Identification of TWIST1 transcriptional targets in the cranial mesoderm [ChIP-Seq]
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ABSTRACT: TWIST1, a basic helix-loop-helix transcription factor is essential for the development of cranial mesoderm and cranial neural crest-derived craniofacial structures. Our previous work showed that, in the absence of TWIST1, some cells within the cranial mesoderm adopt an abnormal epithelial configuration. Here, we show by transcriptome analysis that loss of TWIST1 in the cranial mesoderm is accompanied by a reduction in the expression of genes that are associated with cell-extracellular matrix interactions and the acquisition of mesenchymal characteristics. By comparing the transcriptional profiles of cranial mesoderm-specific Twist1 loss-of-function mutant and control mouse embryos, we identified a set of genes that are both TWIST1-dependent and predominantly expressed in the mesoderm. By ChIP-seq in a cell line model of a TWIST1-dependent mesenchymal state, we identified, among the downstream genes, three direct transcriptional targets of TWIST1: Ddr2, Pcolce and Tgfbi. Our findings show that the mesenchymal properties of the cranial mesoderm is likely to be regulated by a network of TWIST1 targets genes that influence the extracellular matrix and cell-matrix interactions, and collectively they are required for the morphogenesis of the craniofacial structures. Chromatin extracts were subject to chromatin immunoprecipitation with anti TWIST1 monoclonal antibody (ChIP-seq). Input chromatin, not subject to ChIP was used as the negative control. Two independent replicate experiments were performed. Purified, immunoprecipitated DNA was sequenced at Australian Genome Research facility.
Project description:TWIST1, a basic helix-loop-helix transcription factor is essential for the development of cranial mesoderm and cranial neural crest-derived craniofacial structures. Our previous work showed that, in the absence of TWIST1, some cells within the cranial mesoderm adopt an abnormal epithelial configuration. Here, we show by transcriptome analysis that loss of TWIST1 in the cranial mesoderm is accompanied by a reduction in the expression of genes that are associated with cell-extracellular matrix interactions and the acquisition of mesenchymal characteristics. By comparing the transcriptional profiles of cranial mesoderm-specific Twist1 loss-of-function mutant and control mouse embryos, we identified a set of genes that are both TWIST1-dependent and predominantly expressed in the mesoderm. By ChIP-seq in a cell line model of a TWIST1-dependent mesenchymal state, we identified, among the downstream genes, three direct transcriptional targets of TWIST1: Ddr2, Pcolce and Tgfbi. Our findings show that the mesenchymal properties of the cranial mesoderm is likely to be regulated by a network of TWIST1 targets genes that influence the extracellular matrix and cell-matrix interactions, and collectively they are required for the morphogenesis of the craniofacial structures. Cranial neural crest and cranial mesoderm cells were isolated by flow sorting of GFP reporter-labelled cells collected from heads of E9.5 mouse embryos. Three replicates were independently isolated and hybridized to Illumina mouse WG v 2.0 chips
Project description:TWIST1, a basic helix-loop-helix transcription factor is essential for the development of cranial mesoderm and cranial neural crest-derived craniofacial structures. Our previous work showed that, in the absence of TWIST1, some cells within the cranial mesoderm adopt an abnormal epithelial configuration. Here, we show by transcriptome analysis that loss of TWIST1 in the cranial mesoderm is accompanied by a reduction in the expression of genes that are associated with cell-extracellular matrix interactions and the acquisition of mesenchymal characteristics. By comparing the transcriptional profiles of cranial mesoderm-specific Twist1 loss-of-function mutant and control mouse embryos, we identified a set of genes that are both TWIST1-dependent and predominantly expressed in the mesoderm. By ChIP-seq in a cell line model of a TWIST1-dependent mesenchymal state, we identified, among the downstream genes, three direct transcriptional targets of TWIST1: Ddr2, Pcolce and Tgfbi. Our findings show that the mesenchymal properties of the cranial mesoderm is likely to be regulated by a network of TWIST1 targets genes that influence the extracellular matrix and cell-matrix interactions, and collectively they are required for the morphogenesis of the craniofacial structures.
