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.

Primate and human genomes comprise billions of base pairs, but we are unlikely to gain a deeper understanding of brain functions unique to human (including cognitive abilities and psychiatric diseases) merely by comparing linear DNA sequences. Such determinants of species-specific function might instead be found in the so-called “epigenetic” characteristics of genomic regions; differences in the protein-packaged chromatin state in which genomic DNA exists in the cell. Here, we examine neurons from the prefrontal cortex, a brain region closely associated with the evolution of the primate brain, and identify hundreds of short DNA sequences defined by human-specific changes in chromatin structure and function when compared to non-human primates. These changes included species-specific regulation of methylation marks on the histone proteins around which genomic DNA is wrapped. Sequences subject to human-specific epigenetic regulation showed significant spatial clustering, and despite being separated by hundreds of thousands of base pairs on the linear genome, were in direct physical contact with each other through chromosomal looping and other higher order chromatin features. This observation raises the intriguing possibility that coordinated epigenetic regulation via newly derived chromatin features at gene transcription start sites could play an important role in the emergence of human-specific gene expression networks in the brain. Finally, we identified a strong genetic footprint of hominid evolution in a small subset of transcription start sites defined by human-specific gains in histone methylation, with particularly strong enrichment in prefrontal cortex neurons. For example, the base pair sequence of DPP10 (a gene critically important for normal human brain development) not only showed distinct human-specific changes, but also evidence for more recent selective pressures within the human population.

Here, we combine comparative genomics and population genetics with genome-scale comparisons for histone H3-trimethyl-lysine 4 (H3K4me3), an epigenetic mark sharply regulated at transcription start sites (TSS) and the 5′ end of transcriptional units in brain and other tissues [5] – [8] that is stably maintained in brain specimens collected postmortem [7] , [9] . Our rationale to focus on TSS chromatin was also guided by the observation that the human brain, and in particular the cerebral cortex, shows distinct changes in gene expression, in comparison to other primates [10] . While there is emerging evidence for an important role of small RNAs shaping human-specific brain transcriptomes via posttranscriptional mechanisms [11] and increased recruitment of recently evolved genes during early brain development [12] , the role of TSS and other cis-regulatory mechanisms remains unclear. Here, we report that cell type-specific epigenome mapping in prefrontal cortex (PFC, a type of higher order cortex closely associated with the evolution of the primate brain) revealed hundreds of sequences with human-specific H3K4me3 enrichment in neuronal chromatin, as compared to two other anthropoid primates, the chimpanzee and the macaque. These included multiple sites carrying a strong footprint of hominid evolution, including accelerated nucleotide substitution rates specifically in the human branch of the primate tree, regulatory motifs absent in non-human primates and archaic hominins including Homo neanderthalensis and H. denisova, and evidence for adaptive fixations in modern day humans. The findings presented here provide the first insights into human-specific modifications of the neuronal epigenome, including evidence for coordinated epigenetic regulation of sites separated by megabases of interspersed sequence, which points to a significant intersect between evolutionary changes in TSS function, species-specific chromatin landscapes, and epigenetic inheritance.

Cognitive abilities and psychiatric diseases unique to modern humans could be based on genomic features distinguishing our brain cells, including neurons, from those of other primates. Because protein coding sequences for synaptic and other neuron-specific genes are highly conserved across the primate tree [1] , [2] , a significant portion of hominid evolution could be due to DNA sequence changes involving regulatory and non-coding regions at the 5′ end of genes [3] , [4] . Quantifying these differences, however, is ultimately a daunting task, considering that, for example, the chimpanzee–human genome comparison alone reveals close to 35×10 6 single bp and 5×10 6 multi-bp substitutions and insertion/deletion events [3] . While a large majority of these are likely to reflect genetic drift and are deemed “non-consequential” with respect to fitness, the challenge is to identify the small subset of regulatory sequence alterations impacting brain function and behavior.

