Bmi1 facilitates primitive endoderm formation by stabilizing Gata6 during early mouse development

International audienceThe transcription factors Nanog and Gata6 are critical to specify the epiblast versus primitive endoderm (PrE) lineages. However, little is known about the mechanisms that regulate the protein stability and activity of these factors in the developing embryo. Here we uncover an early developmental function for the Polycomb group member Bmi1 in supporting PrE lineage formation through Gata6 protein stabilization. We show that Bmi1 is enriched in the extraembryonic (endoderm [XEN] and trophectodermal stem [TS]) compartment and repressed by Nanog in pluripotent embryonic stem (ES) cells. In vivo, Bmi1 overlaps with the nascent Gata6 and Nanog protein from the eight-cell stage onward before it preferentially cosegregates with Gata6 in PrE progenitors. Mechanistically, we demonstrate that Bmi1 interacts with Gata6 in a Ring finger-dependent manner to confer protection against Gata6 ubiquitination and proteasomal degradation. A direct role for Bmi1 in cell fate allocation is established by loss-of-function experiments in chimeric embryoid bodies. We thus propose a novel regulatory pathway by which Bmi1 action on Gata6 stability could alter the balance between Gata6 and Nanog protein levels to introduce a bias toward a PrE identity in a cell-autonomous manner

Epigenetic regulation of key developmental genes during early mouse development

In undifferentiated ES cells, many Polycomb Repressive Complex 2 (PRC2) target genes carry not only repressive H3K27me3 but are also enriched for conventional indicators of active chromatin including methylated H3K4. This so-called bivalent domain structure is thought to silence key developmental regulators while keeping them poised for future activation (or repression). Consistent with this hypothesis, bivalent genes assemble RNAP II preferentially phosphorylated on Serine 5 residues (poised RNAP II) and are transcribed at low levels. Productive expression is, however, prevented by the action of PRC1. Here, I have focused on the pre-implantation stage of mouse development to evaluate whether bivalent or poised chromatin signatures are indeed specific attributes of emerging pluripotent cells and investigate how the fate of key developmental genes is specified while the first lineage decision event (extra-embryonic lineage formation) occurs. Using blastocyst-derived stem cells and chromatin immunoprecipitation (ChIP), I have shown that lineage-inappropriate genes retain bivalent histone marking in extra-embryonic trophoblast stem (TS). However, and in contrast to ES cells, PRC1 (Ring1B) and poised RNAP II are not recruited to these loci in TS cells, indicating that gene priming is a unique hallmark of pluripotent cells in the early embryo. To investigate the intricate relationship between lineage identity and dynamic chromatin changes, I exploited the potential to convert ES cells into trophoblast-like stem (TSL) cells using a previously established artificial system dependent on doxycycline (Dox) induced repression of an Oct4 transgene. I demonstrated that Suv39h1-mediated H3K9me3 alongside DNA methylation is targeted to PRC2-bound bivalent, lineage-inappropriate genes upon trophectoderm lineage commitment. A change in chromatin conformation was observed upon differentiation of ES cells to TSL cells comparable to that seen in TS cells derived in the traditional manner from the trophectoderm (TE) of blastocyst stage embryos. Most importantly, I have begun to explore when epigenetic differences are specified, at the locus level, from 8-cell stage embryos onwards using newly designed Carrier ChIP technology. This data validated the occurrence of bivalent chromatin domains in vivo and further support the view that alternative strategies operate in the TE to silence key developmental regulators upon blastocyst lineage segregation.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Regulation of pluripotent states in human embryonic stem cells

A growing array of mouse and human pluripotent stem cell lines has been derived from the early embryo as well as from adult cells reprogrammed by ectopic expression of transcription factors – i.e. induced pluripotent stem (iPS) cells. These cell lines share the expression of key pluripotency markers and are able to self-renew and to generate differentiated progenies when induced. Their relationship to each other and whether they correspond to different pluripotent states with distinct developmental capacities and affiliations in vivo remains unclear, however. Profiling chromatin in a particular cell line has proven to be a valuable signature for cell identity and developmental stage. One approach has been to assay the timing of DNA replication across a panel of loci, as an indicator of chromatin accessibility. Of interest, this replication timing profiling was capable of discriminating pluripotent mouse ES (mES) cells from cells with a more restricted differentiation capacity. In this study, I have addressed whether distinct pluripotent states could be reliably discriminated at the chromatin level. In particular, I characterised the replication timing profile of a number of human ES (hES) cell lines alongside mES and mouse epiblast-derived stem (mEpiS) cell lines. I showed that mES cells have a steady and mostly early-replicating profile, regardless of their genetic background. In contrast, the profile of undifferentiated H1, H7 and H9 hES cell lines harboured an increased proportion of late-replicating loci during S-phase. Moreover, hES cell replication profile greatly varied between cultures and cell lines; a level of replication timing variability also observed among mEpiS cells, as opposed to mES cells. These results highlighted that hES and mEpiS cells share a common unstable or transitional state: primed on the verge of differentiation. This view was, however, further challenged by exploring how hES cell cultures could be modulated towards an ES-like versus epiblast-like state under different conditions. In particular, extensive and dynamic shifts of replication timing, from late to early, were consistently observed at many target loci in hES and hiPS cells upon increased Smad2/3 and p300 histone acetyltransferase activity. Importantly, these alterations were reversible and associated with differential gene expression profiles and functional properties of hES cells. Collectively, these data revealed the existence of distinct but interchangeable pluripotent hES cell states and proposed a key role for TGF-β/Activin signalling and the HAT p300 in modulating the balance between a naive versus primed state in hES cell cultures.EThOS - Electronic Theses Online ServiceFCT, PortugalIOG Trust FundGBUnited Kingdo

