Modeling abnormal early development with induced pluripotent stem cells from aneuploid syndromes.

Many human diseases share a developmental origin that manifests during childhood or maturity. Aneuploid syndromes are caused by supernumerary or reduced number of chromosomes and represent an extreme example of developmental disease, as they have devastating consequences before and after birth. Investigating how alterations in gene dosage drive these conditions is relevant because it might help treat some clinical aspects. It may also provide explanations as to how quantitative differences in gene expression determine phenotypic diversity and disease susceptibility among natural populations. Here, we aimed to produce induced pluripotent stem cell (iPSC) lines that can be used to improve our understanding of aneuploid syndromes. We have generated iPSCs from monosomy X [Turner syndrome (TS)], trisomy 8 (Warkany syndrome 2), trisomy 13 (Patau syndrome) and partial trisomy 11;22 (Emanuel syndrome), using either skin fibroblasts from affected individuals or amniocytes from antenatal diagnostic tests. These cell lines stably maintain the karyotype of the donors and behave like embryonic stem cells in all tested assays. TS iPSCs were used for further studies including global gene expression analysis and tissue-specific directed differentiation. Multiple clones displayed lower levels of the pseudoautosomal genes ASMTL and PPP2R3B than the controls. Moreover, they could be transformed into neural-like, hepatocyte-like and heart-like cells, but displayed insufficient up-regulation of the pseudoautosomal placental gene CSF2RA during embryoid body formation. These data support that abnormal organogenesis and early lethality in TS are not caused by a tissue-specific differentiation blockade, but rather involves other abnormalities including impaired placentation.

STARR-seq identifies active, chromatin-masked, and dormant enhancers in pluripotent mouse embryonic stem cells.

BACKGROUND: Enhancers are distal regulators of gene expression that shape cell identity and control cell fate transitions. In mouse embryonic stem cells (mESCs), the pluripotency network is maintained by the function of a complex network of enhancers, that are drastically altered upon differentiation. Genome-wide chromatin accessibility and histone modification assays are commonly used as a proxy for identifying putative enhancers and for describing their activity levels and dynamics. RESULTS: Here, we applied STARR-seq, a genome-wide plasmid-based assay, as a read-out for the enhancer landscape in "ground-state" (2i+LIF; 2iL) and "metastable" (serum+LIF; SL) mESCs. This analysis reveals that active STARR-seq loci show modest overlap with enhancer locations derived from peak calling of ChIP-seq libraries for common enhancer marks. We unveil ZIC3-bound loci with significant STARR-seq activity in SL-ESCs. Knock-out of Zic3 removes STARR-seq activity only in SL-ESCs and increases their propensity to differentiate towards the endodermal fate. STARR-seq also reveals enhancers that are not accessible, masked by a repressive chromatin signature. We describe a class of dormant, p53 bound enhancers that gain H3K27ac under specific conditions, such as after treatment with Nocodazol, or transiently during reprogramming from fibroblasts to pluripotency. CONCLUSIONS: In conclusion, loci identified as active by STARR-seq often overlap with those identified by chromatin accessibility and active epigenetic marking, yet a significant fraction is epigenetically repressed or display condition-specific enhancer activity.

Critical Role for P53 in Regulating the Cell Cycle of Ground State Embryonic Stem Cells.

Mouse embryonic stem cells (ESCs) grown in serum-supplemented conditions are characterized by an extremely short G1 phase due to the lack of G1-phase control. Concordantly, the G1-phase-specific P53-P21 pathway is compromised in serum ESCs. Here, we provide evidence that P53 is activated upon transition of serum ESCs to their pluripotent ground state using serum-free 2i conditions and that is required for the elongated G1 phase characteristic of ground state ESCs. RNA sequencing and chromatin immunoprecipitation sequencing analyses reveal that P53 directly regulates the expression of the retinoblastoma (RB) protein and that the hypo-phosphorylated, active RB protein plays a key role in G1-phase control. Our findings suggest that the P53-P21 pathway is active in ground state 2i ESCs and that its role in the G1-checkpoint is abolished in serum ESCs. Taken together, the data reveal a mechanism by which inactivation of P53 can lead to loss of RB and uncontrolled cell proliferation.

Publisher Correction: Epigenetic modulation of a hardwired 3D chromatin landscape in two naive states of pluripotency.

In the version of the article originally published, extra lines were displayed in Fig. 7. Fig. 7a contained a solid black line that extended into panel b, and Fig. 7c contained two extra scale bars on the left. These have been removed from the figure. The errors have been corrected in the HTML and PDF versions of the article.

Epigenetic modulation of a hardwired 3D chromatin landscape in two naive states of pluripotency.

