SIP1/ZEB2 induces EMT by repressing genes of different epithelial cell–cell junctions

SIP1/ZEB2 is a member of the δEF-1 family of two-handed zinc finger nuclear factors. The expression of these transcription factors is associated with epithelial mesenchymal transitions (EMT) during development. SIP1 is also expressed in some breast cancer cell lines and was detected in intestinal gastric carcinomas, where its expression is inversely correlated with that of E-cadherin. Here, we show that expression of SIP1 in human epithelial cells results in a clear morphological change from an epithelial to a mesenchymal phenotype. Induction of this epithelial dedifferentiation was accompanied by repression of several cell junctional proteins, with concomitant repression of their mRNA levels. Besides E-cadherin, other genes coding for crucial proteins of tight junctions, desmosomes and gap junctions were found to be transcriptionally regulated by the transcriptional repressor SIP1. Moreover, study of the promoter regions of selected genes by luciferase reporter assays and chromatin immunoprecipitation shows that repression is directly mediated by SIP1. These data indicate that, during epithelial dedifferentiation, SIP1 represses in a coordinated manner the transcription of genes coding for junctional proteins contributing to the dedifferentiated state; this repression occurs by a general mechanism mediated by Smad Interacting Protein 1 (SIP1)-binding sites

Detection and validation of copy number variation in X-linked mental retardation

Studies to identify the genetic defects associated with X-linked mental retardation (XLMR) in males have revealed tens of genes important for normal brain development and cognitive functioning in men. Despite extensive efforts in breakpoint cloning of chromosomal rearrangements and mutation screening of candidate genes on the X chromosome, still many XLMR families and sporadic cases remain unsolved. It is now clear that submicroscopic copy number changes on the X chromosome can explain about 5% of these idiopathic cases. Interestingly, beside gene deletions, an increase in gene dosage due to genomic duplications seems to contribute to causality more often than expected. Since larger duplications on the X chromosome are tolerated compared to deletions, they often harbour more than one gene hampering the identification of the causal gene. In contrast to copy number variations (CNVs) on autosomes, most disease-associated CNVs on the X chromosome in males are inherited from their mothers who normally do not present with any clinical symptoms due to non-random X inactivation. Here, we review the different methods applied to study copy number alterations on the X chromosome in patients with cognitive impairment, discuss those CNVs that are associated with disease and elaborate on the genes and mechanisms involved. At the end, we will resume in vivo assays to study the relation of CNVs on the X chromosome and mental disability.status: publishe

Behavior of the different proteins of the cadherin–catenin complex upon SIP1 induction

<p><b>Copyright information:</b></p><p>Taken from "SIP1/ZEB2 induces EMT by repressing genes of different epithelial cell–cell junctions"</p><p>Nucleic Acids Research 2005;33(20):6566-6578.</p><p>Published online 24 Nov 2005</p><p>PMCID:PMC1298926.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Immunofluorescence microscopy of non-induced and induced DLD1Tr21/WTSIP1 cells using antibodies specific for adherens junction components. E-cadherin as well as αE-catenin, p120ctn and β-catenin became nearly undetectable at cell–cell contacts in the SIP1-induced cells. () Western blot analysis of the non-induced and induced DLD1Tr21/WTSIP1 cell line. E-cadherin and αE-catenin were downregulated at the protein level in the SIP1-expressing cells. β-catenin protein levels were unaltered in the SIP1-induced compared to non-induced cells. Protein expression of p120ctn isoform 1 was upregulated and that of isoform 3 downregulated after SIP1 induction (: addition of Dox every 2 days, : washing away Dox from the cell culture medium)

The mitochondrial solute carrier SLC25A5 at Xq24 is a novel candidate gene for non-syndromic intellectual disability

