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

MUC4 (mucin 4, cell surface associated)

Review on MUC4 (mucin 4, cell surface associated), with data on DNA, on the protein encoded, and where the gene is implicated

Tumour growth and resistance to gemcitabine of pancreatic cancer cells are decreased by AP-2α overexpression

International audienceBACKGROUND: Activator protein-2alpha (AP-2alpha) is a transcription factor that belongs to the family of AP-2 proteins that have essential roles in tumorigenesis. Indeed, AP-2alpha is considered as a tumour-suppressor gene in different tissues such as colonic, prostatic or breast epithelial cells. Moreover, AP-2alpha also participates in the control of colon and breast cancer cells sensitivity towards chemotherapeutic drugs. Despite its potential interest, very few data are available regarding the roles of AP-2alpha in pancreatic cancer. METHODS: We have developed a stable pancreatic CAPAN-1 cell line overexpressing AP-2alpha. Consequences of overexpression were studied in terms of in vivo cell growth, gene expression, migration capacity and chemosensitivity. RESULTS: In vivo tumour growth of CAPAN-1 cells overexpressing AP-2alpha was significantly decreased by comparison to control cells. An altered expression pattern of cell cycle-controlling factors (CDK-4, CDK-6, cyclin-G1, p27(kip1) and p57(kip2)) was observed in AP-2alpha-overexpressing clones by microarrays and western blot analysis. Promoter activity and ChIP analysis indicated that AP-2alpha induces p27(kip1) protein levels by direct binding to and transactivation of its promoter. Moreover, AP-2alpha overexpression increased the chemosensitivity of CAPAN-1 cells to low doses of gemcitabine and reduced their in vitro migration capacity. CONCLUSION: Our data suggested that AP-2alpha overexpression could be exploited to decrease in vivo tumour growth of pancreatic cancer cells and to increase their sensitivity to gemcitabine

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

Free-energy distribution of binary protein-protein binding suggests cross-species interactome differences

Major advances in large-scale yeast two hybrid (Y2H) screening have provided a global view of binary protein-protein interactions across species as dissimilar as human, yeast, and bacteria. Remarkably, these analyses have revealed that all species studied have a degree distribution of protein-protein binding that is approximately scale-free (varies as a power law) even though their evolutionary divergence times differ by billions of years. The universal power-law shows only the surface of the rich information harbored by these high-throughput data. We develop a detailed mathematical model of the protein-protein interaction network based on association free energy, the biochemical quantity that determines protein-protein interaction strength. This model reproduces the degree distribution of all of the large-scale Y2H data sets available and allows us to extract the distribution of free energy, the likelihood that a pair of proteins of a given species will bind. We find that across-species interactomes have significant differences that reflect the strengths of the protein-protein interaction. Our results identify a global evolutionary shift: more evolved organisms have weaker binary protein-protein binding. This result is consistent with the evolution of increased protein unfoldedness and challenges the dogma that only specific protein-protein interactions can be biologically functional..Comment: 31 pages, 5 figure