Les gènes TWIST : cibles transcriptionnelles des gènes MYC dans le neuroblastome

The N-MYC gene is amplified in 20-25 % of human neuroblastoma, and this amplification is associated with poor clinical outcome. We previously reported aconstant deregulation of TWIST1 in synergy with N-MYC in aggressive stage IVneuroblastoma harboring N-MYC amplification (Valsesia-Wittmann et al., 2004). We demonstrated here that specifically in neuroblastoma cells, TWIST1 and TWIST2 are negatively or positively regulated depending on Myc oncoproteins dosage, thus being a putative Myc transcriptional target. We confirmed by EMSA that Myc proteins could bind TWIST1 promoter. We further highlighted TWIST1 maintenance of expression strictly Myc dependant. Therefore, we propose that deregulation and amplification of Myc oncoproteins in aggressive neuroblastoma tumors induce selective expression and maintenance of TWIST1 oncogene, responsible forapoptosis resistanceDans les neuroblastomes, le gène N-MYC est amplifié dans 20-25 % des cas,associé à un mauvais pronostic. Au laboratoire, nous avions préalablement montré que la dérégulation de l’expression du gène TWIST1 était corrélée à celle de N-MYC dans les neuroblastomes agressifs de stade IV avec une amplification de N-MYC (Valsesia-Wittmann et al., 2004). Au cours de ma thèse, j’ai pu mettre en évidence que les gènes TWIST1 et TWIST2 étaient régulés positivement ou négativement de façon dose dépendante par les oncoprotéines Myc. De façon intéressante, le maintien de l’expression de TWIST1 est dépendant de l’expression des protéines Myc. Ces résultats suggèrent que la dérégulation et l’amplification des oncoprotéines Myc dans les neuroblastomes N-MYC Amplifiés pourraient permettre l’induction sélective et le maintien de l’expression de l’oncogène TWIST, agissant comme facteur de survie

TWIST genes : transcriptional targets of N-MYC and c-MYC in neuroblastoma

Dans les neuroblastomes, le gène N-MYC est amplifié dans 20-25 % des cas,associé à un mauvais pronostic. Au laboratoire, nous avions préalablement montré que la dérégulation de l’expression du gène TWIST1 était corrélée à celle de N-MYC dans les neuroblastomes agressifs de stade IV avec une amplification de N-MYC (Valsesia-Wittmann et al., 2004). Au cours de ma thèse, j’ai pu mettre en évidence que les gènes TWIST1 et TWIST2 étaient régulés positivement ou négativement de façon dose dépendante par les oncoprotéines Myc. De façon intéressante, le maintien de l’expression de TWIST1 est dépendant de l’expression des protéines Myc. Ces résultats suggèrent que la dérégulation et l’amplification des oncoprotéines Myc dans les neuroblastomes N-MYC Amplifiés pourraient permettre l’induction sélective et le maintien de l’expression de l’oncogène TWIST, agissant comme facteur de survie.The N-MYC gene is amplified in 20-25 % of human neuroblastoma, and this amplification is associated with poor clinical outcome. We previously reported aconstant deregulation of TWIST1 in synergy with N-MYC in aggressive stage IVneuroblastoma harboring N-MYC amplification (Valsesia-Wittmann et al., 2004). We demonstrated here that specifically in neuroblastoma cells, TWIST1 and TWIST2 are negatively or positively regulated depending on Myc oncoproteins dosage, thus being a putative Myc transcriptional target. We confirmed by EMSA that Myc proteins could bind TWIST1 promoter. We further highlighted TWIST1 maintenance of expression strictly Myc dependant. Therefore, we propose that deregulation and amplification of Myc oncoproteins in aggressive neuroblastoma tumors induce selective expression and maintenance of TWIST1 oncogene, responsible forapoptosis resistanc

Histone H1 of Saccharomyces cerevisiae Inhibits Transcriptional Silencing

Eukaryotic genomes contain euchromatic regions, which are transcriptionally active, and heterochromatic regions, which are repressed. These domains are separated by “barrier elements”: DNA sequences that protect euchromatic regions from encroachment by neighboring heterochromatin. To identify proteins that play a role in the function of barrier elements we have carried out a screen in S. cerevisiae. We recovered the gene HHO1, which encodes the yeast ortholog of histone H1, as a high-copy modifier of barrier activity. Histone H1 is a linker histone that binds the outside of nucleosomes and modifies chromatin dynamics. Here we show that Hho1p reinforces the action of several types of barrier elements, and also inhibits silencing on its own

Slug controls stem/progenitor cell growth dynamics during mammary gland morphogenesis.

