RESUMEN
Single-cell transcriptomics coupled with dynamic two-color fluorescence are used by Gehart et al. (2019) to elucidate adult mammalian cell trajectories in real time. The authors' close examination of intestinal enteroendocrine differentiation reveals new lineage features and shifting cell identities, and experiments in organoids uncover specific roles for transcriptional regulators identified by this approach.
Asunto(s)
Organoides , Células Madre , Animales , Diferenciación Celular , Linaje de la Célula , IntestinosRESUMEN
Bivalent promoters in embryonic stem cells (ESCs) carry methylation marks on two lysine residues, K4 and K27, in histone3 (H3). K4me2/3 is generally considered to promote transcription, and Polycomb Repressive Complex 2 (PRC2) places K27me3, which is erased at lineage-restricted genes when ESCs differentiate in culture. Molecular defects in various PRC2 null adult tissues lack a unifying explanation. We found that epigenomes in adult mouse intestine and other self-renewing tissues show fewer and distinct bivalent promoters compared to ESCs. Groups of tissue-specific genes that carry bivalent marks are repressed, despite the presence of promoter H3K4me2/3. These are the predominant genes de-repressed in PRC2-deficient adult cells, where aberrant expression is proportional to the H3K4me2/3 levels observed at their promoters in wild-type cells. Thus, in adult animals, PRC2 specifically represses genes with acquired, tissue-restricted promoter bivalency. These findings provide new insights into specificity in chromatin-based gene regulation.
Asunto(s)
Células Madre Embrionarias/metabolismo , Complejo Represivo Polycomb 2/genética , Regiones Promotoras Genéticas , Animales , Diferenciación Celular/genética , Metilación de ADN , Regulación de la Expresión Génica , Histonas/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/citología , Lisina/metabolismo , Ratones , Ratones Endogámicos C57BL , Complejo Represivo Polycomb 2/metabolismoRESUMEN
The progeny of intestinal stem cells (ISCs) dedifferentiate in response to ISC attrition. The precise cell sources, transitional states, and chromatin remodeling behind this activity remain unclear. In the skin, stem cell recovery after injury preserves an epigenetic memory of the damage response; whether similar memories arise and persist in regenerated ISCs is not known. We addressed these questions by examining gene activity and open chromatin at the resolution of single Neurog3-labeled mouse intestinal crypt cells, hence deconstructing forward and reverse differentiation of the intestinal secretory (Sec) lineage. We show that goblet, Paneth, and enteroendocrine cells arise by multilineage priming in common precursors, followed by selective access at thousands of cell-restricted cis-elements. Selective ablation of the ISC compartment elicits speedy reversal of chromatin and transcriptional features in large fractions of precursor and mature crypt Sec cells without obligate cell cycle re-entry. ISC programs decay and reappear along a cellular continuum lacking discernible discrete interim states. In the absence of gross tissue damage, Sec cells simply reverse their forward trajectories, without invoking developmental or other extrinsic programs, and starting chromatin identities are effectively erased. These findings identify strikingly plastic molecular frameworks in assembly and regeneration of a self-renewing tissue.
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Cromatina , Células Madre , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/genética , Cromatina/metabolismo , Mucosa Intestinal/metabolismo , Intestinos , Ratones , Proteínas del Tejido Nervioso/metabolismoRESUMEN
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.
Asunto(s)
Esófago de Barrett , Factores de Transcripción , Animales , Esófago de Barrett/genética , Esófago de Barrett/metabolismo , Esófago de Barrett/patología , Factor de Transcripción CDX2/genética , Proteínas de Homeodominio/genética , Metaplasia/genética , Ratones , Factores de Transcripción/genéticaRESUMEN
Engineering clinically relevant cells in vitro holds promise for regenerative medicine, but most protocols fail to faithfully recapitulate target cell properties. To address this, we developed CellNet, a network biology platform that determines whether engineered cells are equivalent to their target tissues, diagnoses aberrant gene regulatory networks, and prioritizes candidate transcriptional regulators to enhance engineered conversions. Using CellNet, we improved B cell to macrophage conversion, transcriptionally and functionally, by knocking down predicted B cell regulators. Analyzing conversion of fibroblasts to induced hepatocytes (iHeps), CellNet revealed an unexpected intestinal program regulated by the master regulator Cdx2. We observed long-term functional engraftment of mouse colon by iHeps, thereby establishing their broader potential as endoderm progenitors and demonstrating direct conversion of fibroblasts into intestinal epithelium. Our studies illustrate how CellNet can be employed to improve direct conversion and to uncover unappreciated properties of engineered cells.