Project description:TWIST1, a basic helix-loop-helix transcription factor is essential for the development of cranial mesoderm and cranial neural crest-derived craniofacial structures. Our previous work showed that, in the absence of TWIST1, some cells within the cranial mesoderm adopt an abnormal epithelial configuration. Here, we show by transcriptome analysis that loss of TWIST1 in the cranial mesoderm is accompanied by a reduction in the expression of genes that are associated with cell-extracellular matrix interactions and the acquisition of mesenchymal characteristics. By comparing the transcriptional profiles of cranial mesoderm-specific Twist1 loss-of-function mutant and control mouse embryos, we identified a set of genes that are both TWIST1-dependent and predominantly expressed in the mesoderm. By ChIP-seq in a cell line model of a TWIST1-dependent mesenchymal state, we identified, among the downstream genes, three direct transcriptional targets of TWIST1: Ddr2, Pcolce and Tgfbi. Our findings show that the mesenchymal properties of the cranial mesoderm is likely to be regulated by a network of TWIST1 targets genes that influence the extracellular matrix and cell-matrix interactions, and collectively they are required for the morphogenesis of the craniofacial structures.
Project description:TWIST1, a basic helix-loop-helix transcription factor is essential for the development of cranial mesoderm and cranial neural crest-derived craniofacial structures. Our previous work showed that, in the absence of TWIST1, some cells within the cranial mesoderm adopt an abnormal epithelial configuration. Here, we show by transcriptome analysis that loss of TWIST1 in the cranial mesoderm is accompanied by a reduction in the expression of genes that are associated with cell-extracellular matrix interactions and the acquisition of mesenchymal characteristics. By comparing the transcriptional profiles of cranial mesoderm-specific Twist1 loss-of-function mutant and control mouse embryos, we identified a set of genes that are both TWIST1-dependent and predominantly expressed in the mesoderm. By ChIP-seq in a cell line model of a TWIST1-dependent mesenchymal state, we identified, among the downstream genes, three direct transcriptional targets of TWIST1: Ddr2, Pcolce and Tgfbi. Our findings show that the mesenchymal properties of the cranial mesoderm is likely to be regulated by a network of TWIST1 targets genes that influence the extracellular matrix and cell-matrix interactions, and collectively they are required for the morphogenesis of the craniofacial structures.
Project description:TWIST1, a basic helix-loop-helix transcription factor is essential for the development of cranial mesoderm and cranial neural crest-derived craniofacial structures. Our previous work showed that, in the absence of TWIST1, some cells within the cranial mesoderm adopt an abnormal epithelial configuration. Here, we show by transcriptome analysis that loss of TWIST1 in the cranial mesoderm is accompanied by a reduction in the expression of genes that are associated with cell-extracellular matrix interactions and the acquisition of mesenchymal characteristics. By comparing the transcriptional profiles of cranial mesoderm-specific Twist1 loss-of-function mutant and control mouse embryos, we identified a set of genes that are both TWIST1-dependent and predominantly expressed in the mesoderm. By ChIP-seq in a cell line model of a TWIST1-dependent mesenchymal state, we identified, among the downstream genes, three direct transcriptional targets of TWIST1: Ddr2, Pcolce and Tgfbi. Our findings show that the mesenchymal properties of the cranial mesoderm is likely to be regulated by a network of TWIST1 targets genes that influence the extracellular matrix and cell-matrix interactions, and collectively they are required for the morphogenesis of the craniofacial structures.
Project description:TWIST1, a basic helix-loop-helix transcription factor is essential for the development of cranial mesoderm and cranial neural crest-derived craniofacial structures. Our previous work showed that, in the absence of TWIST1, some cells within the cranial mesoderm adopt an abnormal epithelial configuration. Here, we show by transcriptome analysis that loss of TWIST1 in the cranial mesoderm is accompanied by a reduction in the expression of genes that are associated with cell-extracellular matrix interactions and the acquisition of mesenchymal characteristics. By comparing the transcriptional profiles of cranial mesoderm-specific Twist1 loss-of-function mutant and control mouse embryos, we identified a set of genes that are both TWIST1-dependent and predominantly expressed in the mesoderm. By ChIP-seq in a cell line model of a TWIST1-dependent mesenchymal state, we identified, among the downstream genes, three direct transcriptional targets of TWIST1: Ddr2, Pcolce and Tgfbi. Our findings show that the mesenchymal properties of the cranial mesoderm is likely to be regulated by a network of TWIST1 targets genes that influence the extracellular matrix and cell-matrix interactions, and collectively they are required for the morphogenesis of the craniofacial structures. For microarray analysis of CM-CKO embryos, embryo heads of four genotypes were collected at E8.5 (5-7 somites) and E9.5 (18- 20 somites): CM-CKO (Twist1flox/del; Mesp1Cre/+), CM-Het (Twist1flox/wt; Mesp1Cre/+), Het (Twist1flox/del; Mesp1+/+) and Control (Twist1flox/wt; Mesp1+/+). Sample sizes for E8.5 embryos were as follows: Control, n=4 CM-CKO, n=4; Het, n=3; CM-Hets, n=3).
