Enhancer-associated H3K4 methylation safeguards in vitro germline competence.

Germline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naïve pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1-deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions.

Orphan CpG islands amplify poised enhancer regulatory activity and determine target gene responsiveness.

CpG islands (CGIs) represent a widespread feature of vertebrate genomes, being associated with ~70% of all gene promoters. CGIs control transcription initiation by conferring nearby promoters with unique chromatin properties. In addition, there are thousands of distal or orphan CGIs (oCGIs) whose functional relevance is barely known. Here we show that oCGIs are an essential component of poised enhancers that augment their long-range regulatory activity and control the responsiveness of their target genes. Using a knock-in strategy in mouse embryonic stem cells, we introduced poised enhancers with or without oCGIs within topologically associating domains harboring genes with different types of promoters. Analysis of the resulting cell lines revealed that oCGIs act as tethering elements that promote the physical and functional communication between poised enhancers and distally located genes, particularly those with large CGI clusters in their promoters. Therefore, by acting as genetic determinants of gene-enhancer compatibility, CGIs can contribute to gene expression control under both physiological and potentially pathological conditions.

PRC2 Facilitates the Regulatory Topology Required for Poised Enhancer Function during Pluripotent Stem Cell Differentiation.

Poised enhancers marked by H3K27me3 in pluripotent stem cells have been implicated in the establishment of somatic expression programs during embryonic stem cell (ESC) differentiation. However, the functional relevance and mechanism of action of poised enhancers remain unknown. Using CRISPR/Cas9 technology to engineer precise genetic deletions, we demonstrate that poised enhancers are necessary for the induction of major anterior neural regulators. Interestingly, circularized chromosome conformation capture sequencing (4C-seq) shows that poised enhancers already establish physical interactions with their target genes in ESCs in a polycomb repressive complex 2 (PRC2)-dependent manner. Loss of PRC2 does not activate poised enhancers or induce their putative target genes in undifferentiated ESCs; however, loss of PRC2 in differentiating ESCs severely and specifically compromises the induction of major anterior neural genes representing poised enhancer targets. Overall, our work illuminates an unexpected function for polycomb proteins in facilitating neural induction by endowing major anterior neural loci with a permissive regulatory topology.

Foxd3 Promotes Exit from Naive Pluripotency through Enhancer Decommissioning and Inhibits Germline Specification.

Following implantation, mouse epiblast cells transit from a naive to a primed state in which they are competent for both somatic and primordial germ cell (PGC) specification. Using mouse embryonic stem cells as an in vitro model to study the transcriptional regulatory principles orchestrating peri-implantation development, here we show that the transcription factor Foxd3 is necessary for exit from naive pluripotency and progression to a primed pluripotent state. During this transition, Foxd3 acts as a repressor that dismantles a significant fraction of the naive pluripotency expression program through decommissioning of active enhancers associated with key naive pluripotency and early germline genes. Subsequently, Foxd3 needs to be silenced in primed pluripotent cells to allow re-activation of relevant genes required for proper PGC specification. Our findings therefore uncover a cycle of activation and deactivation of Foxd3 required for exit from naive pluripotency and subsequent PGC specification.

The short non-coding transcriptome of the protozoan parasite Trypanosoma cruzi.

The pathway for RNA interference is widespread in metazoans and participates in numerous cellular tasks, from gene silencing to chromatin remodeling and protection against retrotransposition. The unicellular eukaryote Trypanosoma cruzi is missing the canonical RNAi pathway and is unable to induce RNAi-related processes. To further understand alternative RNA pathways operating in this organism, we have performed deep sequencing and genome-wide analyses of a size-fractioned cDNA library (16-61 nt) from the epimastigote life stage. Deep sequencing generated 582,243 short sequences of which 91% could be aligned with the genome sequence. About 95-98% of the aligned data (depending on the haplotype) corresponded to small RNAs derived from tRNAs, rRNAs, snRNAs and snoRNAs. The largest class consisted of tRNA-derived small RNAs which primarily originated from the 3' end of tRNAs, followed by small RNAs derived from rRNA. The remaining sequences revealed the presence of 92 novel transcribed loci, of which 79 did not show homology to known RNA classes.

Histone acetylation and methylation at sites initiating divergent polycistronic transcription in Trypanosoma cruzi.

Trypanosomes are ancient eukaryotic parasites in which the protein-coding genes, organized in large polycistronic clusters on both strands, are transcribed from as yet unidentified promoters. In an effort to reveal transcriptional initiation sites, we examined the Trypanosoma cruzi genome for histone modification patterns shown to be linked to active genes in various organisms. Here, we show that acetylated and methylated histones were found to be enriched at strand switch regions of divergent gene arrays, not at convergent clusters or intra- and intergenic regions within clusters. The modified region showed a bimodular profile with two peaks centered over the 5'-regions of the gene pair flanking the strand switch region. This pattern, which demarcates polycistronic transcription units originating from bidirectional initiation sites, is likely to be common in kinetoplastid parasites as well as in other organisms with polycistronic transcription. In contrast, no acetylation was found at promoters of the highly expressed rRNA and spliced leader genes or satellite DNA or at tested retrotransposonal elements. These results reveal, for the first time, the presence of specific epigenetic marks in T. cruzi with potential implications for transcriptional regulation; they indicate that both histone modifications and bidirectional transcription are evolutionarily conserved.

Characterization of a Trypanosoma cruzi acetyltransferase: cellular location, activity and structure.

Trypanosomatids are widespread parasites that cause three major tropical diseases. In trypanosomatids, as in most other organisms, acetylation is a common protein modification that is important in multiple, diverse processes. This paper describes a new member of the Trypanosoma cruzi acetyltransferase family. The gene is single copy and orthologs are also present in the other two sequenced trypanosomatids, Trypanosoma brucei and Leishmania major. This protein (TcAT-1) has the essential motifs present in members of the GCN5-related acetyltransferase (GNAT) family, as well as an additional motif also found in some enzymes from plant and animal species. The protein is evolutionarily more closely related to this group of enzymes than to histone acetyltransferases. The native protein has a cytosolic cellular location and is present in all three life-cycle stages of the parasite. The recombinant protein was shown to have autoacetylation enzymatic activity.