Results

H3K4me3 Landscapes across Cell Types and Species The present study focused on the rostral dorsolateral PFC, including cytoarchitectonic Brodmann Area BA10 and the immediately surrounding areas. These brain regions represent a higher association cortex subject to disproportionate morphological expansion during primate evolution [13], and are involved in cognitive operations important for informed choice and creativity [14],[15], among other executive functions. Given that histone methylation in neuronal and non-neuronal chromatin is differentially regulated at thousands of sites genome-wide [7], we avoided chromatin studies in tissue homogenates because glia-to-neuron ratios are 1.4- to 2-fold higher in mature human PFC as compared to chimpanzee and macaque [16]. Instead, we performed cell type-specific epigenome profiling for each of the three primate species, based on NeuN (“neuron nucleus”) antigen-based immunotagging and fluorescence-activated sorting, followed by deep sequencing of H3K4me3-tagged neuronal nucleosomes. Prefrontal H3K4me3 epigenomes from NeuN+ nuclei of 11 humans, including seven children and four adults [7], were compared to four chimpanzees and three macaques of mature age (Table S1). Sample-to-sample comparison, based on a subset of highly conserved Refseq TSS with one mismatch maximum/36bp, consistently revealed the highest correlations between neuronal epigenomes from the same species (Table S2). Strikingly, however, the H3K4me3 landscape in human neurons was much more similar to chimpanzee and macaque neurons, when compared to non-neuronal (NeuN−) cells [7] from the same specimen/donor or to blood (Figure 1A). Therefore, PFC neuronal epigenomes, including their histone methylation landscapes at TSS, carry a species-specific signature, but show an even larger difference when compared to their surrounding glial and other NeuN− cells. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Human-specific signatures of the neuronal epigenome in PFC. (A) Pearson correlation coefficients (R, 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 (n = 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 [19]. (C) The actual co-localization of human-specific H3K4me3 peaks (n = 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 *(**), p<10−3(−4). (D) Representative example of a TSS (PDE4DIP/Myelomegalin (“regulator of brain size”) with species- and cell type-specific H3K4me3 profile. Genome browser tracks showing ChIP-seq H3K4me3 signal at PDE4DIP (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. https://doi.org/10.1371/journal.pbio.1001427.g001

Several Hundred Loci Show Human-Specific Gain, or Loss, of Histone Methylation in PFC Neurons To identify loci with human-specific H3K4me3 enrichment in PFC neurons, we screened 34,639 H3K4me3 peaks that were at least 500 bp long and showed a consistent >2-fold H3K4me3 increase for the 11 humans as compared to the average of the seven chimps and macaques and (ii) minimum length of 500 bp. We identified 410 peaks in the human genome (HG19) with significant enrichment compared to the two non-human primate species (with reads also mapped to HG19) after correcting for false discovery (FDR), and we call these peaks “HP” hereafter for “human-specific peaks” (Figure 1D; Table S3). We had previously reported that infant and child PFC neurons tend to have stronger peaks at numerous loci, compared to the adult [7]. To better age-match the human and non-human primate cohorts, we therefore repeated the analysis with our entire, recently published cohort of nine adult humans without known neurological or psychiatric disease [7],[8]. Using the same set of filter criteria (>2-fold increase in humans compared to chimpanzees and macaques), we identified 425 peaks and 296 of them overlapped with the original 410 HP (Table S3). Furthermore, 345 of the 410 peaks overlapped with the overlapped with the peaks with >1.5-fold increase for nine adult humans (compared to non-human primates; with correction for FDR) (Table S4), indicating that HPs can be detected reliably. To obtain human depleted peaks we used a reciprocal approach where initial peaks were detected in chimpanzee and macaque. For the original cohort of 11 children and adult humans, this resulted in 61 peaks with a significant, at least 2-fold depletion in human PFC neurons (Table S5). 50 peaks defined by human-specific depletion in the mixed cohort of 11 children and adults were part of the total of 177 peaks with >1.5-fold decrease in the cohort of nine adults (compared to each of the two non-human primate species; Table S6). From this, we conclude that at least 471 loci in the genome of PFC neurons show robust human-specific changes (gain, 410; loss, 61) in histone methylation across a very wide postnatal age range. We further explored chimpanzee-specific changes in the H3K4me3 landscape of PFC neurons by comparing human and chimpanzee peaks within the chimpanzee genome. To this end, we constructed a mono-nucleosomal DNA library from chimpanzee PFC to control for input, and mapped the neuronal H3K4me3 datasets from four chimpanzee PFC specimens, and their 11 human counterparts, to the chimpanzee genome (PT2). We identified 551 peaks in the PT2 genome that were subject to >2-fold gain and 337 peaks subject to >2-fold depletion, compared to human regardless of the H3K4me3 level in macaque (Tables S7 and S8). A substantial portion of these PT2-annotated peaks (133 and 40 peaks, respectively) with gain or loss in chimpanzee PFC neurons matched loci with the corresponding, reciprocal changes specific to human PFC neurons in HG19 (410 and 61 peaks as described above). Genetic differences among these genomes and additional, locus-specific differences in nucleosomal organization (leading to differences in background signal in the input libraries) are potential factors that would lead to only partial matching of peaks when species-specific H3K4me3 signals are mapped within the human, or chimpanzee genome, respectively. These findings, taken together, confirm that genome sequence differences in cis are one important factor for the species-specific histone methylation landscapes in PFC neurons.