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Ingeniería Celular/métodos , Biología de Sistemas/métodos , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Ingeniería Celular/normas , Redes Reguladoras de Genes , Macrófagos/citología , Macrófagos/metabolismo , RatonesRESUMEN
Polycomb repressive complex 2 (PRC2) places H3K27me3 at developmental genes and is causally implicated in keeping bivalent genes silent. It is unclear if that silence requires minimum H3K27me3 levels and how the mark transmits faithfully across mammalian somatic cell generations. Mouse intestinal cells lacking EZH2 methyltransferase reduce H3K27me3 proportionately at all PRC2 target sites, but â¼40% uniform residual levels keep target genes inactive. These genes, derepressed in PRC2-null villus cells, remain silent in intestinal stem cells (ISCs). Quantitative chromatin immunoprecipitation and computational modeling indicate that because unmodified histones dilute H3K27me3 by 50% each time DNA replicates, PRC2-deficient ISCs initially retain sufficient H3K27me3 to avoid gene derepression. EZH2 mutant human lymphoma cells also require multiple divisions before H3K27me3 dilution relieves gene silencing. In both cell types, promoters with high basal H3K4me2/3 activate in spite of some residual H3K27me3, compared to less-poised promoters. These findings have implications for PRC2 inhibition in cancer therapy.
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Replicación del ADN , Proteína Potenciadora del Homólogo Zeste 2/fisiología , Silenciador del Gen , Código de Histonas , Regiones Promotoras Genéticas , Animales , Línea Celular Tumoral , Proteína Potenciadora del Homólogo Zeste 2/genética , Histonas/metabolismo , Humanos , Intestinos/citología , Ratones , Complejo Represivo Polycomb 2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Madre/metabolismo , Activación TranscripcionalRESUMEN
Developing and adult tissues use different cis-regulatory elements. Although DNA at some decommissioned embryonic enhancers is hypomethylated in adult cells, it is unknown whether this putative epigenetic memory is complete and recoverable. We find that, in adult mouse cells, hypomethylated CpG dinucleotides preserve a nearly complete archive of tissue-specific developmental enhancers. Sites that carry the active histone mark H3K4me1, and are therefore considered "primed," are mainly cis elements that act late in organogenesis. In contrast, sites decommissioned early in development retain hypomethylated DNA as a singular property. In adult intestinal and blood cells, sustained absence of polycomb repressive complex 2 indirectly reactivates most-and only-hypomethylated developmental enhancers. Embryonic and fetal transcriptional programs re-emerge as a result, in reverse chronology to cis element inactivation during development. Thus, hypomethylated DNA in adult cells preserves a "fossil record" of tissue-specific developmental enhancers, stably marking decommissioned sites and enabling recovery of this epigenetic memory.
Asunto(s)
Metilación de ADN/genética , Elementos de Facilitación Genéticos/genética , Epigenómica , Histonas/genética , Animales , Regulación del Desarrollo de la Expresión Génica/genética , RatonesRESUMEN
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
The cardinal properties of adult tissue stem cells are self-renewal and the ability to generate diverse resident cell types. The daily losses of terminally differentiated intestinal, skin, and blood cells require "professional" stem cells to produce replacements. This occurs by continuous expansion of stem cells and their immediate progeny, followed by coordinated activation of divergent transcriptional programs to generate stable cells with diverse functions. Other tissues turn over slowly, if at all, and vary widely in strategies for facultative stem cell activity or interconversion among mature resident cell types (transdifferentiation). Cell fate potential is programmed in tissue-specific configurations of chromatin, which restrict the complement of available genes and cis-regulatory elements, hence allowing specific cell types to arise. Using as a model the transcriptional and chromatin basis of cell differentiation and dedifferentiation in intestinal crypts, we discuss here how self-renewing and other tissues execute homeostatic and injury-responsive stem cell activity.
