Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Grhl2 regulated genes in the mouse lung epithelial MLE15 cell line

ABSTRACT: The Grainyhead family of transcription factors controls morphogenesis and differentiation of epithelial cell layers in multicellular organisms by regulating cell junction- and proliferation-related genes. Grainyhead-like 2 (Grhl2) is expressed in developing mouse lung epithelium and is required for normal lung organogenesis. The specific epithelial cells expressing Grhl2 and the genes regulated by Grhl2 in normal lungs are mostly unknown. In these studies, we identified the NK2 homeobox 1 transcription factor (Nkx2-1) as a direct transcriptional target of Grhl2. By binding and transcriptional assays, and by confocal microscopy we showed that these two transcription factors form a positive feed-back loop in vivo and in cell lines, and are co-expressed in lung bronchiolar and alveolar type II cells. The morphological changes observed in flattening lung alveolar type II cells in culture are associated with down-regulation of Grhl2 and Nkx2-1. Reduction of Grhl2 in lung epithelial cell lines results in lower expression levels of Nkx2-1 and of known Grhl2 target genes. By microarray analysis we identified that in addition to Cadherin1 and Claudin4, Grhl2 regulates other cell interaction genes such as semaphorins and their receptors, which also play a functional role in developing lung epithelium. Impaired collective cell migration observed in Grhl2 knockdown cell monolayers is associated with reduced expression of these genes and may contribute to the altered epithelial phenotype reported in Grhl2 mutant mice. Thus, Grhl2 functions at the nexus of a novel regulatory network, connecting lung epithelial cell identity, migration and cell-cell interactions. To identify genes regulated by GRHL2 in lung epithelial cells, we performed cDNA microarray analyses in MLE15 cells transduced with Grhl2-shRNA and compared to a non-silencing control. Independent transductions of MLE15 cells using Grhl2-shRNA (n=3) and a non-silencing control (n=2) were analyzed.

ORGANISM(S): Mus musculus

SUBMITTER: Maria Ramirez

PROVIDER: E-GEOD-40729 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

The transcription factors Grainyhead-like 2 and NK2-homeobox 1 form a regulatory loop that coordinates lung epithelial cell morphogenesis and differentiation.

Varma Saaket S Cao Yuxia Y Tagne Jean-Bosco JB Lakshminarayanan Meenakshi M Li Jun J Friedman Thomas B TB Morell Robert J RJ Warburton David D Kotton Darrell N DN Ramirez Maria I MI

The Journal of biological chemistry 20120906 44

The Grainyhead family of transcription factors controls morphogenesis and differentiation of epithelial cell layers in multicellular organisms by regulating cell junction- and proliferation-related genes. Grainyhead-like 2 (Grhl2) is expressed in developing mouse lung epithelium and is required for normal lung organogenesis. The specific epithelial cells expressing Grhl2 and the genes regulated by Grhl2 in normal lungs are mostly unknown. In these studies we identified the NK2-homeobox 1 transcr ...[more]

PMID: 22955271

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Project description:Using a retrovirus-mediated cDNA expression cloning approach we identified the grainyhead-like 2 (GRHL2) transcription factor as novel protooncogene. Overexpression of GRHL2 in NIH3T3 cells induced striking morphological changes, an increase in cell proliferation, anchorage-independent growth, and tumor growth in vivo. By combining a microarray analysis and a phylogenetic footprinting analysis with various biochemical assays we identified the epidermal growth factor receptor family member Erbb3 as a novel GRHL2 target gene. In breast cancer cell lines, shRNA-mediated knockdown of GRHL2 expression or functional inactivation of GRHL2 using dominant-negative GRHL2 proteins induce downregulation of ERBB3 gene expression, a striking reduction in cell proliferation and morphological and phenotypical alterations characteristic of an epithelial-to-mesenchymal transition (EMT), thus implying dual roles of GRHL2 in breast carcinogenesis. Interestingly, we could further demonstrate that expression of GRHL2 is directly suppressed by the transcription factor zinc-finger-enhancer binding protein 1 (ZEB1) which in turn is a direct target for repression by GRHL2, suggesting that the EMT transcription factors GRHL2 and ZEB1 form a double negative regulatory feedback loop in breast cancer cells. Finally, a comprehensive immunohistochemical analysis of GRHL2 expression in primary breast cancers showed loss of GRHL2 expression at the invasive front of primary tumors. A pathophysiological relevance of GRHL2 in breast cancer metastasis is further demonstrated by our finding of a statistically significant association between loss of GRHL2 expression in primary breast cancers and lymph-node metastasis. We thus demonstrate a crucial role of GRHL2 in breast carcinogenesis. To evaluate the role of the GRHL2 transcription factor in carcinogenessis we overexpressed the human GRHL2 protein in NIH3T3 cells. Parental NIH3T3 cells do not express the orthologic protein. Alterations in genome-wide transcriptional profiles were determined by microarray analysis.