Project description:TWIST1, a basic helix-loop-helix transcription factor is essential for the development of cranial mesoderm and cranial neural crest-derived craniofacial structures. Our previous work showed that, in the absence of TWIST1, some cells within the cranial mesoderm adopt an abnormal epithelial configuration. Here, we show by transcriptome analysis that loss of TWIST1 in the cranial mesoderm is accompanied by a reduction in the expression of genes that are associated with cell-extracellular matrix interactions and the acquisition of mesenchymal characteristics. By comparing the transcriptional profiles of cranial mesoderm-specific Twist1 loss-of-function mutant and control mouse embryos, we identified a set of genes that are both TWIST1-dependent and predominantly expressed in the mesoderm. By ChIP-seq in a cell line model of a TWIST1-dependent mesenchymal state, we identified, among the downstream genes, three direct transcriptional targets of TWIST1: Ddr2, Pcolce and Tgfbi. Our findings show that the mesenchymal properties of the cranial mesoderm is likely to be regulated by a network of TWIST1 targets genes that influence the extracellular matrix and cell-matrix interactions, and collectively they are required for the morphogenesis of the craniofacial structures. For microarray analysis of CM-CKO embryos, embryo heads of four genotypes were collected at E8.5 (5-7 somites) and E9.5 (18- 20 somites): CM-CKO (Twist1flox/del; Mesp1Cre/+), CM-Het (Twist1flox/wt; Mesp1Cre/+), Het (Twist1flox/del; Mesp1+/+) and Control (Twist1flox/wt; Mesp1+/+). Sample sizes for E9.5 were as follows: Control, n=3; CM-CKO, n=4; Het, n=4; CM-Het, n=4). RNA was extracted using the RNeasy Micro kit (Qiagen) and samples sent to the Australian Genome Research Foundation for labelling and hybridization.
Project description:During Epithelial-Mesenchymal Transition (EMT), apical-basal polarized epithelial cells are converted to front-to-back polarized mesenchymal cells that only form loose cell-cell adhesions. These phenotypic changes are accompanied by acquisition of increased motility and invasiveness. EMT programs are orchestrated by pleiotropic transcription factors (TFs), such as Twist1 and Snail1 and effect morphogenetic steps during embryogenesis, including mesoderm formation and neural crest migration. EMTs have also been implicated in the acquisition of aggressive traits by carcinoma cells, including the ability to complete several steps of the metastatic cascade as well as propagation of the tumor by single cells (clonogenicity), a defining trait of tumor-initiating or cancer stem cells. However, the molecular links between the expression of EMT-TFs, the process of EMT and acquisition of clonogenicity remain obscure. Using inducible Twist1 or Snail1 expressed in CD24-positive mammary epithelial cells, we show that clonal growth in anchorage independence and EMT are induced sequentially and independently: clonogenic potential is induced prior to EMT and requires transient TF-activity. By contrast, EMT depends on continuous TF-activation over a longer period. In 3D-collagen assays, continuous Twist1 activity suppresses colony formation, whereas transient activation induces highly invasive growth independently of EMT. In conclusion, our results demonstrate that transient Twist1 activation suffices to drive tumor progression of CD24-positive breast epithelial cells, assessed by invasive as well as anchorage-independent clonal growth, whereas chronic Twist1 exposure can suppress these traits of aggressive tumor cells. We performed gene expression microarray analysis on CD24high and CD24negative populations derived from HMLE-Twist1-ER or HMLE-Snail1-ER cell lines upon various culture conditions