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Plasticidad de la Célula/genética , Plasticidad de la Célula/fisiología , Epigénesis Genética/genética , Epigénesis Genética/fisiología , Intestinos/fisiología , Células Madre/fisiología , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Linaje de la Célula/genética , Linaje de la Célula/fisiología , HumanosRESUMEN
Compacted chromatin and nucleosomes are known barriers to gene expression; the nature and relative importance of other transcriptional constraints remain unclear, especially at distant enhancers. Polycomb repressor complex 2 (PRC2) places the histone mark H3K27me3 predominantly at promoters, where its silencing activity is well documented. In adult tissues, enhancers lack H3K27me3, and it is unknown whether intergenic H3K27me3 deposits affect nearby genes. In primary intestinal villus cells, we identified hundreds of tissue-restricted enhancers that require the transcription factor (TF) CDX2 to prevent the incursion of H3K27me3 from adjoining areas of elevated basal marking into large well-demarcated genome domains. Similarly, GATA1-dependent enhancers exclude H3K27me3 from extended regions in erythroid blood cells. Excess intergenic H3K27me3 in both TF-deficient tissues is associated with extreme mRNA deficits, which are significantly rescued in intestinal cells lacking PRC2. Explaining these observations, enhancers show TF-dependent binding of the H3K27 demethylase KDM6A. Thus, in diverse cell types, certain genome regions far from promoters accumulate H3K27me3, and optimal gene expression depends on enhancers clearing this repressive mark. These findings reveal new "anti-repressive" function for hundreds of tissue-specific enhancers.
Asunto(s)
Elementos de Facilitación Genéticos/fisiología , Regulación de la Expresión Génica , Genoma/genética , Histonas/metabolismo , Intestino Delgado/metabolismo , Factores de Transcripción/metabolismo , Animales , Factor de Transcripción CDX2/genética , Células Eritroides/metabolismo , Femenino , Histona Demetilasas/metabolismo , Intestino Delgado/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Unión ProteicaRESUMEN
Pancreatic ductal adenocarcinoma (PDA) is a lethal, therapy-resistant cancer that thrives in a highly desmoplastic, nutrient-deprived microenvironment. Several studies investigated the effects of depriving PDA of either glucose or glutamine alone. However, the consequences on PDA growth and metabolism of limiting both preferred nutrients have remained largely unknown. Here, we report the selection for clonal human PDA cells that survive and adapt to limiting levels of both glucose and glutamine. We find that adapted clones exhibit increased growth in vitro and enhanced tumor-forming capacity in vivo. Mechanistically, adapted clones share common transcriptional and metabolic programs, including amino acid use for de novo glutamine and nucleotide synthesis. They also display enhanced mTORC1 activity that prevents the proteasomal degradation of glutamine synthetase (GS), the rate-limiting enzyme for glutamine synthesis. This phenotype is notably reversible, with PDA cells acquiring alterations in open chromatin upon adaptation. Silencing of GS suppresses the enhanced growth of adapted cells and mitigates tumor growth. These findings identify nongenetic adaptations to nutrient deprivation in PDA and highlight GS as a dependency that could be targeted therapeutically in pancreatic cancer patients.
Asunto(s)
Carcinoma Ductal Pancreático/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/genética , Línea Celular Tumoral , Estabilidad de Enzimas , Glutamato-Amoníaco Ligasa/genética , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/genéticaRESUMEN
BACKGROUND & AIMS: Tissue metaplasia is uncommon in adults because established cis-element programs resist rewiring. In Barrett's esophagus, the distal esophageal mucosa acquires a predominantly intestinal character, with notable gastric features, and is predisposed to developing invasive cancers. We sought to understand the chromatin underpinnings of Barrett's metaplasia and why it commonly displays simultaneous gastric and intestinal properties. METHODS: We profiled cis-regulatory elements with active histone modifications in primary human biopsy materials using chromatin immunoprecipitation followed by DNA sequencing. Mutations in Barrett's esophagus were examined in relation to tissue-specific enhancer landscapes using a random forest machine-learning algorithm. We also profiled open chromatin at single-cell resolution in primary Barrett's biopsy specimens using the assay for transposase-accessible chromatin. We used 1- and 2-color immunohistochemistry to examine protein expression of tissue-restricted genes. RESULTS: Barrett's esophagus bears epigenome fingerprints of human stomach and intestinal columnar, but not esophageal squamous, epithelia. Mutational patterns were best explained as arising on the epigenome background of active gastric cis-elements, supporting the view that adjoining stomach epithelium is a likely tissue source. Individual cells in Barrett's metaplasia coexpress gastric and intestinal genes, reflecting concomitant chromatin access at enhancers ordinarily restricted to one or the other epithelium. Protein expression of stomach-specific mucins; CLDN18; and a novel gastric marker, ANXA10, showed extensive tissue and subclonal heterogeneity of dual stomach-intestinal cell states. CONCLUSIONS: These findings reveal mixed and dynamic tissue-restricted chromatin states and phenotypic heterogeneity in Barrett's esophagus. Pervasive intragland variation argues against stem-cell governance of this phenotype.
