RESUMEN
After acquiring competence for selected cell fates, embryonic primordia may remain plastic for variable periods before tissue identity is irrevocably determined (commitment). We investigated the chromatin basis for these developmental milestones in mouse endoderm, a tissue with recognizable rostro-caudal patterning and transcription factor (TF)-dependent interim plasticity. Foregut-specific enhancers are as accessible and active in early midgut as in foregut endoderm, and intestinal enhancers and identity are established only after ectopic cis-regulatory elements are decommissioned. Depletion of the intestinal TF CDX2 before this cis element transition stabilizes foregut enhancers, reinforces ectopic transcriptional programs, and hence imposes foregut identities on the midgut. Later in development, as the window of chromatin plasticity elapses, CDX2 depletion weakens intestinal, without strengthening foregut, enhancers. Thus, midgut endoderm is primed for heterologous cell fates, and TFs act on a background of shifting chromatin access to determine intestinal at the expense of foregut identity. Similar principles likely govern other fate commitments.
Asunto(s)
Endodermo/metabolismo , Elementos de Facilitación Genéticos , Mucosa Intestinal/metabolismo , Intestinos/embriología , Transcripción Genética , Animales , Factor de Transcripción CDX2/genética , Factor de Transcripción CDX2/metabolismo , Cromatina/metabolismo , Endodermo/embriología , Intestinos/anatomía & histología , RatonesRESUMEN
As embryos mature, cells undergo remarkable transitions that are accompanied by shifts in transcription factor regulatory networks. Mechanisms driving developmental transitions are incompletely understood. The embryonic intestine transitions from a rapidly proliferating tube with pseudostratified epithelium prior to murine embryonic day (E) 14.5 to an exquisitely folded columnar epithelium in fetal stages. We sought to identify factors driving mouse fetal intestinal maturation by mining chromatin accessibility data for transcription factor motifs. ATAC-seq accessible regions shift during tissue maturation, with CDX2 transcription factor motifs abundant at chromatin-accessible regions of the embryo. Hepatocyte nuclear factor 4 (HNF4) transcription factor motifs are the most abundant in the fetal stages (>E16.5). Genetic inactivation of Hnf4a and its paralog Hnf4g revealed that HNF4 factors are redundantly required for fetal maturation. CDX2 binds to and activates Hnf4 gene loci to elevate HNF4 expression at fetal stages. HNF4 and CDX2 transcription factors then occupy shared genomic regulatory sites to promote chromatin accessibility and gene expression in the maturing intestine. Thus, HNF4 paralogs are key components of an intestinal transcription factor network shift during the embryonic to fetal transition.
Asunto(s)
Cromatina/metabolismo , Feto/embriología , Factor Nuclear 4 del Hepatocito/metabolismo , Intestinos/embriología , Animales , Factor de Transcripción CDX2/metabolismo , Línea Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Modelos Biológicos , MorfogénesisRESUMEN
Lineage-restricted transcription factors, such as the intestine-specifying factor CDX2, often have dual requirements across developmental time. Embryonic loss of CDX2 triggers homeotic transformation of intestinal fate, whereas adult-onset loss compromises crucial physiological functions but preserves intestinal identity. It is unclear how such diverse requirements are executed across the developmental continuum. Using primary and engineered human tissues, mouse genetics, and a multi-omics approach, we demonstrate that divergent CDX2 loss-of-function phenotypes in embryonic versus adult intestines correspond to divergent CDX2 chromatin-binding profiles in embryonic versus adult stages. CDX2 binds and activates distinct target genes in developing versus adult mouse and human intestinal cells. We find that temporal shifts in chromatin accessibility correspond to these context-specific CDX2 activities. Thus, CDX2 is not sufficient to activate a mature intestinal program; rather, CDX2 responds to its environment, targeting stage-specific genes to contribute to either intestinal patterning or mature intestinal function. This study provides insights into the mechanisms through which lineage-specific regulatory factors achieve divergent functions over developmental time.
