Transcription factor-mediated intestinal metaplasia and the role of a shadow enhancer.

Barrett's esophagus (BE) and gastric intestinal metaplasia are related premalignant conditions in which areas of human stomach epithelium express mixed gastric and intestinal features. Intestinal transcription factors (TFs) are expressed in both conditions, with unclear causal roles and cis-regulatory mechanisms. Ectopic CDX2 reprogrammed isogenic mouse stomach organoid lines to a hybrid stomach-intestinal state transcriptionally similar to clinical metaplasia; squamous esophageal organoids resisted this CDX2-mediated effect. Reprogramming was associated with induced activity at thousands of previously inaccessible intestine-restricted enhancers, where CDX2 occupied DNA directly. HNF4A, a TF recently implicated in BE pathogenesis, induced weaker intestinalization by binding a novel shadow Cdx2 enhancer and hence activating Cdx2 expression. CRISPR/Cas9-mediated germline deletion of that cis-element demonstrated its requirement in Cdx2 induction and in the resulting activation of intestinal genes in stomach cells. dCas9-conjugated KRAB repression mapped this activity to the shadow enhancer's HNF4A binding site. Altogether, we show extensive but selective recruitment of intestinal enhancers by CDX2 in gastric cells and that HNF4A-mediated ectopic CDX2 expression in the stomach occurs through a conserved shadow cis-element. These findings identify mechanisms for TF-driven intestinal metaplasia and a likely pathogenic TF hierarchy.

Inducible histone K-to-M mutations are dynamic tools to probe the physiological role of site-specific histone methylation in vitro and in vivo.

Development and differentiation are associated with profound changes to histone modifications, yet their in vivo function remains incompletely understood. Here, we generated mouse models expressing inducible histone H3 lysine-to-methionine (K-to-M) mutants, which globally inhibit methylation at specific sites. Mice expressing H3K36M developed severe anaemia with arrested erythropoiesis, a marked haematopoietic stem cell defect, and rapid lethality. By contrast, mice expressing H3K9M survived up to a year and showed expansion of multipotent progenitors, aberrant lymphopoiesis and thrombocytosis. Additionally, some H3K9M mice succumbed to aggressive T cell leukaemia/lymphoma, while H3K36M mice exhibited differentiation defects in testis and intestine. Mechanistically, induction of either mutant reduced corresponding histone trimethylation patterns genome-wide and altered chromatin accessibility as well as gene expression landscapes. Strikingly, discontinuation of transgene expression largely restored differentiation programmes. Our work shows that individual chromatin modifications are required at several specific stages of differentiation and introduces powerful tools to interrogate their roles in vivo.

The RNA Helicase DDX6 Controls Cellular Plasticity by Modulating P-Body Homeostasis.

Post-transcriptional mechanisms have the potential to influence complex changes in gene expression, yet their role in cell fate transitions remains largely unexplored. Here, we show that suppression of the RNA helicase DDX6 endows human and mouse primed embryonic stem cells (ESCs) with a differentiation-resistant, "hyper-pluripotent" state, which readily reprograms to a naive state resembling the preimplantation embryo. We further demonstrate that DDX6 plays a key role in adult progenitors where it controls the balance between self-renewal and differentiation in a context-dependent manner. Mechanistically, DDX6 mediates the translational suppression of target mRNAs in P-bodies. Upon loss of DDX6 activity, P-bodies dissolve and release mRNAs encoding fate-instructive transcription and chromatin factors that re-enter the ribosome pool. Increased translation of these targets impacts cell fate by rewiring the enhancer, heterochromatin, and DNA methylation landscapes of undifferentiated cell types. Collectively, our data establish a link between P-body homeostasis, chromatin organization, and stem cell potency.

Direct Reprogramming of Mouse Fibroblasts into Functional Skeletal Muscle Progenitors.

Skeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse fibroblasts into expandable induced myogenic progenitor cells (iMPCs). iMPCs express key skeletal muscle stem and progenitor cell markers including Pax7 and Myf5 and give rise to dystrophin-expressing myofibers upon transplantation in vivo. Notably, a subset of transplanted iMPCs maintain Pax7 expression and sustain serial regenerative responses. Similar to satellite cells, iMPCs originate from Pax7+ cells and require Pax7 itself for maintenance. Finally, we show that myogenic progenitor cell lines can be established from muscle tissue following small-molecule exposure alone. This study thus reports on a robust approach to derive expandable myogenic stem/progenitor-like cells from multiple cell types.

Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells.

