Epigenome comparisons reveal linkage between gene expression and postnatal remodeling of chromatin domain topology.

Substantial evidence has accumulated linking epigenome change to alterations in stem cell function during postnatal development and aging. Yet much remains to be learned about causal relationships, and large gaps remain in our understanding of epigenome-transcriptome interactions. Here we investigate structural features of large histone H3K27me3-enriched regions in human stem cell-like monocytes and their dendritic cell derivatives, where the H3K27me3 modification is considered to demarcate Polycomb (PcG) domains. Both differentiation- and postnatal development-related change are explored, initially by confirming expected reciprocal relationships between transcript abundance and span of PcG domains overlapping transcribed regions. PcG-associated postnatal transcriptome change specific to the stem cell-like monocytes is found to be incompletely explained by conventional measures of PcG region structure. To address this, we introduce algorithms that quantify local nucleosome-scale conservation of PcG-region topology. It is shown that topology-based comparisons can reveal broad statistical linkage between postnatal gene down-regulation and epigenome remodeling; further, such comparisons provide access to a previously unexplored dimension of epigenome architecture.

Epigenetic regulation of hematopoiesis by DNA methylation.

During embryonic development, cell type-specific transcription factors promote cell identities, while epigenetic modifications are thought to contribute to maintain these cell fates. Our understanding of how genetic and epigenetic modes of regulation work together to establish and maintain cellular identity is still limited, however. Here, we show that DNA methyltransferase 3bb.1 (dnmt3bb.1) is essential for maintenance of hematopoietic stem and progenitor cell (HSPC) fate as part of an early Notch-runx1-cmyb HSPC specification pathway in the zebrafish. Dnmt3bb.1 is expressed in HSPC downstream from Notch1 and runx1, and loss of Dnmt3bb.1 activity leads to reduced cmyb locus methylation, reduced cmyb expression, and gradual reduction in HSPCs. Ectopic overexpression of dnmt3bb.1 in non-hematopoietic cells is sufficient to methylate the cmyb locus, promote cmyb expression, and promote hematopoietic development. Our results reveal an epigenetic mechanism supporting the maintenance of hematopoietic cell fate via DNA methylation-mediated perdurance of a key transcription factor in HSPCs.

DNA methylome and transcriptome sequencing in human ovarian granulosa cells links age-related changes in gene expression to gene body methylation and 3'-end GC density.

Diminished ovarian function occurs early and is a primary cause for age-related decline in female fertility; however, its underlying mechanism remains unclear. This study investigated the roles that genome and epigenome structure play in age-related changes in gene expression and ovarian function, using human ovarian granulosa cells as an experimental system. DNA methylomes were compared between two groups of women with distinct age-related differences in ovarian functions, using both Methylated DNA Capture followed by Next Generation Sequencing (MethylCap-seq) and Reduced Representation Bisulfite Sequencing (RRBS); their transcriptomes were investigated using mRNA-seq. Significant, non-random changes in transcriptome and DNA methylome features are observed in human ovarian granulosa cells as women age and their ovarian functions deteriorate. The strongest correlations between methylation and the age-related changes in gene expression are not confined to the promoter region; rather, high densities of hypomethylated CpG-rich regions spanning the gene body are preferentially associated with gene down-regulation. This association is further enhanced where CpG regions are localized near the 3'-end of the gene. Such features characterize several genes crucial in age-related decline in ovarian function, most notably the AMH (Anti-Müllerian Hormone) gene. The genome-wide correlation between the density of hypomethylated intragenic and 3'-end regions and gene expression suggests previously unexplored mechanisms linking epigenome structure to age-related physiology and pathology.

Postnatal development- and age-related changes in DNA-methylation patterns in the human genome.

Alterations in DNA methylation have been reported to occur during development and aging; however, much remains to be learned regarding post-natal and age-associated epigenome dynamics, and few if any investigations have compared human methylome patterns on a whole genome basis in cells from newborns and adults. The aim of this study was to reveal genomic regions with distinct structure and sequence characteristics that render them subject to dynamic post-natal developmental remodeling or age-related dysregulation of epigenome structure. DNA samples derived from peripheral blood monocytes and in vitro differentiated dendritic cells were analyzed by methylated DNA Immunoprecipitation (MeDIP) or, for selected loci, bisulfite modification, followed by next generation sequencing. Regions of interest that emerged from the analysis included tandem or interspersed-tandem gene sequence repeats (PCDHG, FAM90A, HRNR, ECEL1P2), and genes with strong homology to other family members elsewhere in the genome (FZD1, FZD7 and FGF17). Our results raise the possibility that selected gene sequences with highly homologous copies may serve to facilitate, perhaps even provide a clock-like function for, developmental and age-related epigenome remodeling. If so, this would represent a fundamental feature of genome architecture in higher eukaryotic organisms.

Developmental and epigenetic regulation of the human TLR3 gene.

The receptor encoded by the human TLR3 gene recognizes double-strand RNAs (dsRNAs) associated with viral infection. TLR3 expression is strongly activated upon differentiation of monocytes to dendritic cells, and can be further stimulated by the dsRNA analog polyinosine:polycytosine (PI:C). We report evidence for developmental regulation of the TLR3 gene. In dendritic cells derived from cord blood, both differentiation- and PI:C-associated TLR3 transcriptional activation are impaired as compared to cells from adults. Consistent with relative expression patterns, chromatin states and remodeling differ between newborn and adult samples. TLR3 expression in newborn dendritic cells exhibits heterocellularity and allelic imbalance (skewing), features characteristic of cis-acting epigenetic control. These findings reveal a new source for variability in innate immune system function and provide a model for further study of perinatal epigenetic transitions during development.

Mapping development-related and age-related chromatin remodeling by a high throughput ChIP-HPLC approach.

Common to numerous differentiation pathways in vertebrate organisms is the regulation of key genes through epigenetic mechanisms. Less well studied is to what extent cells of a given differentiation state, but examined at different points within the life history of an organism, are distinct at the level of the epigenome. A few instances of such variation have been reported, and it would be of considerable value to have at hand a means to characterize additional examples more efficiently. We describe an integrated approach to this task, and further present evidence for regions of age-related histone H4 acetylation change extending over tens to hundreds of kilobases. Broad similarity between two distinct regions of such change suggests a previously unsuspected link between developmental programs and aging.

Semirandom sampling to detect differentiation-related and age-related epigenome remodeling.

With completion of the human genome project, patterns of higher order chromatin structure can be easily related to other features of genome organization. A well-studied aspect of chromatin, histone H4 acetylation, is examined here on the basis of its role in setting competence for gene activation. Three applications of a new hybrid genome sampling-chromatin immunoprecipitation strategy are described. The first explores aspects of epigenome architecture in human fibroblasts. A second focuses on chromatin from HL-60 promyelocytic leukemia cells before and after differentiation into macrophage-like cells. A third application explores age-related epigenome change. In the latter, acetylation patterns are compared in human skin fibroblast chromatin from donors of various ages. Two sites are reported at which observed histone H4 acetylation differences suggest decreasing acetylation over time. The sites, located in chromosome 4p16.1 and 4q35.2 regions, appear to remodel during late fetal-early child development and from preadolescence through adult life, respectively.