Human-Specific H3K4me3 Peaks in PFC Neurons Overlap with DNA Methylation Signatures in the Male Germline Both catalytic and non-catalytic subunits of H3K4 methyltransferase complex are associated with transgenerational epigenetic inheritance in the worm, Caenorhabditis elegans, and other simple model organisms [17], and furthermore, H3K4me3 and other epigenetic markings such as DNA cytosine methylation are readily detectable in non-somatic (“germline”-related) cells such as sperm, potentially passing on heritable information to human offspring [18]. Therefore, we wanted to explore whether a subset of the 410 loci with at least 2-fold H3K4me3 enrichment in human neurons are subject to species-specific epigenetic regulation in germ tissue. To this end, we screened a human and chimpanzee sperm database on DNA methylation [19], in order to find out which, if any of the 410 sequences with human-specific H3K4me3 gain in brain overlap with a set of >70,000 sequences defined by very low, or non-detectable DNA methylation in human and chimpanzee sperm (termed (DNA) “hypomethylated regions” in [19]). Of note, the genome-wide distribution of H3K4me3 and DNA cytosine methylation is mutually exclusive in germ and embryonic stem cells, and gains in DNA methylation generally are associated with loss of H3K4me3 in differentiated tissues [20],[21]. Unsurprisingly therefore, 300/410 HP peaks in brain matched a DNA hypomethylated sequence in sperm of both species. Strikingly, however, 90/410, or approximately 22% of HP were selectively (DNA) hypomethylated in human but not in chimpanzee sperm (Table S3), a ratio that is approximately 4-fold higher than the expected 5.7% based on 10,000 simulations (p<0.00001; see also Text S1) (Figure 1B). Conversely, the portion of HP lacking DNA hypomethylation in male germ cells of either species altogether (18/410 or 4%), or with selective hypomethylation in chimpanzee sperm (2/410 or 0.5%), showed a significant, 5-fold underrepresentation in our dataset (Figure 1B). Thus, approximately one-quarter of the 410 loci with human-specific gain in histone methylation in PFC neurons also carry species-specific DNA methylation signatures in sperm, with extremely strong bias towards human (DNA) hypomethylated regions (22%) compared to chimpanzee-specific (DNA) hypomethylated regions (0.5%). In striking contrast, fewer than ten of the 61 loci with human-specific H3K4me3 depletion in PFC neurons showed species-specific differences in sperm DNA methylation between species (six human- and three chimpanzee-specific DNA hypomethylated regions; Table S5).

