1,721,077 research outputs found
Sex-reversing mutations affect the architecture of SRY-DNA complexes
No full textThe testis determining factor, SRY, is a DNA binding protein that causes a large distortion of its DNA target sites. We have analysed the biochemical properties of the DNA binding domains (HMG-boxes) of mutant SRY proteins from five patients with complete gonadal dysgenesis. The mutant proteins fall into three categories: two bind and bend DNA almost normally, two bind inefficiently but bend DNA normally and one binds DNA with almost normal affinity but produces a different angle. The mutations with moderate effect on complex formation can be transmitted to male progeny, the ones with severe effects on either binding or bending are de novo. The angle induced by SRY depends on the exact DNA sequence and thus adds another level of discrimination in target site recognition. These data suggest that the exact spatial arrangement of the nucleoprotein complex organized by SRY is essential for sex determination
Mouse Dax1 expression is consistent with a role in sex determination as well as in adrenal and hypothalamus function.
No full textDisruption of the LF-A1 and LF-B1 binding sites in the human alpha-1-antitrypsin gene has a differential effect during development in transgenic mice.
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Evolutionary conservation in the DNA-binding and -bending properties of HMG-boxes from SRY proteins of primates
No full textMammalian sex determination is caused by the Y-chromosome gene SRY, which encodes a protein containing a DNA-binding domain (HMG-box) of about 70 amino acids (aa). The HMG-box is very conserved in a wide variety of mammals; conversely, the flanking non-box regions show a high degree of aa sequence divergence, even between closely related species, The HMG-box of human SRY binds sequence-specifically to linear DNA and produces a sharp bend; it also interacts with high affinity to kinked DNA structures irrespective of their sequences. Point mutations associated with sex reversal in XY human females fall within the HMG-box and either affect the affinity for DNA or modify the geometry of the DNA-protein complex. Here, we show that the DNA-binding and -bending properties of the HMG-boxes of SRY from human and seven different primates are extremely similar to each other. Together with other data, this suggests that the inability of mouse and human SRY to substitute for each other is due to differences in the conserved HMG-box, rather than the non-conserved flanking sequences
Disruption of the LF-A1 and LF-B1 binding sites in the human alpha-1-antitrypsin gene has a differential effect during development in transgenic mice.
No full textPrevious work in transfected cell lines and in nuclear extracts has led to the identification of two cis-acting elements important for transcription of the human alpha-1-antitrypsin (A1AT) gene, which bind to two liver specific trans-acting factors, LF-A1 and LF-B1. Mutations EM3 and PM1, which abolish the binding of LF-A1 and LF-B1 respectively, drastically reduce transcription activity of the A1AT gene in vitro and in cell culture. The same mutants have now been introduced in a larger DNA context and their effect has been tested in transgenic mice. A stretch of DNA was constructed which carries two transcriptional units: 18 kb of the human retinol binding protein (RBP) gene, driving the expression of the bacterial chloramphenicol acetyl transferase, linked to 17.5 kb containing the entire A1AT coding sequence with additional 5' and 3' flanking sequences. Transcription from the RBP promoter was shown to predominate in liver, and could be used as an internal marker of 'active copy number'. Mutations in the A1AT gene promoter were introduced by homologous recombination in bacterial cells. The results show that base pair substitutions in the binding site for LF-A1 and LF-B1 drastically reduce transcription in non-hepatic adult tissues, yolk sac, and fetal liver, whereas only LF-B1 binding site mutations have a marked, albeit variable, effect in adult liver
Region-specific roles of SOX9 in forebrain oligodendrogenesis and development of the dentate gyrus