The mechanisms underlying enhancer activation and the extent to which enhancer-promoter rewiring contributes to spatiotemporal gene expression are not well understood. Using integrative and time-resolved analyses we show that the extensive transcriptome and epigenome resetting during the conversion between 'serum' and '2i' states of mouse embryonic stem cells (ESCs) takes place with minimal enhancer-promoter rewiring that becomes more evident in primed-state pluripotency. Instead, differential gene expression is strongly linked to enhancer activation via H3K27ac. Conditional depletion of transcription factors and allele-specific enhancer analysis reveal an essential role for Esrrb in H3K27 acetylation and activation of 2i-specific enhancers. Restoration of a polymorphic ESRRB motif using CRISPR-Cas9 in a hybrid ESC line restores ESRRB binding and enhancer H3K27ac in an allele-specific manner but has no effect on chromatin interactions. Our study shows that enhancer activation in serum- and 2i-ESCs is largely driven by transcription factor binding and epigenetic marking in a hardwired network of chromatin interactions.

The non-coding variant rs1800734 enhances DCLK3 expression through long-range interaction and promotes colorectal cancer progression.

Genome-wide association studies have identified a great number of non-coding risk variants for colorectal cancer (CRC). To date, the majority of these variants have not been functionally studied. Identification of allele-specific transcription factor (TF) binding is of great importance to understand regulatory consequences of such variants. A recently developed proteome-wide analysis of disease-associated SNPs (PWAS) enables identification of TF-DNA interactions in an unbiased manner. Here we perform a large-scale PWAS study to comprehensively characterize TF-binding landscape that is associated with CRC, which identifies 731 allele-specific TF binding at 116 CRC risk loci. This screen identifies the A-allele of rs1800734 within the promoter region of MLH1 as perturbing the binding of TFAP4 and consequently increasing DCLK3 expression through a long-range interaction, which promotes cancer malignancy through enhancing expression of the genes related to epithelial-to-mesenchymal transition.

Dynamic Reorganization of Extremely Long-Range Promoter-Promoter Interactions between Two States of Pluripotency.

Serum-to-2i interconversion of mouse embryonic stem cells (mESCs) is a valuable in vitro model for early embryonic development. To assess whether 3D chromatin organization changes during this transition, we established Capture Hi-C with target-sequence enrichment of DNase I hypersensitive sites. We detected extremely long-range intra- and inter-chromosomal interactions between a small subset of H3K27me3 marked bivalent promoters involving the Hox clusters in serum-grown cells. Notably, these promoter-mediated interactions are not present in 2i ground-state pluripotent mESCs but appear upon their further development into primed-like serum mESCs. Reverting serum mESCs to ground-state 2i mESCs removes these promoter-promoter interactions in a spatiotemporal manner. H3K27me3, which is largely absent at bivalent promoters in ground-state 2i mESCs, is necessary, but not sufficient, to establish these interactions, as confirmed by Capture Hi-C on Eed(-/-) serum mESCs. Our results implicate H3K27me3 and PRC2 as critical players in chromatin alteration during priming of ESCs for differentiation.

The oncogene c-Jun impedes somatic cell reprogramming.

Oncogenic transcription factors are known to mediate the conversion of somatic cells to tumour or induced pluripotent stem cells (iPSCs). Here we report c-Jun as a barrier for iPSC formation. c-Jun is expressed by and required for the proliferation of mouse embryonic fibroblasts (MEFs), but not mouse embryonic stem cells (mESCs). Consistently, c-Jun is induced during mESC differentiation, drives mESCs towards the endoderm lineage and completely blocks the generation of iPSCs from MEFs. Mechanistically, c-Jun activates mesenchymal-related genes, broadly suppresses the pluripotent ones, and derails the obligatory mesenchymal to epithelial transition during reprogramming. Furthermore, inhibition of c-Jun by shRNA, dominant-negative c-Jun or Jdp2 enhances reprogramming and replaces Oct4 among the Yamanaka factors. Finally, Jdp2 anchors 5 non-Yamanaka factors (Id1, Jhdm1b, Lrh1, Sall4 and Glis1) to reprogram MEFs into iPSCs. Our studies reveal c-Jun as a guardian of somatic cell fate and its suppression opens the gate to pluripotency.

H3K9 methylation is a barrier during somatic cell reprogramming into iPSCs.

The induction of pluripotent stem cells (iPSCs) by defined factors is poorly understood stepwise. Here, we show that histone H3 lysine 9 (H3K9) methylation is the primary epigenetic determinant for the intermediate pre-iPSC state, and its removal leads to fully reprogrammed iPSCs. We generated a panel of stable pre-iPSCs that exhibit pluripotent properties but do not activate the core pluripotency network, although they remain sensitive to vitamin C for conversion into iPSCs. Bone morphogenetic proteins (BMPs) were subsequently identified in serum as critical signaling molecules in arresting reprogramming at the pre-iPSC state. Mechanistically, we identified H3K9 methyltransferases as downstream targets of BMPs and showed that they function with their corresponding demethylases as the on/off switch for the pre-iPSC fate by regulating H3K9 methylation status at the core pluripotency loci. Our results not only establish pre-iPSCs as an epigenetically stable signpost along the reprogramming road map, but they also provide mechanistic insights into the epigenetic reprogramming of cell fate.