Cryosectioning the intestinal crypt-villus axis: An ex vivo method to study the dynamics of epigenetic modifications from stem cells to differentiated cells

AbstractThe intestinal epithelium is a particularly attractive biological adult model to study epigenetic mechanisms driving adult stem cell renewal and cell differentiation. Since epigenetic modifications are dynamic, we have developed an original ex vivo approach to study the expression and epigenetic profiles of key genes associated with either intestinal cell pluripotency or differentiation by isolating cryosections of the intestinal crypt-villus axis. Gene expression, DNA methylation and histone modifications were studied by qRT-PCR, methylation-specific PCR and micro-chromatin immunoprecipitation, respectively. Using this approach, it was possible to identify segment-specific methylation and chromatin profiles. We show that (i) expression of intestinal stem cell markers (Lgr5, Ascl2) exclusively in the crypt is associated with active histone marks, (ii) promoters of all pluripotency genes studied and transcription factors involved in intestinal cell fate (Cdx2) harbour a bivalent chromatin pattern in the crypts and (iii) expression of differentiation markers (Muc2, Sox9) along the crypt-villus axis is associated with DNA methylation. Hence, using an original model of cryosectioning along the crypt-villus axis that allows in situ detection of dynamic epigenetic modifications, we demonstrate that regulation of pluripotency and differentiation markers in healthy intestinal mucosa involves different and specific epigenetic mechanisms

Transcript Profiling of Elf5+/− Mammary Glands during Pregnancy Identifies Novel Targets of Elf5

Background: Elf5, an epithelial specific Ets transcription factor, plays a crucial role in the pregnancy-associated development of the mouse mammary gland. Elf5 2/2 embryos do not survive, however the Elf5 +/2 mammary gland displays a severe pregnancy-associated developmental defect. While it is known that Elf5 is crucial for correct mammary development and lactation, the molecular mechanisms employed by Elf5 to exert its effects on the mammary gland are largely unknown. Principal Findings: Transcript profiling was used to investigate the transcriptional changes that occur as a result of Elf5 haploinsufficiency in the Elf5 +/2 mouse model. We show that the development of the mouse Elf5 +/2 mammary gland is delayed at a transcriptional and morphological level, due to the delayed increase in Elf5 protein in these glands. We also identify a number of potential Elf5 target genes, including Mucin 4, whose expression, is directly regulated by the binding of Elf5 to an Ets binding site within its promoter. Conclusion: We identify novel transcriptional targets of Elf5 and show that Muc4 is a direct target of Elf5, further elucidatin

Complex interplay between β-catenin signalling and Notch effectors in intestinal tumorigenesis

International audienceAims The activation of β-catenin signalling is a key step in intestinal tumorigenesis. Interplay between the β-catenin and Notch pathways during tumorigenesis has been reported, but the mechanisms involved and the role of Notch remain unclear. Methods Notch status was analysed by studying expression of the Notch effector Hes1 and Notch ligands/receptors in human colorectal cancer (CRC) and mouse models of Apc mutation. A genetic approach was used, deleting the Apc and RBP-J or Atoh1 genes in murine intestine. CRC cell lines were used to analyse the control of Hes1 and Atoh1 by β-catenin signalling. Results Notch signalling was found to be activated downstream from β-catenin. It was rapidly induced and maintained throughout tumorigenesis. Hes1 induction was mediated by β-catenin and resulted from both the induction of the Notch ligand/receptor and Notch-independent control of the Hes1 promoter by β-catenin. Surprisingly, the strong phenotype of unrestricted proliferation and impaired differentiation induced by acute Apc deletion in the intestine was not rescued by conditional Notch inactivation. Hyperactivation of β-catenin signalling overrode the forced differention induced by Notch inhibition, through the downregulation of Atoh1, a key secretory determinant factor downstream of Notch. This process involves glycogen synthase kinase 3 β (GSK3β) and proteasome-mediated degradation. The restoration of Atoh1 expression in CRC cell lines displaying β-catenin activation was sufficient to increase goblet cell differentiation, whereas genetic ablation of Atoh1 greatly increased tumour formation in Apc mutant mice. Conclusion Notch signalling is a downstream target of β-catenin hyperactivation in intestinal tumorigenesis. However, its inhibition had no tumour suppressor effect in the context of acute β-catenin activation probably due to the downregulation of Atoh1. This finding calls into question the use of γ-secretase inhibitors for the treatment of CRC and suggests that the restoration of Atoh1 expression in CRC should be considered as a therapeutic approach