Asunto(s)
Esófago de Barrett/genética , Esófago de Barrett/patología , Plasticidad de la Célula , Ensamble y Desensamble de Cromatina , Epigenoma , Mucosa Esofágica/patología , Células Madre/patología , Linaje de la Célula , Secuenciación de Inmunoprecipitación de Cromatina , Análisis Mutacional de ADN , Elementos de Facilitación Genéticos , Epigenómica , Predisposición Genética a la Enfermedad , Humanos , Inmunohistoquímica , Metaplasia , Mutación , Fenotipo , Análisis de la Célula IndividualRESUMEN
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
Tissues may adopt diverse strategies to establish specific transcriptional programs in daughter lineages. In intestinal crypts, enhancers for genes expressed in both major cell types appear broadly permissive in stem and specified progenitor cells. In blood, another self-renewing tissue, it is unclear when chromatin becomes permissive for transcription of genes expressed in distinct terminal lineages. Using chromatin immunoprecipitation (ChIP) combined with deep sequencing (ChIP-seq) to profile activating histone marks, we studied enhancer dynamics in primary mouse blood stem, progenitor, and specified cells. Stem and multipotent progenitor cells show scant H3K4me2 marking at enhancers bound by specific transcription factors in their committed progeny. Rather, enhancers are modulated dynamically and serially, with substantial loss and gain of H3K4me2, at each cellular transition. Quantitative analysis of these dynamics accurately modeled hematopoiesis according to Waddington's notion of epigenotypes. Delineation of enhancers in terminal blood lineages coincides with cell specification, and enhancers active in single lineages show well-positioned H3K4me2- and H3K27ac-marked nucleosomes and DNaseI hypersensitivity in other cell types, revealing limited lineage fidelity. These findings demonstrate that enhancer chronology in blood cells differs markedly from that in intestinal crypts. Chromatin dynamics in hematopoiesis provide a useful foundation to consider classical observations such as cellular reprogramming and multilineage locus priming.
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Células Sanguíneas/citología , Células Sanguíneas/metabolismo , Elementos de Facilitación Genéticos/genética , Hematopoyesis/genética , Animales , Diferenciación Celular , Linaje de la Célula , Epigénesis Genética , Histonas/metabolismo , Ratones , Células Madre/citología , Células Madre/metabolismoRESUMEN
To fulfill the lifelong need to supply diverse epithelial cells, intestinal stem cells (ISCs) rely on executing accurate transcriptional programs. This review addresses the mechanisms that control those programs. Genes that define cell behaviors and identities are regulated principally through thousands of dispersed enhancers, each individually <1 kb long and positioned from a few to hundreds of kilobases away from transcription start sites, upstream or downstream from coding genes or within introns. Wnt, Notch, and other epithelial control signals feed into these cis-regulatory DNA elements, which are also common loci of polymorphisms and mutations that confer disease risk. Cell-specific gene activity requires promoters to interact with the correct combination of signal-responsive enhancers. We review the current state of knowledge in ISCs regarding active enhancers, the nucleosome modifications that may enable appropriate and hinder inappropriate enhancer-promoter contacts, and the roles of lineage-restricted transcription factors.