H3K4 Methylation Sites with Human-Specific Gain Physically Interact in Megabase-Scale Higher Order Chromatin Structures and Provide an Additional Layer for Transcriptional Regulation We noticed that, at numerous chromosomal loci, HP tended to group in pairs or clusters (Table S3). There were more than 245 (163) from the total of 410 HP spaced less than 1 (or 0.5) Mb apart, which is a highly significant, 2- (or 3-) fold enrichment compared to random distribution within the total pool of 34,639 peaks (Figure 1C; Text S1). Therefore, sequences with human-specific gain in H3K4me3 in PFC neurons appear to be co-regulated with neighboring sequences on the same chromosome that are decorated with the same type of histone modification. Likewise, the actual number of human-depleted peaks within one 1 Mb (n = 6) was higher than what is expected from random distribution (n = 2.6), (p = 0.051), albeit no firm conclusions can be drawn due to the smaller sample size (n = 61). This type of non-random distribution due to pairing or clustering of the majority of human-enriched sequences broadly resonates with the recently introduced concept of Mb-sized topological domains as a pervasive feature of genome organization, including increased physical interactions of sequences carrying the same set of epigenetic decorations within a domain [22]. Of note, H3K4 trimethylation of nucleosomes is linked to the RNA polymerase II transcriptional initiation complex, and sharply increased around TSS and broadly correlated with “open chromatin” and gene expression activity [5],[6]. Therefore, we reasoned that a subset of human-enriched “paired” H3K4me3 peaks could engage in chromatin loopings associated with transcriptional regulation. This is a very plausible hypothesis given that promoters and other regulatory sequences involved in transcriptional regulation are often tethered together in loopings and other higher order chromatin [23],[24]. To explore this, we screened a database obtained on chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) for RNA polymerase II, a technique designed to detect chromosomal loopings bound by the Pol II complex [25]. Indeed, we identified at least three interactions that matched to our H3K4me3 peaks with human-specific gain in PFC neurons (Table S9), including a loop interspersed by approximately 2.5 Mb of sequence in chromosome 16p11.2–12.2. This is a risk locus for microdeletions that are linked to a wide spectrum of neurodevelopmental disease including autism spectrum disorder (ASD), intellectual disability (ID), attention deficit hyperactivity disorder (ADHD), seizures, and schizophrenia [26]–[31]. We were able to validate this interaction by chromosome conformation capture (3C), a technique for mapping long range physical interactions between chromatin segments [32], in 2/2 human PFC specimens and also in a human embryonic kidney (HEK) cell line (Figure 2). We conclude that human-specific H3K4me3 peaks spaced as far apart as 1 Mb are potentially co-regulated and physically interact via chromatin loopings and other higher order chromatin structures. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. H3K4me3 landscapes and higher order chromatin at the psychiatric susceptibility locus, 16p11.2. (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. https://doi.org/10.1371/journal.pbio.1001427.g002

Association of Human-Specific H3K4-Methylation Sites with Disease The aforementioned human-specific gains in histone methylation at DPP10 and the emergence of human RNA de novo at this locus could reflect a phylogenetically driven reorganization of neuronal functions that may have contributed not only to the emergence of human-specific executive and social-emotional functions, but also for increased susceptibility for developmental brain disease [42]. In this context, we noticed that the 33 neuHP (which are defined by two criteria which are (i) human-specific gain compared to non-human primates and (ii) high H3K4me3 in PFC neurons but not their surrounding non-neuronal cells) included multiple genes conferring susceptibility to neurological disease. Three loci, including DPP10 on chromosome 2q14.1 and two genes in close proximity on chromosome 3p26.3, CNTN4 and CHL1, both encoding cell adhesion molecules [34],[43]–[45], confer very strong susceptibility to autism, schizophrenia, and related disease. Other disease-associated loci with human-specific gain selectively in PFC neurons include ADCYAP1, a schizophrenia [46],[47] and movement disorder gene [48] that is part of a cAMP-activating pathway also implicated in posttraumatic stress [49]. PDE4DIP (MYOMEGALIN) (Figure 1D) encodes a centrosomal regulator of brain size and neurogenesis [50] that in some studies was 9-fold higher expressed in human as compared to chimpanzee cortex [51],[52]. SORCS1 is implicated in beta amyloid processing and Alzheimer