Mucin Muc2 Deficiency and Weaning Influences the Expression of the Innate Defense Genes Reg3β, Reg3γ and Angiogenin-4

Background Mucin Muc2 is the structural component of the intestinal mucus layer. Absence of Muc2 leads to loss of this layer allowing direct bacterial-epithelial interactions. We hypothesized that absence of the mucus layer leads to increased expression of innate defense peptides. Specifically, we aimed to study the consequence of Muc2 deficiency (Muc2-/-) on the expression of regenerating islet-derived protein 3 beta (Reg3ß), regenerating islet-derived protein 3 gamma (Reg3¿), and angiogenin-4 (Ang4) in the intestine shortly before and after weaning. Methods Intestinal tissues of Muc2-/- and wild-type (WT) mice were collected at postnatal day 14 (P14, i.e. pre-weaning) and P28 (i.e. post-weaning). Reg3ß, Reg3¿, and Ang4 expression was studied by quantitative real-time PCR, Western-blot, in situ hybridization, and immunohistochemistry. Results Reg3ß and Reg3¿ were expressed by diverging epithelial cell types; namely enterocytes, Paneth cells, and goblet cells. Additionally, Ang4 expression was confined to Paneth cells and goblet cells. Expression of Reg3ß, Reg3¿, and Ang4 differed between WT and Muc2-/- mice before and after weaning. Interestingly, absence of Muc2 strongly increased Reg3ß and Reg3¿ expression in the small intestine and colon. Finally, morphological signs of colitis were only observed in the distal colon of Muc2-/- mice at P28, where and when expression levels of Reg3ß, Reg3¿, and Ang4 were the lowest. Conclusions Expression of Reg3 proteins and Ang4 by goblet cells point to an important role for goblet cells in innate defense. Absence of Muc2 results in up-regulation of Reg3ß and Reg3¿ expression, suggesting altered bacterial-epithelial signaling and an innate defense response in Muc2-/- mice. The inverse correlation between colitis development and Reg3ß, Reg3¿, and Ang4 expression levels might point toward a role for these innate defense peptides in regulating intestinal inflammatio

Analysis of the proximal promoter of the human colon-specific B4GALNT2 (Sda synthase) gene: B4GALNT2 is transcriptionally regulated by ETS1

13siopenBackground: The Sda antigen and corresponding biosynthetic enzyme B4GALNT2 are primarily expressed in normal colonic mucosa and are down-regulated to a variable degree in colon cancer tissues. Although their expression profile is well studied, little is known about the underlying regulatory mechanisms. Methods: To clarify the molecular basis of Sda expression in the human gastrointestinal tract, we investigated the transcriptional regulation of the human B4GALNT2 gene. The proximal promoter region was delineated using luciferase assays and essential trans-acting factors were identified through transient overexpression and silencing of several transcription factors. Results: A short cis-regulatory region restricted to the −72 to +12 area upstream of the B4GALNT2 short-type transcript variant contained the essential promoter activity that drives the expression of the human B4GALNT2 regardless of the cell type. We further showed that B4GALNT2 transcriptional activation mostly requires ETS1 and to a lesser extent SP1. Conclusions: Results presented herein are expected to provide clues to better understand B4GALNT2 regulatory mechanisms.openWavelet-Vermuse C.; Groux-Degroote S.; Vicogne D.; Cogez V.; Venturi G.; Trinchera M.; Brysbaert G.; Krzewinski-Recchi M.-A.; Bachir E.H.; Schulz C.; Vincent A.; Van Seuningen I.; Harduin-Lepers A.Wavelet-Vermuse, C.; Groux-Degroote, S.; Vicogne, D.; Cogez, V.; Venturi, G.; Trinchera, M.; Brysbaert, G.; Krzewinski-Recchi, M. -A.; Bachir, E. H.; Schulz, C.; Vincent, A.; Van Seuningen, I.; Harduin-Lepers, A