Morphogenesis results from the coordination of distinct cell signaling pathways controlling migration, differentiation, apoptosis, and proliferation, along stem/progenitor cell dynamics. To decipher this puzzle, we focused on epithelial-mesenchymal transition (EMT) "master genes". EMT has emerged as a unifying concept, involving cell-cell adhesion, migration and apoptotic pathways. EMT also appears to mingle with stemness. However, very little is known on the physiological role and relevance of EMT master-genes. We addressed this question during mammary morphogenesis. Recently, a link between Slug/Snai2 and stemness has been described in mammary epithelial cells, but EMT master genes actual localization, role and targets during mammary gland morphogenesis are not known and we focused on this basic question.Using a Slug-lacZ transgenic model and immunolocalization, we located Slug in a distinct subpopulation covering about 10-20% basal cap and duct cells, mostly cycling cells, coexpressed with basal markers P-cadherin, CK5 and CD49f. During puberty, Slug-deficient mammary epithelium exhibited a delayed development after transplantation, contained less cycling cells, and overexpressed CK8/18, ER, GATA3 and BMI1 genes, linked to luminal lineage. Other EMT master genes were overexpressed, suggesting compensation mechanisms. Gain/loss-of-function in vitro experiments confirmed Slug control of mammary epithelial cell luminal differentiation and proliferation. In addition, they showed that Slug enhances specifically clonal mammosphere emergence and growth, cell motility, and represses apoptosis. Strikingly, Slug-deprived mammary epithelial cells lost their potential to generate secondary clonal mammospheres.We conclude that Slug pathway controls the growth dynamics of a subpopulation of cycling progenitor basal cells during mammary morphogenesis. Overall, our data better define a key mechanism coordinating cell lineage dynamics and morphogenesis, and provide physiological relevance to broadening EMT pathways

Slug controls cell proliferation, apoptosis, motility and cell lineage commitment.

<p>A-B. CommaDβ cells were transfected with control (Ct) and Slug expression vector (Slug), or with si control (siCt) and two distinct anti-Slug siRNA (siSlug1 and siSlug2). After fixation, DAPI staining and immulocalization (A), average percentage of cells expressing KI67 or caspase 3 were calculated and reported (B). C. Slug controls mammary epithelial cell motility. Cell motility was estimated using a wound healing assay in confluent CommaDβ cells. Ability to repopulate the wound area was estimated by measuring total uncovered substrate area after 48 h, reported to 0 h. Cells were transfected as indicated with Slug full-length cDNA (duplicate Slug1 and Slug2), control vector (duplicate Ct1 and Ct2), and with anti-Slug siRNA (siSlug1 and siSlug2), and two distinct controls siCt1 and siCt2. Experiment was repeated three times. D. Slug controls mammary epithelial cell commitment. Basal/Luminal differentiation phenotype was evaluated by immunofluorescence using cytokeratin expression pattern. CK5 (basal) was found to be significantly overexpressed in cells overexpressing Slug (Slug) as compared to control (Ct), when CK8 (luminal) was downregulated in cells overexpressing Slug.</p

Slug regulates mammary epithelial cell differentiation pathways.

<p>We quantified expression levels of various genes involved in mammary epithelial cell differentiation in WT and KO mammary glands from same litter mice. Two KO mice from the same litter were used for the 5 weeks study and the expression level averaged. A. EMT-master genes from Snail and Twist families were screened at both stages. B. Basal (CK14) and luminal (CK 8, 18, 19) cytokeratin genes were screened to evaluate the relative epithelial fraction and putative regulation at 5 weeks. Genes reflecting proliferation (PCNA1), stem/progenitor or luminal phenotype (Bmi1, CD133, Gata 3, Elf5, Muc1, ER, PR) were also screened at 5 weeks. Because CK average expression level was significantly increased, we related the expression levels from strictly epithelial genes (Gata3, Elf5, Muc1) to the mean CK expression level. Finally, members of the Sox family of transcription factors were also screened. Gray areas cover gene expression ratios considered as not significantly modified (ratio between 0.5 and 1.5). C. PCNA1 expression was quantified by calculating the percentage of cells expressing PCNA1 among 1–500 cells in growing ducts paraffin sections from 5–8 weeks old mice, showing a strong decrease in SlugKO cells (*Student test, p<0,05). D. PCNA1 and CK5 were visualized by double-immunofluorescence including DAPI co-staining on paraffin sections from wild-type (WT) or Slug-deficient mouse (KO) ducts, as indicated. E. ER was colocalized with CK5 and DAPI to show a relative overexpression in Slug-deficient mouse (KO) ducts. Arrows points to ER+ CK5- cells identified as luminal cells.</p

Slug, Twist and Zeb1 localization during mammary gland morphogenesis.