Asunto(s)
Factor de Transcripción CDX2/metabolismo , Cromatina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Intestinos/embriología , Animales , Factor de Transcripción CDX2/genética , Sistemas CRISPR-Cas , Diferenciación Celular , Linaje de la Célula , Femenino , Humanos , Mucosa Intestinal/metabolismo , Ratones , Ratones Noqueados , Mutación , Células Madre Pluripotentes/citología , Unión Proteica , Dominios Proteicos , Transactivadores/metabolismoRESUMEN
BACKGROUND & AIMS: Functions of intestinal stem cells (ISCs) are regulated by diet and metabolic pathways. Hepatocyte nuclear factor 4 (HNF4) family are transcription factors that bind fatty acids. We investigated how HNF4 transcription factors regulate metabolism and their functions in ISCs in mice. METHODS: We performed studies with Villin-CreERT2;Lgr5-EGFP-IRES-CreERT2;Hnf4αf/f;Hnf4γCrispr/Crispr mice, hereafter referred to Hnf4αγDKO. Mice were given tamoxifen to induce Cre recombinase. Mice transgenic with only Cre alleles (Villin-CreERT2, Lgr5-EGFP-IRES-CreERT2, Hnf4α+/+, and Hnf4γ+/+) or mice given vehicle were used as controls. Crypt and villus cells were isolated, incubated with fluorescently labeled fatty acids or glucose analog, and analyzed by confocal microscopy. Fatty acid oxidation activity and tricarboxylic acid (TCA) cycle metabolites were measured in cells collected from the proximal half of the small intestine of Hnf4αγDKO and control mice. We performed chromatin immunoprecipitation and gene expression profiling analyses to identify genes regulated by HNF4 factors. We established organoids from duodenal crypts, incubated them with labeled palmitate or acetate, and measured production of TCA cycle metabolites or fatty acids. Acetate, a precursor of acetyl coenzyme A (CoA) (a product of fatty acid ß-oxidation [FAO]), or dichloroacetate, a compound that promotes pyruvate oxidation and generation of mitochondrial acetyl-CoA, were used for metabolic intervention. RESULTS: Crypt cells rapidly absorbed labeled fatty acids, and messenger RNA levels of Lgr5+ stem cell markers (Lgr5, Olfm4, Smoc2, Msi1, and Ascl2) were down-regulated in organoids incubated with etomoxir, an inhibitor of FAO, indicating that FAO was required for renewal of ISCs. HNF4A and HNF4G were expressed in ISCs and throughout the intestinal epithelium. Single knockout of either HNF4A or HNF4G did not affect maintenance of ISCs, but double-knockout of HNF4A and HNF4G resulted in ISC loss; stem cells failed to renew. FAO supports ISC renewal, and HNF4 transcription factors directly activate FAO genes, including Acsl5 and Acsf2 (encode regulators of acyl-CoA synthesis), Slc27a2 (encodes a fatty acid transporter), Fabp2 (encodes fatty acid binding protein), and Hadh (encodes hydroxyacyl-CoA dehydrogenase). In the intestinal epithelium of Hnf4αγDKO mice, expression levels of FAO genes, FAO activity, and metabolites of TCA cycle were all significantly decreased, but fatty acid synthesis transcripts were increased, compared with control mice. The contribution of labeled palmitate or acetate to the TCA cycle was reduced in organoids derived from Hnf4αγDKO mice, compared with control mice. Incubation of organoids derived from double-knockout mice with acetate or dichloroacetate restored stem cells. CONCLUSIONS: In mice, the transcription factors HNF4A and HNF4G regulate the expression of genes required for FAO and are required for renewal of ISCs.