Concomitant activation of the Wnt pathway and suppression of Mapk signalling by two small molecule inhibitors (2i) in the presence of leukaemia inhibitory factor (LIF) (hereafter termed 2i/L) induces a naive state in mouse embryonic stem (ES) cells that resembles the inner cell mass (ICM) of the pre-implantation embryo. Since the ICM exists only transiently in vivo, it remains unclear how sustained propagation of naive ES cells in vitro affects their stability and functionality. Here we show that prolonged culture of male mouse ES cells in 2i/L results in irreversible epigenetic and genomic changes that impair their developmental potential. Furthermore, we find that female ES cells cultured in conventional serum plus LIF medium phenocopy male ES cells cultured in 2i/L. Mechanistically, we demonstrate that the inhibition of Mek1/2 is predominantly responsible for these effects, in part through the downregulation of DNA methyltransferases and their cofactors. Finally, we show that replacement of the Mek1/2 inhibitor with a Src inhibitor preserves the epigenetic and genomic integrity as well as the developmental potential of ES cells. Taken together, our data suggest that, although short-term suppression of Mek1/2 in ES cells helps to maintain an ICM-like epigenetic state, prolonged suppression results in irreversible changes that compromise their developmental potential.

Sox2 Suppresses Gastric Tumorigenesis in Mice.

Sox2 expression marks gastric stem and progenitor cells, raising important questions regarding the genes regulated by Sox2 and the role of Sox2 itself during stomach homeostasis and disease. By using ChIP-seq analysis, we have found that the majority of Sox2 targets in gastric epithelial cells are tissue specific and related to functions such as endoderm development, Wnt signaling, and gastric cancer. Unexpectedly, we found that Sox2 itself is dispensable for gastric stem cell and epithelial self-renewal, yet Sox2(+) cells are highly susceptible to tumorigenesis in an Apc/Wnt-driven mouse model. Moreover, Sox2 loss enhances, rather than impairs, tumor formation in Apc-deficient gastric cells in vivo and in vitro by inducing Tcf/Lef-dependent transcription and upregulating intestinal metaplasia-associated genes, providing a mechanistic basis for the observed phenotype. Together, these data identify Sox2 as a context-dependent tumor suppressor protein that is dispensable for normal tissue regeneration but restrains stomach adenoma formation through modulation of Wnt-responsive and intestinal genes.

Activation of Nrf2 in keratinocytes causes chloracne (MADISH)-like skin disease in mice.

The transcription factor Nrf2 is a key regulator of the cellular stress response, and pharmacological Nrf2 activation is a promising strategy for skin protection and cancer prevention. We show here that prolonged Nrf2 activation in keratinocytes causes sebaceous gland enlargement and seborrhea in mice due to upregulation of the growth factor epigen, which we identified as a novel Nrf2 target. This was accompanied by thickening and hyperkeratosis of hair follicle infundibula. These abnormalities caused dilatation of infundibula, hair loss, and cyst development upon aging. Upregulation of epigen, secretory leukocyte peptidase inhibitor (Slpi), and small proline-rich protein 2d (Sprr2d) in hair follicles was identified as the likely cause of infundibular acanthosis, hyperkeratosis, and cyst formation. These alterations were highly reminiscent to the phenotype of chloracne/"metabolizing acquired dioxin-induced skin hamartomas" (MADISH) patients. Indeed, SLPI, SPRR2, and epigen were strongly expressed in cysts of MADISH patients and upregulated by dioxin in human keratinocytes in an NRF2-dependent manner. These results identify novel Nrf2 activities in the pilosebaceous unit and point to a role of NRF2 in MADISH pathogenesis.

Aurora kinase-A deficiency during skin development impairs cell division and stratification.

Aurora kinase-A (Aurora-A) promotes timely entry into mitosis, centrosome maturation, and formation of bipolar spindles. To address the role of Aurora-A in skin development and homeostasis, we interbred a floxed Aurora-A (Aurora-A(fl)) mouse with the Cre-deleter strain, K14.Cre. Aurora-A(fl/fl);Krt14.Cre (Aurora-A(-/-)) mice died shortly after birth. These mice had translucent skin, and histological evaluation showed that the dorsal skin was very thin and fragile with frank erosions. Although the expression of the basal layer marker keratin 14 and the differentiation marker keratin 1 was evident in Aurora-A(-/-) epidermis, there was a marked reduction in the number of suprabasal layers and basal keratinocytes. Dye exclusion assays also showed defects in barrier function. Unlike wild-type cells, Aurora-A(-/-) basal progenitors were delayed in forming two layers at embryonic day (E)13.5 when embryonic skin begins to stratify. Increased numbers of mitotic cells, apoptotic bodies, and polyploid keratinocytes were evident in Aurora-A(-/-) epidermis, indicating that a deficiency in Aurora-A promotes aberrant mitosis, mitotic slippage, and cell death. Finally, Aurora-A(-/-) keratinocytes displayed centrosomal abnormalities that included centrosomes located at nonapical sites in basal cells. Thus, the deletion of Aurora-A in the developing epidermis alters centrosome function of basal keratinocytes and markedly impairs their ability to divide and stratify.