Full text linkDuring central nervous system (CNS) development, expression of the SOXE transcription factor SOX9 is up-regulated in neuroepithelial progenitors (NEPs) by E11.5 in the mouse and it is required for their induction and maintenance, as well as for their switch from neurogenesis to gliogenesis. To better characterize its role, Sox9 was conditionally deleted earlier and specifically in the CNS using Sox1Cre, which is active before the onset of Sox9 expression. Oligodendrocyte precursors are initially missing in Sox9fl/fl;Sox1Cre/+ embryos, but recover perinatally. This is due to functional compensation by SOX8, a SOXE member closely related to SOX9, because Sox9fl/fl; Sox1Cre/+;Sox8-/- embryos do not recover oligodendrocytes. Therefore, SOX8 may allow late embryonic oligodendrogenesis in absence of SOX9, by a yet unknown mechanism. The origin of the recovered oligodendrocytes in Sox9fl/fl;Sox1Cre/+ embryos is currently being investigated with in utero electroporation. Sox9fl/fl;Sox1Cre/+ embryos also show defective development of the archicortex, the embryonic structure giving rise to the dentate gyrus (DG). Generation of mature granule neurons and their progenitors is unaffected; however, these fail to reach the developing DG and accumulate close to the ventricle, suggesting a migration defect. In Sox9fl/fl;Sox1Cre/+ mutants, we observed a dramatic reduction of the radial glial scaffold and its astrocytic progenitors, which originate from the adjacent cortical hem (CH) and support granule neuron progenitors migration during DG development. Consequently, in adult Sox9fl/fl;Sox1Cre/+ mice, DG size and neural stem cell numbers are significantly reduced, which may explain their defective memory forming abilities. Therefore, these results suggest that SOX9 is required in the archicortex for formation of the radial glial scaffold, and consequently for the migration of DG granule neurons and their progenitors. This hypothesis is further confirmed by analysis of Sox9fl/fl;NestinCre embryos, where radial glial scaffold and consequently granule neuron progenitor migration, are less affected compared to Sox9fl/fl;Sox1Cre/+. Because Nestin-Cre is acting later in the archicortex compared to Sox1Cre, this highlights an early critical requirement of SOX9 for induction of astrocyte progenitors in the archicortex and, subsequently, DG development. CH-specific deletion of Sox9 is currently being performed to assess this hypothesis. In conclusion, analysis of Sox9 deletion during forebrain development highlights unique temporal- and region-specific functions of this transcription factors for specification of glial cells
Identification and characterisation of novel Wnt regulators in colorectal cancer
Full text linkColorectal cancer (CRC) is a multistep mutagenic process that provides tumour cells with a growth advantage for clonal expansion. The Wnt signalling pathway is crucial for regulating ISC maintenance and differentiation, whilst aberrant Wnt activation is a hallmark of human CRC. Recent advances in genome-scale analysis of large cohorts of CRCs have uncovered a large number of novel mutations that had not been previously characterised. Interestingly, hierarchical clustering analyses of the related expression data reveals a cluster of differentially expressed genes that is enriched in the previously reported ISC markers, Wnt targets and/or Wnt inhibitors. We hypothesised that some of the genes within the cluster are expressed at the intestinal crypt bottoms to antagonise Wnt signalling to form part of a negative feedback mechanism for ISC homeostasis. The aim of my project was to screen for novel Wnt regulators within this gene cluster. Comprehensive analysis of the cluster identified two potential Wnt regulators: the SH3 domain-binding protein 4 (SH3BP4) and the serine/threonine kinase ZAK/MAP3K20. Here we show that both Sh3bp4 and Zak are expressed at the ISC compartment and are enriched in Apc-mutated tumours. Deletion of Zak enhances radiation-induced intestinal regeneration, suggesting a growth inhibitory role of ZAK in the intestine. Loss of SH3BP4 in vivo exacerbates the Apcmin tumour phenotype by promoting the total number of adenomas and high-grade dysplasia. Mechanistically, ZAK and SH3BP4 suppresses Wnt signalling downstream of -catenin phosporylation. The data revealed novel roles for both ZAK and SH3BP4 in intestinal homeostasis and tumourigenesis via Wnt/-catenin signalling regulation. Taken together, the findings highlight the crucial role that these newly identified genes play in Wnt negative feedback mechanisms in intestinal regeneration and tumourigenesis, which may provide new insights into targeted therapeutic intervention
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