Asunto(s)
Ácidos Grasos/metabolismo , Factor Nuclear 4 del Hepatocito/fisiología , Intestino Delgado/citología , Células Madre/metabolismo , Animales , Duodeno/citología , Proteínas de Unión a Ácidos Grasos/metabolismo , Mucosa Intestinal/citología , Ratones , Ratones Noqueados , Organoides/metabolismo , Oxidación-ReducciónRESUMEN
During development, the embryo transitions from a metabolism favoring glycolysis to a metabolism favoring mitochondrial respiration. How metabolic shifts regulate developmental processes, or how developmental processes regulate metabolic shifts, remains unclear. To test the requirement of mitochondrial function in developing endoderm-derived tissues, we genetically inactivated the mitochondrial transcription factor, Tfam, using the Shh-Cre driver. Tfam mutants did not survive postnatally, exhibiting defects in lung development. In the developing intestine, TFAM-loss was tolerated until late fetal development, during which the process of villus elongation was compromised. While progenitor cell populations appeared unperturbed, markers of enterocyte maturation were diminished and villi were blunted. Loss of TFAM was also tested in the adult intestinal epithelium, where enterocyte maturation was similarly dependent upon the mitochondrial transcription factor. While progenitor cells in the transit amplifying zone of the adult intestine remained proliferative, intestinal stem cell renewal was dependent upon TFAM, as indicated by molecular profiling and intestinal organoid formation assays. Taken together, these studies point to critical roles for the mitochondrial regulator TFAM for multiple aspects of intestinal development and maturation, and highlight the importance of mitochondrial regulators in tissue development and homeostasis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas del Grupo de Alta Movilidad/fisiología , Mucosa Intestinal/metabolismo , Animales , Técnicas de Cultivo de Célula/métodos , Autorrenovación de las Células/fisiología , Proteínas de Unión al ADN/genética , Feto/metabolismo , Regulación de la Expresión Génica/genética , Glucólisis/genética , Glucólisis/fisiología , Proteínas del Grupo de Alta Movilidad/genética , Mucosa Intestinal/embriología , Mucosa Intestinal/crecimiento & desarrollo , Ratones , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Organogénesis/genética , Organogénesis/fisiología , Organoides/metabolismo , Células Madre/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The brush border is comprised of microvilli surface protrusions on the apical surface of epithelia. This specialized structure greatly increases absorptive surface area and plays crucial roles in human health. However, transcriptional regulatory networks controlling brush border genes are not fully understood. Here, we identify that hepatocyte nuclear factor 4 (HNF4) transcription factor is a conserved and important regulator of brush border gene program in multiple organs, such as intestine, kidney and yolk sac. Compromised brush border gene signatures and impaired transport were observed in these tissues upon HNF4 loss. By ChIP-seq, we find HNF4 binds and activates brush border genes in the intestine and kidney. H3K4me3 HiChIP-seq identifies that HNF4 loss results in impaired chromatin looping between enhancers and promoters at gene loci of brush border genes, and instead enhanced chromatin looping at gene loci of stress fiber genes in the intestine. This study provides comprehensive transcriptional regulatory mechanisms and a functional demonstration of a critical role for HNF4 in brush border gene regulation across multiple murine epithelial tissues.
Asunto(s)
Regulación de la Expresión Génica , Factor Nuclear 4 del Hepatocito/genética , Mucosa Intestinal/metabolismo , Riñón/metabolismo , Microvellosidades/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Saco Vitelino/metabolismo , Animales , Epitelio/metabolismo , Perfilación de la Expresión Génica/métodos , Factor Nuclear 4 del Hepatocito/metabolismo , Humanos , Intestinos/ultraestructura , Riñón/ultraestructura , Ratones Noqueados , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Receptores Citoplasmáticos y Nucleares/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BMP/SMAD signaling is a crucial regulator of intestinal differentiation1-4. However, the molecular underpinnings of the BMP pathway in this context are unknown. Here, we characterize the mechanism by which BMP/SMAD signaling drives enterocyte differentiation. We establish that the transcription factor HNF4A acts redundantly with an intestine-restricted HNF4 paralog, HNF4G, to activate enhancer chromatin and upregulate the majority of transcripts enriched in the differentiated epithelium; cells fail to differentiate on double knockout of both HNF4 paralogs. Furthermore, we show that SMAD4 and HNF4 function via a reinforcing feed-forward loop, activating each other's expression and co-binding to regulatory elements of differentiation genes. This feed-forward regulatory module promotes and stabilizes enterocyte cell identity; disruption of the HNF4-SMAD4 module results in loss of enterocyte fate in favor of progenitor and secretory cell lineages. This intersection of signaling and transcriptional control provides a framework to understand regenerative tissue homeostasis, particularly in tissues with inherent cellular plasticity5.