disease [53],[54] and attention deficit hyperactivity disorder [55], which again are considered human-specific neurological conditions [10]. Because four of 33, or 12% of neuHP overlapped with neurodevelopmental susceptibility genes (CNTN4, CHL1, DPP10, SORCS1), we then checked whether the entire set of 410 human-specific peaks is enriched for genes and loci conferring genetic risk for autism, intellectual disability, and related neurological disease with onset in early childhood. However, there was only minimal overlap with the Simons Foundation Autism Research Initiative database (SFARI) [56], and Human unidentified Gene Encoded protein database (HuGE) for pervasive developmental disorder (including autism) associated polymorphism [57], and recent reference lists for mental retardation and/or autism-related genes (each of these databases five or fewer of the human-enriched peaks) [58]. Likewise, there was minimal, and non-significant overlap with the set of 61 human- and 337 chimpanzee-depleted peaks, or the 551 chimpanzee-enriched in PFC neurons (five or fewer of peaks/database). None of the lists of peaks with human- or chimpanzee-specific gain or loss of H3K4me3 revealed statistical significance for any associations with the Gene Ontology (GO) database. We conclude that DNA sequences subject to differential histone methylation in human or chimpanzee PFC neurons are, as a group, not clustered together into specific cellular signaling pathways or functions. Table 1 presents examples of disease-associated genes associated with human-specific gain, or loss of H3K4-trimethylation. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Examples of disease-associated genes with human-specific gain or loss of H3K4 trimethylation in PFC neurons. https://doi.org/10.1371/journal.pbio.1001427.t001

Evolutionary Footprints at Sites Defined by Human-Specific Histone Methylation We then asked whether the subset of DNA sequences with species- and cell type-specific epigenetic regulation, including the neuHP peaks mentioned above carry a strong footprint of hominid evolution. Indeed, nucleotide substitution analysis revealed that both DPP10 peaks DPP10 -1/2, as well as ADCYAP1, CHL1, CNTN4, NRSN2, and SIRPA show a significantly elevated rate, with 2- to 5-fold increase specifically in the human branch of the primate tree, when compared to four other anthropoid primate species (Pan troglodytes, Gorilla gorilla, Pongo abelii, Macaca mulatta) (Table S11). The finding that both DPP10 peaks, DPP10-1 and -2 showed a significant, >4-fold increase in nucleotide substitution rates in the human branch of the primate tree—indicating “co-evolution” (or coordinated loss of constraint)—is very plausible given that chromatin structures surrounding these DNA sequences are in direct physical contact (discussed above), reflecting a potential functional interaction and shared regulatory mechanisms between peaks. To further confirm the role of phylogenetic factors in the emergence of human-specific H3K4me3 peaks, we focused on the set of 33 neuHP and calculated the total number of human-specific sequence alterations (HSAs), in a comparative genome analyses across five primates (H. sapiens, P. troglodytes, G. gorilla, P. abelii, M. mulatta). We recorded altogether 1,519 HSAs, with >90% as single nucleotide substitutions, five >100 bp INDELs, one (Alu) retrotransposon-like element at TRIB3 pseudokinase consistent with a role of mobile elements in primate evolution [3], and gain or loss of hundreds of regulatory motifs (Table S12). When compared to a group of (neuronal) H3K4me3 peaks showing minimal changes between the three primate species (Table S13), the neuHP, as a group, showed a significant, 2.5-fold increase in the number of HSA (20.08±5.52 HSAs versus 8.36±2.44 HSAs per 1-kb sequence, p = 2.4e−06, Wilcoxon rank sum test; Figure S3). The findings further confirm that genetic differences related to speciation indeed could play a major role for changes in the brain's histone methylation landscape, particularly for H3K4me3 peaks that are highly specific for human neurons (neuHP). Interestingly, none of the above loci showed evidence for accelerated evolution of neighboring protein coding sequences (Table S11), reaffirming the view that protein coding sequences for synaptic and other neuron-specific genes are extremely conserved across the primate tree [1],[2]. These DNA sequence alterations at sites of neuron-restricted H3K4me3 peaks (with human-specific gain) point, at least for this subset of loci, to a strong evolutionary footprint before the split of human–chimpanzee lineage several million years ago [3]. Next, we wanted to find out whether there is also evidence for more recent selective pressures at these loci. Indeed, a subset of neuHP contain H. sapiens-specific sequences not only absent in rodents, anthropoid primates, but