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.

In higher eukaryotes, histone methylation is involved in maintaining cellular identity during somatic development. As most nucleosomes are replaced by protamines during spermatogenesis, it is unclear whether histone modifications function in paternal transmission of epigenetic information. Here we show that two modifications important for Trithorax- and Polycomb-mediated gene regulation have methylation-specific distributions at regulatory regions in human spermatozoa. Histone H3 Lys4 dimethylation (H3K4me2) marks genes that are relevant in spermatogenesis and cellular homeostasis. In contrast, histone H3 Lys27 trimethylation (H3K27me3) marks developmental regulators in sperm, as in somatic cells. However, nucleosomes are only moderately retained at regulatory regions in human sperm. Nonetheless, genes with extensive H3K27me3 coverage around transcriptional start sites in particular tend not to be expressed during male and female gametogenesis or in preimplantation embryos. Promoters of orthologous genes are similarly modified in mouse spermatozoa. These data are compatible with a role for Polycomb in repressing somatic determinants across generations, potentially in a variegating manner.

The adult mouse mammary epithelium contains self-sustained cell lineages that form the inner luminal and outer basal cell layers, with stem and progenitor cells contributing to its proliferative and regenerative potential. A key issue in breast cancer biology is the effect of genomic lesions in specific mammary cell lineages on tumour heterogeneity and progression. The impact of transforming events on fate conversion in cancer cells of origin and thus their contribution to tumour heterogeneity remains largely elusive. Using in situ genetic lineage tracing and limiting dilution transplantation, we have unravelled the potential of PIK3CA(H1047R), one of the most frequent mutations occurring in human breast cancer, to induce multipotency during tumorigenesis in the mammary gland. Here we show that expression of PIK3CA(H1047R) in lineage-committed basal Lgr5-positive and luminal keratin-8-positive cells of the adult mouse mammary gland evokes cell dedifferentiation into a multipotent stem-like state, suggesting this to be a mechanism involved in the formation of heterogeneous, multi-lineage mammary tumours. Moreover, we show that the tumour cell of origin influences the frequency of malignant mammary tumours. Our results define a key effect of PIK3CA(H1047R) on mammary cell fate in the pre-neoplastic mammary gland and show that the cell of origin of PIK3CA(H1047R) tumours dictates their malignancy, thus revealing a mechanism underlying tumour heterogeneity and aggressiveness.

Cellular differentiation entails reprogramming of the transcriptome from a pluripotent to a unipotent fate. This process was suggested to coincide with a global increase of repressive heterochromatin, which results in a reduction of transcriptional plasticity and potential. Here we report the dynamics of the transcriptome and an abundant heterochromatic histone modification, dimethylation of histone H3 at lysine 9 (H3K9me2), during neuronal differentiation of embryonic stem cells. In contrast to the prevailing model, we find H3K9me2 to occupy over 50% of chromosomal regions already in stem cells. Marked are most genomic regions that are devoid of transcription and a subgroup of histone modifications. Importantly, no global increase occurs during differentiation, but discrete local changes of H3K9me2 particularly at genic regions can be detected. Mirroring the cell fate change, many genes show altered expression upon differentiation. Quantitative sequencing of transcripts demonstrates however that the total number of active genes is equal between stem cells and several tested differentiated cell types. Together, these findings reveal high prevalence of a heterochromatic mark in stem cells and challenge the model of low abundance of epigenetic repression and resulting global basal level transcription in stem cells. This suggests that cellular differentiation entails local rather than global changes in epigenetic repression and transcriptional activity.

In mammalian somatic cells, PRC1 and PRC2 proteins are transcriptional repressors that function in large multi-protein complexes and that modify chromatin by monoubiquitinating histone H2A at lysine 119 (H2AK119u1) and tri-methylating H3 at lysine 27 (H3K27me3), respectively 9,12 . At day 12.5 of embryonic development (E12.5), we observed in PGCs marked by Cdh1 (E-cadherin) staining 13 nuclear localization of PRC1 components Rnf2/Ring1b, Mel18/Pcgf2 and Rybp ( Fig. 1a; Supplementary Fig. 1) as well as a robust H2AK119u1 signal suggesting the presence of catalytically active PRC1 complexes (Fig. 1a). To address the function of PRC1 in PGC development ( Supplementary Fig. 2), we conditionally deleted Rnf2 in PGCs from E9.5 onwards by Fig. 3, 6a). This strategy resulted in ~90% deletion efficiency at E11.5 ( Supplementary Fig. 4). At E12.5, Rnf2, Mel18, Rybp and H2AK119u1 were lost in PGCs of Rnf2 cko but not Rnf2 F/Δ embryos indicating that complex stability and catalytic activity of PRC1 is regulated by Rnf2 in PGCs at E12.5 ( Fig. 1a; Supplementary Fig. 4). In contrast, Ezh2 and H3K27me3 levels were similar in Rnf2 cko versus control PGCs suggesting globally unaltered PRC2 function in Rnf2 cko PGCs ( Supplementary Fig. 5).To study the fate of Rnf2-deficient PGCs, we analyzed expression of the pluripotency and germ cell marker Oct4 in whole gonads of E10.5 to E13.5 embryos ( Fig. 1b; Supplementary Fig. 2). We observed a strong reduction of Oct4-positive PGCs specifically in female Rnf2 cko embryos but not in male Rnf2 cko or control embryos, starting around E12.5 of gestation (Fig. 1b, c). In contrast, double deficiency of Ring1 and Rnf2 caused a strong reduction of Oct4-positive PGCs already at E11.5 in both sexes ( Supplementary Fig. 6b, c), indicating an essential role for PRC1 in PGCs after their migration into the embryonic gonad ( Supplementary Fig. 2) 11.To further dissect the role of Rnf2 in regulating Oct4 expression versus PGC development, we assessed in Rnf2 cko embryos co-expression of Oct4 and Rnf2 at E12.5 in Cdh1-positive PGCs (Fig. 1d). The number of PGCs lacking detectable Rnf2 protein was strongly reduced in female but not male gonads, despite that gonads of opposite sexes harbored comparable numbers of Rnf2-deficient PGCs at E12.0 ( Supplementary Fig. 4a, data not shown). We further observed a pronounced downregulation of Oct4 protein in female and some male Rnf2-deficient PGCs (Fig. 1d).These data indicate that Rnf2 contributes to maintaining Oct4 expression particularly in female PGCs beginning between E12.0 and E12.5. At E11.5, gonads still possess the potential to develop either into ovaries or testes while they are committed to a sex-specific differentiation process two days later.To relate aberrant expression in Rnf2 ∆ PGCs to changes occurring during normal PGC differentiation, we profiled expression in Rnf2 + PGCs isolated between E11.5 and E13.5. In this developmental period, 6-fold more genes were up-regulated in female (810) than male (132) Rnf2 + PGCs, with an additional 196 ge...