Asunto(s)
Diferenciación Celular/fisiología , Epigénesis Genética , Intestinos/citología , Células Madre/fisiología , Animales , Regulación de la Expresión Génica , HumanosRESUMEN
Cells differentiate when transcription factors bind accessible cis-regulatory elements to establish specific gene expression programs. In differentiating embryonic stem cells, chromatin at lineage-restricted genes becomes sequentially accessible, probably by means of 'pioneer' transcription factor activity, but tissues may use other strategies in vivo. Lateral inhibition is a pervasive process in which one cell forces a different identity on its neighbours, and it is unclear how chromatin in equipotent progenitors undergoing lateral inhibition quickly enables distinct, transiently reversible cell fates. Here we report the chromatin and transcriptional underpinnings of differentiation in mouse small intestine crypts, where notch signalling mediates lateral inhibition to assign progenitor cells into absorptive or secretory lineages. Transcript profiles in isolated LGR5(+) intestinal stem cells and secretory and absorptive progenitors indicated that each cell population was distinct and the progenitors specified. Nevertheless, secretory and absorptive progenitors showed comparable levels of H3K4me2 and H3K27ac histone marks and DNase I hypersensitivity--signifying accessible, permissive chromatin-at most of the same cis-elements. Enhancers acting uniquely in progenitors were well demarcated in LGR5(+) intestinal stem cells, revealing early priming of chromatin for divergent transcriptional programs, and retained active marks well after lineages were specified. On this chromatin background, ATOH1, a secretory-specific transcription factor, controls lateral inhibition through delta-like notch ligand genes and also drives the expression of numerous secretory lineage genes. Depletion of ATOH1 from specified secretory cells converted them into functional enterocytes, indicating prolonged responsiveness of marked enhancers to the presence or absence of a key transcription factor. Thus, lateral inhibition and intestinal crypt lineage plasticity involve interaction of a lineage-restricted transcription factor with broadly permissive chromatin established in multipotent stem cells.
Asunto(s)
Diferenciación Celular/genética , Cromatina/genética , Cromatina/metabolismo , Regulación de la Expresión Génica , Intestino Delgado/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Linaje de la Célula/genética , Desoxirribonucleasa I/metabolismo , Elementos de Facilitación Genéticos/genética , Enterocitos/citología , Enterocitos/metabolismo , Femenino , Histonas/metabolismo , Intestino Delgado/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores Notch/metabolismo , Células Madre/citología , Células Madre/metabolismo , Transcripción GenéticaRESUMEN
The stomach, an organ derived from foregut endoderm, secretes acid and enzymes and plays a key role in digestion. During development, mesenchymal-epithelial interactions drive stomach specification, patterning, differentiation and growth through selected signaling pathways and transcription factors. After birth, the gastric epithelium is maintained by the activity of stem cells. Developmental signals are aberrantly activated and stem cell functions are disrupted in gastric cancer and other disorders. Therefore, a better understanding of stomach development and stem cells can inform approaches to treating these conditions. This Review highlights the molecular mechanisms of stomach development and discusses recent findings regarding stomach stem cells and organoid cultures, and their roles in investigating disease mechanisms.
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Células Madre/citología , Gastropatías/patología , Estómago/embriología , Animales , Tipificación del Cuerpo , Endodermo/embriología , Humanos , Transducción de Señal , Estómago/anatomía & histología , Gastropatías/congénitoRESUMEN
Diverse functions of the homeodomain transcription factor BARX1 include Wnt-dependent, non-cell autonomous specification of the stomach epithelium, tracheo-bronchial septation, and Wnt-independent expansion of the spleen primordium. Tight spatio-temporal regulation of Barx1 levels in the mesentery and stomach mesenchyme suggests additional roles. To determine these functions, we forced constitutive BARX1 expression in the Bapx1 expression domain, which includes the mesentery and intestinal mesenchyme, and also examined Barx1(-/)(-) embryos in further detail. Transgenic embryos invariably showed intestinal truncation and malrotation, in part reflecting abnormal left-right patterning. Ectopic BARX1 expression did not affect intestinal epithelium, but intestinal smooth muscle developed with features typical of the stomach wall. BARX1, which is normally restricted to the developing stomach, drives robust smooth muscle expansion in this organ by promoting proliferation of myogenic progenitors at the expense of other sub-epithelial cells. Undifferentiated embryonic stomach and intestinal mesenchyme showed modest differences in mRNA expression and BARX1 was sufficient to induce much of the stomach profile in intestinal cells. However, limited binding at cis-regulatory sites implies that BARX1 may act principally through other transcription factors. Genes expressed ectopically in BARX1(+) intestinal mesenchyme and reduced in Barx1(-/-) stomach mesenchyme include Isl1, Pitx1, Six2 and Pitx2, transcription factors known to control left-right patterning and influence smooth muscle development. The sum of evidence suggests that potent BARX1 functions in intestinal rotation and stomach myogenesis occur through this small group of intermediary transcription factors.