OCT-1 is over-expressed in intestinal metaplasia and intestinal gastric carcinomas and binds to, but does not transactivate, CDX2 in gastric cells

Intestinal metaplasia (IM) is a preneoplastic lesion of the stomach in which there is transdifferentiation of the gastric mucosa to an intestinal phenotype. The caudal-related homeobox gene CDX2 encodes an intestine-specific transcription factor crucial for the regulation of proliferation and differentiation of intestinal cells. In addition, CDX2 is involved in the induction of IM in the stomach. The aim of this study was to access the putative involvement of OCT-1 in the induction of CDX2 expression de novo in gastric mucosa leading to the onset of IM. OCT-1 protein expression was evaluated by immunohistochemistry in 31 biopsies with chronic gastritis, 15 biopsies with foci of IM and adjacent gastric mucosa and 42 gastric carcinomas. Furthermore, we evaluated OCT-1 binding by electrophoretic mobility shift assay and activation of the CDX2 promoter by co-transfecting a CDX2 promoter/reporter construct with an OCT-1 expression vector in two gastric carcinoma cell lines, GP220 and MKN45. Our results show that OCT-1 is expressed in chronic gastritis, particularly when it is adjacent to IM and is expressed in 87% of IM foci. Furthermore, 74% of the gastric carcinomas were positive for OCT-1 and a strong association was observed between OCT-1 expression and intestinal-type carcinoma. We identified that OCT-1 binds to the CDX2 promoter, although we could not see a transactivation effect in gastric carcinoma cell lines. In conclusion, we observed increased OCT-1 expression in IM and in intestinal gastric carcinomas and identified the capacity of OCT-1 to bind to the CDX2 promoter, although we could not demonstrate a direct effect of OCT-1 in the transactivation of CDX2. Copyright (c) 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

Transcriptional regulation of the 11p15 mucin genes. towards new biological tools in human therapy, in inflammatory diseases and cancer?

International audienc

Qu’est ce que l’épigénétique ? Stratégies d’étude et application au cancer

International audienc

Régulation des gènes de mucines humaines et murines par le TGF-b et par les facteurs de transcription impliqués dans la différenciation gastro-intestinale

LILLE2-BU Santé-Recherche (593502101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Comment on: Functional MUC4 suppress epithelial-mesenchymal transition in lung adenocarcinoma metastasis. Gao L, Liu J, Zhang B, Zhang H, Wang D, Zhang T, Liu Y, Wang C. Tumour Biol. 2013, in press

Comment onFunctional MUC4 Suppress Epithelial-Mesenchymal Transition in Lung Adenocarcinoma MetastasisL Gao et al. Tumour Biol 35 (2), 1335-41. Feb 2014. PMID 24037917.The mucin MUC4 is a high molecular weight membrane-bound transmembrane glycoprotein that is frequently detected in invasive and metastatic cancer. The overexpression of M …International audienceGao and collaborators (Tumour Biol, 2013) have investigated the role of mucin 4 (MUC4) in lung cancer and have concluded that a loss of MUC4 results in epithelial mesenchymal transition via beta-catenin nuclear translocation and that MUC4 expression is correlated with a risk of lymph node metastasis in a cohort of 20 lung adenocarcinoma patients. This surprising analysis is contradictory to most of the scientific knowledge and literature regarding MUC4 contribution in epithelial cancers that is very hardly discussed in their manuscript