Senescence impairs successful reprogramming to pluripotent stem cells

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by overexpressing combinations of factors such as Oct4, Sox2, Klf4, and c-Myc. Reprogramming is slow and stochastic, suggesting the existence of barriers limiting its efficiency. Here we identify senescence as one such barrier. Expression of the four reprogramming factors triggers senescence by up-regulating p53, p16INK4a, and p21CIP1. Induction of DNA damage response and chromatin remodeling of the INK4a/ARF locus are two of the mechanisms behind senescence induction. Crucially, ablation of different senescence effectors improves the efficiency of reprogramming, suggesting novel strategies for maximizing the generation of iPS cells

Evolution of an amniote-specific mechanism for modulating ubiquitin signalling via phosphoregulation of the E2 enzyme UBE2D3

Genetic variation in the enzymes that catalyze posttranslational modification of proteins is a potentially important source of phenotypic variation during evolution. Ubiquitination is one such modification that affects turnover of virtually all of the proteins in the cell in addition to roles in signaling and epigenetic regulation. UBE2D3 is a promiscuous E2 enzyme, which acts as an ubiquitin donor for E3 ligases that catalyze ubiquitination of developmentally important proteins. We have used protein sequence comparison of UBE2D3 orthologs to identify a position in the C-terminal α-helical region of UBE2D3 that is occupied by a conserved serine in amniotes and by alanine in anamniote vertebrate and invertebrate lineages. Acquisition of the serine (S138) in the common ancestor to modern amniotes created a phosphorylation site for Aurora B. Phosphorylation of S138 disrupts the structure of UBE2D3 and reduces the level of the protein in mouse embryonic stem cells (ESCs). Substitution of S138 with the anamniote alanine (S138A) increases the level of UBE2D3 in ESCs as well as being a gain of function early embryonic lethal mutation in mice. When mutant S138A ESCs were differentiated into extraembryonic primitive endoderm, levels of the PDGFRα and FGFR1 receptor tyrosine kinases were reduced and primitive endoderm differentiation was compromised. Proximity ligation analysis showed increased interaction between UBE2D3 and the E3 ligase CBL and between CBL and the receptor tyrosine kinases. Our results identify a sequence change that altered the ubiquitination landscape at the base of the amniote lineage with potential effects on amniote biology and evolution.</p

Evolution of an amniote-specific mechanism for modulating ubiquitin signalling via phosphoregulation of the E2 enzyme UBE2D3

Genetic variation in the enzymes that catalyse post-translational modification of proteins is a potentially important source of phenotypic variation during evolution. Ubiquitination is one such modification that affects turnover of virtually all of the proteins in the cell in addition to roles in signalling and epigenetic regulation. UBE2D3 is a promiscuous E2 enzyme, which acts as a ubiquitin donor for E3 ligases that catalyse ubiquitination of developmentally important proteins. We have used protein sequence comparison of UBE2D3 orthologues to identify a position in the C-terminal α-helical region of UBE2D3 that is occupied by a conserved serine in amniotes and by alanine in anamniote vertebrate and invertebrate lineages. Acquisition of the serine (S138) in the common ancestor to modern amniotes created a phosphorylation site for Aurora B. Phosphorylation of S138 disrupts the structure of UBE2D3 and reduces the level of the protein in mouse ES cells (ESCs). Substitution of S138 with the anamniote alanine (S138A) increases the level of UBE2D3 in ESCs as well as being a gain of function early embryonic lethal mutation in mice. When mutant S138A ESCs were differentiated into extra-embryonic primitive endoderm (PrE), levels of the PDGFRα and FGFR1 receptor tyrosine kinases (RTKs) were reduced and PreE differentiation was compromised. Proximity ligation analysis showed increased interaction between UBE2D3 and the E3 ligase CBL and between CBL and the RTKs. Our results identify a sequence change that altered the ubiquitination landscape at the base of the amniote lineage with potential effects on amniote biology and evolution

MicroRNA Regulation of Cbx7 Mediates a Switch of Polycomb Orthologs during ESC Differentiation

The Polycomb Group (PcG) of chromatin modifiers regulates pluripotency and differentiation. Mammalian genomes encode multiple homologs of the Polycomb repressive complex 1 (PRC1) components, including five orthologs of the Drosophila Polycomb protein (Cbx2, Cbx4, Cbx6, Cbx7, and Cbx8). We have identified Cbx7 as the primary Polycomb ortholog of PRC1 complexes in embryonic stem cells (ESCs). The expression of Cbx7 is downregulated during ESC differentiation, preceding the upregulation of Cbx2, Cbx4, and Cbx8, which are directly repressed by Cbx7. Ectopic expression of Cbx7 inhibits differentiation and X chromosome inactivation and enhances ESC self-renewal. Conversely, Cbx7 knockdown induces differentiation and derepresses lineage-specific markers. In a functional screen, we identified the miR-125 and miR-181 families as regulators of Cbx7 that are induced during ESC differentiation. Ectopic expression of these miRNAs accelerates ESC differentiation via regulation of Cbx7. These observations establish a critical role for Cbx7 and its regulatory miRNAs in determining pluripotency