<p>A. At 3 weeks, Slug expression (blue X-Gal staining) is visible in the primary duct on a mammary gland wholemount (a, arrowhead), and section (b). Lower sectioning level (c) show Slug expression in polarizing basal epithelial cells during initial tubulogenesis (arrowhead). B. During mammary tubulogenesis at 6 weeks, Slug is found in growing tubules in wholemount (a) and tubule sections (b–c) involving epithelial cells (c, arrow) and peri-tubular mesenchymal cells (b, arrow and c, arrowhead). Cell location is better defined at higher magnification, including basal epithelial (arrow) and mesenchymal (arrowhead) cells (c). Tubule terminal end buds (TEB) sections also show expression by mostly basal epithelial cap cells (d–e, arrows). Co-labeling using DAPI (blue) and antibodies against Slug (red) and Pcad (P-cad, green) demonstrates a basal localization for Slug in 6 weeks (f) and adult (g) tubules. In addition, Twist (h) and Zeb1 (i) were located in tubules from 10 weeks-old mammary glands. Colabeling with CK5 show that only stromal cells express Twist (arrow). Conversely, CK8 colabeling show that Zeb1 is expressed by basal and luminal epithelial cells (arrows).</p

Mammary glands from Slug-deficient mice exhibit several defects.

<p>A. Mammary glands from wild type (WT) or SlugKO (KO) mice were analyzed in wholemount preparations during tubulogenesis (6 weeks old mice). KO mammary gland displayed a growth delay visible (double arrows) downstream and upstream from the primary duct (arrow). More than 6 mice were examined with a similar phenotype. Size bar = 1 mm. B. At later stages (8 weeks), wholemount preparations show a recovery linked to overbranching visible at higher magnification (inserts). C–D. Orthotopic grafts combining explants from wild type (WT) or SlugKO (KO) were examined 15 weeks after transplantation during estrus cycle (C), including diestrus phase (D). A significant increase (*Student test, p<0.05) in the terminal branching pattern is found in explants from KO mammary glands at all stages. E. Mammary gland phenotype was quantified by several ways. Average length of 12 primary ducts was estimated based on wholemount preparations from three 6 weeks old-mammary glands. In addition, 5 SlugKO and 9 WT (10 weeks old) mammary glands were weighed and compared. We also determined the number of branching nodes, discriminating between primary and secondary branching, significantly more abundant in KO explants (*Student test, p<0.05). F. Finally, we quantified in the explants the increase in the number of terminal buds in homozygous (black, KO) as compared to wild-type (white, WT) transplants, as determined in 5 KO explants from mammary glands at diestrus stage or estrus phases. Transplanted mammary glands from Slug-deficient mice also express extraneous branching. Internodal segments were sorted into short segments (1–4 mm), medium segments (5–7 mm) and longer segments (8–15 mm). Short segments, resulting from an increase in branching nodes generation, were clearly predominant in KO transplants (*Student test, p<0,05).</p

Slug controls mammosphere growth.

<p>A. Epithelial cells isolated from mammary glands were seeded individually in low-adherence culture wells. All wells were screened after 3 days. Wells containing 1–4 live cells (microsphere) were enumerated. After three weeks, Some of the microspheres evolved into mammospheres that were monitored. Values are reported as percentage compared to the number of live seeded cells or to the number of microspheres at 3 days. Bar = 50 µm. B. Cells from wild-type (WT) or Slug-deficient mouse (KO) were cloned to grow into primary microspheres and mammospheres. For secondary mammospheres, 10–20 primary mammospheres were pooled and enzymatically dissociated before individual cloning and further screening. Experiment was repeated three times. C. CommaDβ cells were transfected with control (siCt) or two distinct anti-Slug siRNA (siSlug1 and siSlug2) and seeded individually in 96 well low-adherence dishes. All wells were screened after 3 days. Microspheres and mammospheres were reported separately after 3 weeks. A significant decrease in viability was observed after 3 days in cells transfected with anti-Slug siRNA. This deficit was even stronger three weeks later (*Student test, p<0.05). CommaDβ cells were also transfected with control (Ct) or Slug full-length cDNA (Slug) expression vectors and seeded individually in 96 well dishes as previously. The number of mammospheres showed a significant and early increase in survival and growth for Slug-overexpressing mammospheres (*Student test, p<0.05).</p