Loss-of-function mutations in several different neuronal pathways have been related to intellectual disability (ID). Such mutations often are found on the X chromosome in males since they result in functional null alleles. So far, microdeletions at Xq24 reported in males always have been associated with a syndromic form of ID due to the loss of UBE2A. Here, we report on overlapping microdeletions at Xq24 that do not include UBE2A or affect its expression, in patients with non-syndromic ID plus some additional features from three unrelated families. The smallest region of overlap, confirmed by junction sequencing, harbors two members of the mitochondrial solute carrier family 25, SLC25A5 and SLC25A43. However, identification of an intragenic microdeletion including SLC25A43 but not SLC25A5 in a healthy boy excluded a role for SLC25A43 in cognition. Therefore, our findings point to SLC25A5 as a novel gene for non-syndromic ID. This highly conserved gene is expressed ubiquitously with high levels in cortex and hippocampus, and a presumed role in mitochondrial exchange of ADP/ATP. Our data indicate that SLC25A5 is involved in memory formation or establishment, which could add mitochondrial processes to the wide array of pathways that regulate normal cognitive functions.status: publishe

Point mutation I634A in the glucocorticoid receptor causes embryonic lethality by reduced ligand binding

The glucocorticoid (GC) receptor (GR) is essential for normal development and in the initiation of inflammation. Healthy GRdim/dim mice with reduced dimerization propensity due to a point mutation (A465T) at the dimer interface of the GR DNA-binding domain (DBD) (here GRD/D) have previously helped to define the functions of GR monomers and dimers. Since GRD/D retains residual dimerization capacity, here we generated the dimer-nullifying double mutant GRD+L/D+L mice, featuring an additional mutation (I634A) in the ligand-binding domain (LBD) of GR. These mice are perinatally lethal, as are GRL/L mice (these mice have the I634A mutation but not the A465T mutation), displaying improper lung and skin formation. Using embryonic fibroblasts, high and low doses of dexamethasone (Dex), nuclear translocation assays, RNAseq, dimerization assays, and ligand-binding assays (and Kd values), we found that the lethal phenotype in these mice is due to insufficient ligand binding. These data suggest there is some correlation between GR dimerization potential and ligand affinity. We conclude that even a mutation as subtle as I634A, at a position not directly involved in ligand interactions sensu stricto, can still influence ligand binding and have a lethal outcome

Ubiquitin Ligase HUWE1 Regulates Axon Branching through the Wnt/beta-Catenin Pathway in a Drosophila Model for Intellectual Disability

Contains fulltext : 126190.pdf (publisher's version ) (Open Access)We recently reported that duplication of the E3 ubiquitin ligase HUWE1 results in intellectual disability (ID) in male patients. However, the underlying molecular mechanism remains unknown. We used Drosophila melanogaster as a model to investigate the effect of increased HUWE1 levels on the developing nervous system. Similar to the observed levels in patients we overexpressed the HUWE1 mRNA about 2-fold in the fly. The development of the mushroom body and neuromuscular junctions were not altered, and basal neurotransmission was unaffected. These data are in agreement with normal learning and memory in the courtship conditioning paradigm. However, a disturbed branching phenotype at the axon terminals of the dorsal cluster neurons (DCN) was detected. Interestingly, overexpression of HUWE1 was found to decrease the protein levels of dishevelled (dsh) by 50%. As dsh as well as Fz2 mutant flies showed the same disturbed DCN branching phenotype, and the constitutive active homolog of beta-catenin, armadillo, could partially rescue this phenotype, our data strongly suggest that increased dosage of HUWE1 compromises the Wnt/beta-catenin pathway possibly by enhancing the degradation of dsh

HUWE1 affects DCN axon branching.

<p>(A-C’) Axon projections of the DCN in the optic lobe, visualized via staining against mCD8-GFP. Lo = lobula, Me = Medulla. (A) Representative image of a control brain: w;UAS-mCD8-GFP/+;control^VK31/atoGal4-14a,UAS-LacZ. (A’) Magnification of the branching area in the white square shown in panel A. (B,C) Overexpression of HUWE1 in w;UAS-HUWE1^VK37/UAS-mCD8-GFP;atoGal4-14a,UAS-LacZ/+ and w;UAs-mCD8-GFP/+;UAS-HUWE1^VK31/atoGal4-14a,UAS-LacZ flies does not affect axon number in the medulla, but leads to increased axon branching at the 3^rd branching point. (B’,C’) Magnification of the branching area in the white squares shown in panels B and C. </p