Project description:Healthy placental development is essential for reproductive success; failure of the feto-maternal interface results in preeclampsia and intrauterine growth retardation. We found that grainyhead-like 2 (GRHL2), a CP2-type transcription factor, is highly expressed in chorionic trophoblast cells, including basal chorionic trophoblast (BCT) cells located at the chorioallantoic interface in murine placentas. Placentas from Grhl2-deficient mouse embryos displayed defects in BCT cell polarity and basement membrane integrity at the chorioallantoic interface, as well as a severe disruption of labyrinth branchingmorphogenesis.Selective Grhl2 inactivation only in epiblastderived cells rescued all placental defects but phenocopied intraembryonic defects observed in global Grhl2 deficiency, implying the importance of Grhl2 activity in trophectoderm-derived cells. ChIPseq identified 5282 GRHL2 binding sites in placental tissue. By integrating these data with placental gene expression profiles, we identified direct and indirect Grhl2 targets and found a marked enrichment of GRHL2 binding adjacent to genes downregulated in Grhl2−/− placentas, which encoded known regulators of placental development and epithelial morphogenesis. These genes included that encoding the serine protease inhibitor Kunitz type 1 (Spint1), which regulates BCT cell integrity and labyrinth formation. In human placenta, we found that human orthologs of murine GRHL2 and its targets displayed co-regulation and were expressed in trophoblast cells in a similar domain as in mouse placenta. Our data indicate that a conserved Grhl2-coordinated gene network controls trophoblast branching morphogenesis, thereby facilitating development of the site of feto-maternal exchange. This might have implications for syndromes related to placental dysfunction. In vivo genome-wide examination of binding sites of the transcription factor GRHL2 by ChIP-seq using wild-type murine E17.5 placenta tissue. Two samples in total: one GRHL2 ChIP sample and one IgG ChIP sample using wild-type placentas tissue as antibody control.

Project description:The airways of the human lung are lined by an epithelium made up of ciliated and secretory luminal cells and undifferentiated p63+ Krt5+ basal cells. The integrity of this epithelium and its ability to act as a selective barrier are critical for normal lung function. In other epithelia there is evidence that transcription factors of the evolutionarily conserved grainyheadlike (GRHL) family play key roles in co-ordinating the expression of numerous proteins required for epithelial morphogenesis, differentiation, remodeling and repair. However, little is known about their function in the adult lung. Here, we focus on the role of GRHL2 in primary human bronchial epithelial (HBE) cells, using either shRNA or a dominant negative protein (DN-GRHL2) to inhibit its function. We follow changes in epithelial phenotype, and in gene transcription using RNA-seq or microarray analysis, both in undifferentiated basal cells and in cells differentiating in air-liquid interface culture into a mucociliary epithelium with transepithelial electrical resistance. We identify several hundreds of genes that are directly or indirectly regulated by GRHL2. Using ChIP-seq to map sites of GRHL2 binding in the basal cells we identify 7,687 potential primary targets, and confirm that GRHL2 binding is strongly enriched near GRHL-regulated genes. Different subsets of the large cohort of potential GRHL2 targets appear to be active in basal and differentiated cells. Taken together, the results strongly support the hypothesis that GRHL2 plays a key role in regulating many physiological functions of human airway epithelium, including those involving cell adhesion, polarity and morphogenesis. Frozen primary human bronchial epithelial (HBE) cells were obtained from three donors and grown to confluence in standard culture conditions. Cells were crosslinked with formaldehyde and collected in aliquots of ~20M cells per donor. ChIP-seq for GRHL2 (Antibody: Sigma HPA004820) was then performed relative to an input control for each donor.

Dataset Information

Grhl2 regulated genes in the mouse lung epithelial MLE15 cell line

Publications

The transcription factors Grainyhead-like 2 and NK2-homeobox 1 form a regulatory loop that coordinates lung epithelial cell morphogenesis and differentiation.

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