even in extinct members of the genus homo, including H. neanderthalensis and H. denisova [59]. Some of the ancestral alleles (including MIAT, SIRPA, NRSN) shared with archaic hominins exhibit very low frequencies at 0%–3% in all modern populations, and therefore it remains possible that positive selection for newly derived alleles contributed to their high population frequencies in modern humans (Table S14). However, for the entire set of neuHP that are defined by high H3K4me3 levels in PFC neurons (but not non-neurons), the number of HSAs that emerged after the human lineage was split from H. denisova or H. neanderthalensis were 3.31% and 1.75%, respectively, which is approximately 2-fold lower as compared to 32 control H3K4me3 peaks with minimal differences among the three primate species (5.03% and 3.77%). The 2-fold difference in the number of H. sapiens-specific alleles (neuHP compared to control peaks) showed a strong trend toward significant (p = 0.067) for the Denisova, and reached the level of significance (p = 0.034) for the Neanderthal genome (based on permutation test with 10,000 simulations [60]). Taken together, these results suggest that at least a subset of the TSS regions with H3K4me3 enrichment in human (compared to non-human primates) were exposed to evolutionary driven DNA sequence changes on a lineage of the common ancestor of H. sapiens and the archaic hominins, but subsequently were stabilized in more recent human evolution, after splitting from other hominins. To further test whether or not there were recent, perhaps even ongoing selective pressures at loci defined by human-specific gain in H3K4me3 peaks of PFC neurons, we searched for overlap among the peaks in our study with hundreds of candidate regions in the human genome showing evidence of selection during the past 10–100,000 years from other studies. These loci typically extend over several kb, and were identified in several recent studies on the basis of criteria associated with a “selective sweep,” which describes the elimination of genetic variation in sequences surrounding an advantageous mutation while it becomes fixed [61]–[64]. However, screening of the entire set of 410 human gain and 61 human depleted H3K4me3 sequences against nine datasets for putative selection in humans [65] revealed only five loci with evidence for recent sweeps (Table S15). One of these matched to the neuHP on chromosome 2q14.1, corresponding to the second DPP10 (DPP10-2) peak (see above). In independent analyses, using the 1,000 genome database, we further confirmed recent adaptive fixations around DPP10-2 (Table S16), as well as two other loci, POLL and TSPAN4. While it is presently extremely difficult to determine how much of the genome has been affected by positive selection (of note, a recent metanalysis of 21 recent studies using total genomic scans for positive selection using human polymorphism data revealed unexpectedly minimal overlap between studies [65]), we conclude that the overwhelming majority of loci associated with human-specific H3K4me3 gain or loss in PFC neurons (compared to non-human primates) indeed does not show evidence for more recent selective pressures. To provide an example on altered chromatin function due to an alteration in a regulatory DNA sequence that occurred after the human lineage split from the common ancestor with non-human primates, we focused on a change in a GATA-1 motif (A/TGATTAG) within a portion of DPP10-2 found in human, within an otherwise deeply conserved sequence across many mammalian lineages (Table S17). Gel shift assays demonstrate that the human-specific sequence harboring the novel GATA-1 site showed much higher affinity to HeLa nuclear protein extracts, compared to the chimpanzee/other mammal sequence (Figure 4C). The emergence of a novel GATA-1 motif at DPP10 is unlikely to reflect a systemic trend because the motif overall was lost, rather than gained in neuHP (10/355 versus 4/375, χ2 p = 0.053). Therefore, evolutionary and highly specific changes in a small subset of regulatory motifs at DPP10 and other loci could potentially result in profound changes in nuclear protein binding at TSS and other regulatory sequences, thereby affecting histone methylation and epigenetic control of gene expression in humans, compared to other mammals including monkeys and great apes. Of note, potentially important changes in chromatin structure and function due to human-specific sequence alterations at a single nucleotide within an otherwise highly conserved mammalian sequence will be difficult to “capture” by comparative genome analyses alone. For example, when the total set of 410 HP was crosschecked against a database of 202 sequences with evidence for human-specific accelerated evolution in loci that are highly conserved between rodent and primate lineages [66], only one of 410 HP matched (Table S15).