To gain insight into the function of DNA methylation at cis-regulatory regions and its impact on gene expression, we measured methylation, RNA polymerase occupancy and histone modifications at 16,000 promoters in primary human somatic and germline cells. We find CpG-poor promoters hypermethylated in somatic cells, which does not preclude their activity. This methylation is present in male gametes and results in evolutionary loss of CpG dinucleotides, as measured by divergence between humans and primates. In contrast, strong CpG island promoters are mostly unmethylated, even when inactive. Weak CpG island promoters are distinct, as they are preferential targets for de novo methylation in somatic cells. Notably, most germline-specific genes are methylated in somatic cells, suggesting additional functional selection. These results show that promoter sequence and gene function are major predictors of promoter methylation states. Moreover, we observe that inactive unmethylated CpG island promoters show elevated levels of dimethylation of Lys4 of histone H3, suggesting that this chromatin mark may protect DNA from methylation.

Cytosine methylation is required for mammalian development and is often perturbed in human cancer. To determine how this epigenetic modification is distributed in the genomes of primary and transformed cells, we used an immunocapturing approach followed by DNA microarray analysis to generate methylation profiles of all human chromosomes at 80-kb resolution and for a large set of CpG islands. In primary cells we identified broad genomic regions of differential methylation with higher levels in gene-rich neighborhoods. Female and male cells had indistinguishable profiles for autosomes but differences on the X chromosome. The inactive X chromosome (Xi) was hypermethylated at only a subset of gene-rich regions and, unexpectedly, overall hypomethylated relative to its active counterpart. The chromosomal methylation profile of transformed cells was similar to that of primary cells. Nevertheless, we detected large genomic segments with hypomethylation in the transformed cell residing in gene-poor areas. Furthermore, analysis of 6,000 CpG islands showed that only a small set of promoters was methylated differentially, suggesting that aberrant methylation of CpG island promoters in malignancy might be less frequent than previously hypothesized.

Cellular differentiation entails loss of pluripotency and gain of lineage- and cell-type-specific characteristics. Using a murine system that progresses from stem cells to lineage-committed progenitors to terminally differentiated neurons, we analyzed DNA methylation and Polycomb-mediated histone H3 methylation (H3K27me3). We show that several hundred promoters, including pluripotency and germline-specific genes, become DNA methylated in lineage-committed progenitor cells, suggesting that DNA methylation may already repress pluripotency in progenitor cells. Conversely, we detect loss and acquisition of H3K27me3 at additional targets in both progenitor and terminal states. Surprisingly, many neuron-specific genes that become activated upon terminal differentiation are Polycomb targets only in progenitor cells. Moreover, promoters marked by H3K27me3 in stem cells frequently become DNA methylated during differentiation, suggesting context-dependent crosstalk between Polycomb and DNA methylation. These data suggest a model how de novo DNA methylation and dynamic switches in Polycomb targets restrict pluripotency and define the developmental potential of progenitor cells.

DNA methylation is extensively reprogrammed during the early phases of mammalian development, yet genomic targets of this process are largely unknown. We optimized methylated DNA immunoprecipitation for low numbers of cells and profiled DNA methylation during early development of the mouse embryonic lineage in vivo. We observed a major epigenetic switch during implantation at the transition from the blastocyst to the postimplantation epiblast. During this period, DNA methylation is primarily targeted to repress the germline expression program. DNA methylation in the epiblast is also targeted to promoters of lineage-specific genes such as hematopoietic genes, which are subsequently demethylated during terminal differentiation. De novo methylation during early embryogenesis is catalyzed by Dnmt3b, and absence of DNA methylation leads to ectopic gene activation in the embryo. Finally, we identify nonimprinted genes that inherit promoter DNA methylation from parental gametes, suggesting that escape of post-fertilization DNA methylation reprogramming is prevalent in the mouse genome.

SummaryLike other bacteria, Bacillus subtilis possesses a family of homologous small acidic proteins (CspB, CspC and CspD, identity > 70%) that are strongly induced in response to cold shock. We show that deletion of cspC or cspD genes did not result in a detectable phenotype; in contrast, csp double mutants exhibited severe reduction in cellular growth at 15ЊC as well as at 37ЊC, including impairment of survival during the stationary phase. Two-dimensional gel analysis showed that protein synthesis was deregulated in csp double mutants and that the loss of one or two CSPs led to an increase in the synthesis of the remaining CSP(s) at 37ЊC and after cold shock, suggesting that CSPs down-regulate production of members from this protein family. A cspB/C/D triple mutant (64BCDbt) could only be generated in the presence of cspB in trans on a plasmid that was not lost, in spite of lack of antibiotic pressure, indicating that a minimum of one csp gene is essential for viability of B. subtilis. After cold shock, synthesis of CspB in 64BCDbt was drastically lower than in wild-type cells accompanied by cessation in growth and strong reduction in general protein synthesis. As CspB, CspC and CspD are shown to bind to RNA in a co-operative and interactive manner, CSPs are suggested to function as RNA chaperones facilitating the initiation of translation under optimal and low temperatures.

Epigenetic reprogramming, characterized by loss of cytosine methylation and histone modifications, occurs during mammalian development in primordial germ cells (PGCs), yet the targets and kinetics of this process are poorly characterized. Here we provide a map of cytosine methylation on a large portion of the genome in developing male and female PGCs isolated from mouse embryos. We show that DNA methylation erasure is global and affects genes of various biological functions. We also reveal complex kinetics of demethylation that are initiated at most genes in early PGC precursors around embryonic day 8.0-9.0. In addition, besides intracisternal A-particles (IAPs), we identify rare LTR-ERV1 retroelements and single-copy sequences that resist global methylation erasure in PGCs as well as in preimplantation embryos. Our data provide important insights into the targets and dynamics of DNA methylation reprogramming in mammalian germ cells.

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Diabetic cardiomyopathy is a complication of type 2 diabetes, with known contributions of lifestyle and genetics. We develop environmentally and genetically driven in vitro models of the condition using human-induced-pluripotent-stem-cell-derived cardiomyocytes. First, we mimic diabetic clinical chemistry to induce a phenotypic surrogate of diabetic cardiomyopathy, observing structural and functional disarray. Next, we consider genetic effects by deriving cardiomyocytes from two diabetic patients with variable disease progression. The cardiomyopathic phenotype is recapitulated in the patient-specific cells basally, with a severity dependent on their original clinical status. These models are incorporated into successive levels of a screening platform, identifying drugs that preserve cardiomyocyte phenotype in vitro during diabetic stress. In this work, we present a patient-specific induced pluripotent stem cell (iPSC) model of a complex metabolic condition, showing the power of this technique for discovery and testing of therapeutic strategies for a disease with ever-increasing clinical significance.