Epigenetic layers and players underlying neurodevelopment

Abstract

Epigenetic mechanisms convey information above and beyond the sequence of DNA, so it is predicted that they are critical in the complex regulation of brain development and explain the long-lived effects of environmental cues on pre- and early post-natal brain development. Neurons have a complex epigenetic landscape that changes dynamically with transcriptional activity in early life. Here, we summarize progress in our understanding of the discrete layers of the dynamic methylome, chromatin proteome, noncoding RNAs, chromatin loops, and long-range interactions in neuronal development and maturation. Many neurodevelopmental disorders have genetic alterations in these epigenetic modifications or regulators, and these human genetics lessons have demonstrated the importance of these epigenetic players and the epigenetic layers that transcriptional events lay down in the early brain.

Figure 1. The context of sequence, transcription, and location in DNA methylation

For the purpose of demonstrating the importance of genetic context in interpreting DNA methylation levels, genes are broken down into identifiable parts, with promoters separated as either CpG islands or non-CpG islands (non-CGI), and parts of the “gene body” (defined as the genomic locus from transcription start to end sites) separated as exons, introns, or non-promoter CpG islands. The methylation levels for each of these genomic locations are color-coded according to the heat map shown at the bottom, with red representing the highest methylation levels and white representing the lowest. Grey shading represents unrepresented or unknown categories. Rows with headings labeled in blue represent different subcategories of genes or controlling regions with distinct methylation patterns from other genes in the genome. Rows with headings labeled in purple represent the different whole genome landscape categories of partially methylated domains (PMDs, <70% methylated CpGs) [12], highly methylated domains (HMDs, >70% methylated CpG at non-CGIs) [3], or neuronal HMDs (N-HMDs, HMD in neuronal SH-SY5Y cells, but PMD in IMR90 fibroblasts and placenta) [24].

Figure 2. Chromatin states defined by histone modifications and protein binding

A. A color-coded guide to the chromatin state maps derived from hidden Markov model (HMM) segmentation of histone modifications and CTCF binding sites in non-neuronal human cell lines from the human ENCODE project [36]. Histone methylation and acetylation was analyzed by ChIP-seq using antibodies to the specific modifications and sites listed. Transcript analysis was determined by RNA-seq on the same cell lines. Genes involved in developmental and tissue-specific functions exhibit H3K27 trimethylation (H3K27me3) blocks characteristic of polycomb-repressed genes (dark grey) and have inactive but “poised” bivalent promoter states (purple) of both silent (H3K27me3) and active (H3K4me3) histone modifications in human embryonic stem cells (hESCs). B. Two examples of chromatin state maps combined with PMD state maps in the UCSC Genome Browser for neurologically relevant genes. PMDs mapped from HMM analysis of MethylC-seq data in fibroblast (IMR90) or neuronal (SH-SY5Y) cell lines [3] are shown as black bars, with gaps at CpG islands (green) because they were removed from the PMD analysis. ENCODE tracks (middle) include annotated genes and chromatin state segmentation by HMM color coded as in A. While neuronal tissue was not included in the preliminary ENCODE analysis of chromatin states, a track of “Brain H3K4me3” is shown at the bottom that identifies active genes in human adult brain. Notice the difference in chromatin states between regions covered by tissue-specific PMDs (BDNF, MYOD1, KCNC1, inactive poised promoter, polycomb-repressed, and heterochromatin) compared to genes with HMD in all tissues (METT5D1, KF18A, active promoter and transcribed). Also, notice the difference in chromatin states at the MYOD1 locus in human skeletal muscle myotubule (HSMM) cells compared to embryonic stem cells (H1 hESCs, inactive/poised promoter) and liver (HepG2) or umbilical vein epithelial cells (HUVEC) which have the H3K27me3 marks of polycomb repression. Both MYOD1 and KCNC1 are transcriptionally active in brain (H3K4me3 promoter peaks) and are within a neuronal highly methylated domain (N-HMD, defined as PMD in IMR90 but HMD in SH-SY5Y cells). In contrast, BDNF is inactive but poised for transcriptional activity differentially at different promoters and in cell lines and the alternative BDNF promoters are within a PMD in both SH-SY5Y and IMR90 cells. The nuclear lamina, a heterochromatic protein matrix at the nuclear periphery made of lamin and scaffold proteins, overlaps with both PMDs and light grey “off the map” locations in the chromatin state maps. CTCF (blue) shows multiple distinct sites genome-wide that are both intergenic and close to promoters and ubiquitous as well as tissue-specific.

Figure 3. Noncoding RNA as an epigenetic layer and player

A. LncRNAs can function to demarcate a nuclear subdomain by forming and “RNA cloud” (red), as seen for Xist and Kcnq1ot1, in order to coat chromosomes or genomic regions (blue) and regulate transcription [60]. Loci coated by the RNA cloud are most often silenced via recruitment of repressive chromatin complexes, but RNA clouds may also upregulate transcription [80]. B. LncRNAs (red) also act as molecular decoys either for DNA binding transcription factors (Gas5) or miRNAs (PTENP1). C. LncRNAs can have multiple protein interacting partners and thereby act as a molecular scaffold for larger complexes. D. In recent years a novel role for lncRNAs as epigenetic “players” has been described. Many lncRNAs were found to interact with the repressive chromatin modifier PRC2 [64, 79], and thereby regulate the chromatin structure of large domains, or even of whole chromosomes. Recently, activating lncRNAs were also described that bind to the Mediator complex and facilitate enhancer-promoter loops [3]. This function may potentially overlap with enhancer RNAs (eRNAs) which are transcribed bidirectionally from enhancers [68]. E. RNA also functions as an epigenetic “layer” by modifying the structure of the DNA or regulating sense transcription via antisense transcription. Such a role has been described at the neurodevelopmentally critical PWS/AS locus on chromosome 15q11-q13, where RNA:DNA hybrid (R-loop) formation at the Prader-Willi imprinting control region (PWS-ICR) protects against DNA methylation [8], and processive transcription to produce UBE3A-ATS silences paternal UBE3A in neurons [89]. Similarly, eRNAs may act as molecular signals to mark active enhancers in response to stimulation [68].

Figure 4. The epigenetic layer of chromatin looping is composed of at least three sublayers

A. At the sub-layer of specific transcription units, enhancers and other distal units make contact with gene promoters such as Arc, a neuronal factor required for the regulation of AMPA receptors, via kb scale chromatin loops. Pol II (red) is recruited by enhancers where transcription initiates before transfer by looping to the Arc promoter [68]. B. Chromatin domains up to 1 Mb organize chromosomes into a series of functional units in mammalian cells. The insulating factor CTCF (green) regulates formation and maintenance of this intermediate layer in the HoxA locus and other domains genome wide [96]. C. Long range, multi-megabase chromatin loop interactions regulate gene expression between distant loci above the domain layer. At this layer, the PWS-IC (red) contacts the locus encoding CHRNA7 (blue) via a 7 megabase looping interaction via MeCP2 and potentially CTCF (green) and cohesion (red) [98, 99]. This long-range interaction modulates expression of CHRNA7 and other neurologic genes in 15q11–13 [98].