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Vólvulo Intestinal/patología , Intestinos/anomalías , Intestinos/embriología , Desarrollo de Músculos , Músculo Liso/embriología , Estómago/embriología , Factores de Transcripción/metabolismo , Animales , Proliferación Celular , Epitelio/metabolismo , Mucosa Gástrica/metabolismo , Marcación de Gen , Proteínas de Homeodominio/genética , Mucosa Intestinal/metabolismo , Vólvulo Intestinal/genética , Mesenterio/metabolismo , Mesodermo/metabolismo , Ratones , Desarrollo de Músculos/genética , Músculo Liso/metabolismo , Especificidad de Órganos , Factores de Transcripción/deficiencia , Factores de Transcripción/genéticaRESUMEN
Distinct groups of transcription factors (TFs) assemble at tissue-specific cis-regulatory sites, implying that different TF combinations may control different genes and cellular functions. Within such combinations, TFs that specify or maintain a lineage and are therefore considered master regulators may play a key role. Gene enhancers often attract these tissue-restricted TFs, as well as TFs that are expressed more broadly. However, the contributions of the individual TFs to combinatorial regulatory activity have not been examined critically in many cases in vivo. We address this question using a genetic approach in mice to inactivate the intestine-specifying and intestine-restricted factor CDX2 alone or in combination with its more broadly expressed partner factors, GATA4 and HNF4A. Compared with single mutants, each combination produced significantly greater defects and rapid lethality through distinct anomalies. Intestines lacking Gata4 and Cdx2 were deficient in crypt cell replication, whereas combined loss of Hnf4a and Cdx2 specifically impaired viability and maturation of villus enterocytes. Integrated analysis of TF binding and of transcripts affected in Hnf4a;Cdx2 compound-mutant intestines indicated that this TF pair controls genes required to construct the apical brush border and absorb nutrients, including dietary lipids. This study thus defines combinatorial TF activities, their specific requirements during tissue homeostasis, and modules of transcriptional targets in intestinal epithelial cells in vivo.
Asunto(s)
Factor de Transcripción GATA4/metabolismo , Regulación de la Expresión Génica , Factor Nuclear 4 del Hepatocito/metabolismo , Proteínas de Homeodominio/metabolismo , Mucosa Intestinal/metabolismo , Factores de Transcripción/metabolismo , Secuencias de Aminoácidos , Animales , Sitios de Unión , Factor de Transcripción CDX2 , Diferenciación Celular , Inmunoprecipitación de Cromatina , Enterocitos/citología , Perfilación de la Expresión Génica , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
BACKGROUND: High-level physical activity is associated with lower colorectal cancer (CRC) mortality, likely through insulin sensitization. Insulin receptor substrate 1 (IRS1) is a mediator of insulin and insulin-like growth factor (IGF) signaling pathways, and its down-regulation is associated with insulin resistance. Therefore, we hypothesized that tumor IRS1 expression status might modify cellular sensitivity to insulin and IGF, and the prognostic association of physical activity. METHODS: We assessed IRS1 expression level in 371 stage I-III rectal and colon cancers in the Nurses' Health Study and the Health Professionals Follow-up Study by immunohistochemistry. In survival analysis, Cox proportional hazards model was used to assess an interaction between post-diagnosis physical activity (ordinal scale of sex-specific quartiles Q1 to Q4) and IRS1 expression (ordinal scale of negative, low, and high), controlling for potential confounders, including microsatellite instability, CpG island methylator phenotype, long interspersed nucleotide element-1 (LINE-1) methylation level, and KRAS, BRAF, and PIK3CA mutation status. RESULTS: There was a statistically significant interaction between post-diagnosis physical activity and tumor IRS1 expression in CRC-specific mortality analysis (P interaction = 0.005). Multivariable hazard ratio (95% confidence interval) for higher post-diagnosis physical activity (Q3-Q4 vs. Q1-Q2) was 0.15 (0.02-1.38) in the IRS1-negative group, 0.45 (0.19-1.03) in the IRS1-low group, and 1.32 (0.50-3.53) in the IRS1-high group. CONCLUSIONS: The association of post-diagnosis physical activity with colorectal carcinoma patient survival may differ by tumor IRS1 expression level. If validated, tumor IRS1 expression status may serve as a predictive marker to identify subgroups of patients who might gain greater survival benefit from an increased level of exercise.