogenin-enriched population over the course of differentiation. Paradoxically, SUV420H1_i1 could not be expressed in C2C12 cells, which suggests it is under post-transcriptional or post-translational control.These data indicate that SUV420H proteins differ substantially in their localization and activity. Importantly, SUV420H2 can induce a transition from H4K20me1 to H4K20me3 in regions of constitutive heterochromatin that is sufficient to enhance myogenic differentiation, suggesting it can act an as epigenetic ‘switch’ in this process

Maternal immune activation alters behavior in adult offspring, with subtle changes in the cortical transcriptome and epigenome

Maternal immune activation during prenatal development, including treatment with the viral RNA mimic, polyriboinosinic–polyribocytidilic acid (poly IC), serves as a widely used animal model to induce behavioral deficits reminiscent of schizophrenia and related disease. Here, we report that massive cytokine activation after a single dose of poly IC in the prenatal period is associated with lasting working memory deficits in adult offspring. To explore whether dysregulated gene expression in cerebral cortex, contributes to cognitive dysfunction, we profiled the cortical transcriptome, and in addition, mapped the genome-wide distribution of trimethylated histone H3-lysine 4 (H3K4me3), an epigenetic mark sharply regulated at the 5′ end of transcriptional units. However, deep sequencing-based H3K4me3 mapping and mRNA profiling by microarray did not reveal significant alterations in mature cerebral cortex after poly IC exposure at embryonic days E17.5 or E12.5. At a small set of genes (including suppressor of cytokine signaling Socs3), H3K4me3 was sensitive to activation of cytokine signaling in primary cultures from fetal forebrain but adult cortex of saline- and poly IC-exposed mice did not show significant differences. A limited set of transcription start sites (TSS), including Disrupted-in-Schizophrenia 1 (Disc1), a schizophrenia risk gene often implicated in gene–environment interaction models, showed altered H3K4me3 after prenatal poly IC but none of these differences survived after correcting for multiple comparisons. We conclude that prenatal poly IC is associated with cognitive deficits later in life, but without robust alterations in epigenetic regulation of gene expression in the cerebral cortex

Epigenetics in the human brain

Many cellular constituents in the human brain permanently exit from the cell cycle during pre- or early postnatal development, but little is known about epigenetic regulation of neuronal and glial epigenomes during maturation and aging, including changes in mood and psychosis spectrum disorders and other cognitive or emotional disease. Here, we summarize the current knowledge base as it pertains to genome organization in the human brain, including the regulation of DNA cytosine methylation and hydroxymethylation, and a subset of (altogether \u3e100) residue-specific histone modifications associated with gene expression, and silencing and various other functional chromatin states. We propose that high-resolution mapping of epigenetic markings in postmortem brain tissue or neural cultures derived from induced pluripotent cells (iPS), in conjunction with transcriptome profiling and whole-genome sequencing, will increasingly be used to define the molecular pathology of specific cases diagnosed with depression, schizophrenia, autism, or other major psychiatric disease. We predict that these highly integrative explorations of genome organization and function will provide an important alternative to conventional approaches in human brain studies, which mainly are aimed at uncovering group effects by diagnosis but generally face limitations because of cohort size

Human-specific signatures of the neuronal epigenome in PFC.

<p>(A) Pearson correlation coefficients (<i>R</i>, mean ± standard deviation [SD]) for sample-to-sample comparison of H3K4me3 ChIP-seq normalized tag counts within Refseq promoters, revealing cell type- and species-specific signatures. (B) Expected (blue)/observed (red) counts of human-specific H3K4me3 peaks (<i>n</i> = 410) overlapping with DNA hypomethylated regions in human (H)/chimpanzee (P) sperm. Notice 4-fold enrichment for loci with human-only (H+,P−) DNA hypomethylation in dataset <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001427#pbio.1001427-Molaro1" target="_blank">[19]</a>. (C) The actual co-localization of human-specific H3K4me3 peaks (<i>n</i> = 410) within 1- or 0.5-Mb genomic distance is 2–3-fold higher than expected (based on average distribution of entire set of 34,639 H3K4me3 peaks *⁽**⁾, <i>p</i><10⁻³⁽⁻⁴⁾. (D) Representative example of a TSS (<i>PDE4DIP/Myelomegalin</i> (“<i>regulator of brain size</i>”) with species- and cell type-specific H3K4me3 profile. Genome browser tracks showing ChIP-seq H3K4me3 signal at <i>PDE4DIP</i> (chromosome 1) locus, annotated to HG19/PT2/RM2 genomes as indicated. Green/blue/black tracks from PFC neuronal (NeuN+) nuclei of 11 humans/four chimpanzees/three macaques as indicated. Red tracks, non-neuronal (NeuN−) human PFC nuclei. Notice much stronger PDE4DIP peaks in human neurons.</p

H3K4me3 landscapes and higher order chromatin at the psychiatric susceptibility locus, 16p11.2.

<p>(Top) UCSC genome browser window track for approximately 1 Mb of human chr16: 21,462,663–22,499,013, with H3K4me3 ChIP-seq tracks from neuronal chromatin (PFC) of three primate species, as indicated. Notice human-enriched H3K4me3 peaks at chr16:21,512,663–21,514,196 and chr16:22,448,157–22,449,013 (marked by arrows) flanking numerous peaks common to all 3 species. (Bottom) Rectangles and thin arrows mark 3C HindIII restriction fragments and primers from 3C assays. Notice positive interaction of sequences captured by primers 2 and 7, agarose gels shows representative 196-bp PCR product for 3C from two PFC specimens (a,b), HEK cells, and no ligase and water controls.</p

Examples of disease-associated genes with human-specific gain or loss of H3K4 trimethylation in PFC neurons.

<p>ADHD, attention deficit hyperactivity disorder; LTD, long-term depression; NGF, nerve growth factor; PTSD, post-traumatic stress disorder.</p

H3K4me3 landscapes and higher order chromatin at <i>DPP10</i> (2q14.1).

<p>(A) (Top) Genome browser tracks showing ChIP-seq H3K4me3 signal at <i>DPP10</i> locus annotated to HG19 and RM2 genomes. Data expressed as normalized tag densities, averaged for 11 humans, four chimpanzees, and three macaques as indicated (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001427#pbio.1001427.s001" target="_blank">Figure S1</a> for comparative annotation for each of the 18 specimens in HG19 at <i>DPP10</i>/2q14.1, and for the non-human primates also for the homologous loci in their respective genomes, PT2 and RM2). Human-specific peak <i>DPP10</i>-1 (1,455 bp) and <i>DPP10</i>-2 (3,808 bp) marked by arrows and shown at higher resolution in boxes, as indicated. (Bottom) Rectangles and arrows mark Hind III restriction fragments and primers from <i>DPP10</i>-1/2 (PK1, 2) and control regions (CR1-3) for 3C assays (human). Dotted lines connect primer pairs with sequence-verified product, indicating physical interaction of the corresponding fragments. Agarose gels for representative PCR products from 3C with (+) or without (−) DNA ligase (human primers 6,17: 282 bp; 6,18: 423 bp; 8,15: 160 bp; 9,15: 130 bp). (B) Rectangles and arrows mark Hind III restriction fragments and primers for corresponding DPP10 sequences in RM2, for macaque brain 3C. Macaque primers 6,12:298 bp, 8,12:154 bp. Notice positive interaction of PK1 with PK2 and neighboring CR2, but with not CR1 or CR3. Notice no signal in PFC 3C assays without DNA ligase and no signal in all 3C assays from H9 pluripotent (H9ESC) and differentiated (DIFF) cell cultures.</p