The BAF complex does not mediate GLI repression (RNA-Seq)
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE224505 | GEO | 2024/02/03
REPOSITORIES: GEO
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Project description:Purpose: This study seeks to identify SMARCC1-bound regions in the mouse limb. Methods: To identify SMARCC1 bound regions in the limb, we performed Cut&Run on pooled anterior and posterior wild-type forelimbs at E11.5 (43-44s; 3 replicates each) and on whole forelimb pairs at E9.5 (21-24s; 2 replicates). We identified 28,082 SMARCC1-bound regions in the anterior forelimb at E11.5, 42,530 regions in the E11.5 posterior limb, and 10,792 SMARCC1-bound regions at E9.5 (FDR<0.05). Results: We find that SMARCC1 is bound to most limb enhancer regions at late stages (E11.5). Additionally, We find that SMARCC1 is bound to the majority of HH-responsive GLI enhancers at E11.5 but only a small portion of GLI enhancers at E9.5.
2024-02-03 | GSE224507 | GEO
Project description:Purpose: This study seeks to determine whether GLI3 is required to recruit the SMARCC1 complex to GLI enhancers in the limb. Methods: To determine if Gli3 is required to recruit SMARCC1 to its anhancers, we performed differential chromatin binding to compare SMARCC1 binding in control and Gli3 mutants. We performed Cut&Run for SMARCC1 binding on individually genotyped E11.5 (40-43s) anterior forelimb pairs from control (Gli3+/+; 3 replicates) and Gli3 mutant (Gli3-/-; 4 replicates) embryos. Results: We found that there is no major difference in SMARCC1 binding in Gli3-mutants compared to controls.
2024-02-03 | GSE224508 | GEO
Project description:Purpose: This study seeks to determine whether Smarcc1 mediates GLI repression through chromatin compaction in the limb. Methods: To determine if Smarcc1 mediated GLI repression through chormatin compaction, we used genomic approached to determine changes in chromatin accessibility in control and Smarcc1-conditional mutants. We performed ATAC-seq on individually genotyped anterior forelimbs at E11.5 (40-48s) on control (2 replicates) and PrxCre/+;Smarcc1c/c (3 replicates) embryos. From this experiment we identified ~10,000 differentially accessible regions (FDR<0.05). Results: We find that SMARCC1 is required to maintain chromatin accessibility at thousands of regions within the mouse limb, including most HH-responsive GLI enhancers
2024-02-03 | GSE224506 | GEO
Project description:Purpose: Most Hedgehog responsive gene expression is mediated through GLI de-repression. Additionally GLI -repression is proposed to play roles in limb pre-patterning before HH pathway activation. This study evaluates if GLI repression is established prior to HH pathway activation. Methods: To determine if GLI-repression is established prior to pathway activation, we used genomic approaches to study GLI-mediated repression using the mouse developing limb as a model. We identified pre-HH (E9.25, 21-23S) and post-HH (E10.5, 32-25S) GLI3 binding regions using CUT&RUN for endogenous FLAG-tagged GLI3 proteins. Using a combination of ChIP-seq, CUT&RUN, CUT&Tag, ATAC-seq and RNA-seq, we tested whether loss of Gli3 prior to HH signaling was able to de-repress genes and enhancers, as it does after HH signaling. Results: Prior to HH signaling, GLI3 binds to poised, accessible regions with histone deacetylase (HDAC) proteins, similar to post-HH signaling. Despite GLI3 binding to most regions as it does in the post-HH limb, loss of Gli3 is unable to prematurely active target genes or enhancers. Furthermore, we find that GLI3-dependent chromatin compaction does not occur until roughly 10 hours after HH signaling would have normally been induced. Collectively, these results support that GLI repressor proteins are inert prior to HH pathway activation.
2022-01-21 | GSE178838 | GEO
Project description:Aberrant activation of Hedgehog (HH) signaling has been identified as a key etiologic factor of many human malignancies. Signal strength, target gene specificity, and oncogenic activity of HH signaling profoundly depend on interactions with other pathways such as epidermal growth factor receptor-mediated signaling which has been shown to cooperate with HH/GLI in basal cell carcinoma and pancreatic cancer. We demonstrate that the human medulloblastoma cell line Daoy possesses a fully inducible endogenous HH pathway. Treatment of Daoy cells with Sonic Hedgehog or Smoothened agonist induced expression of GLI1 protein and prevented processing of GLI3 to its repressor form. To study interactions between HH- and EGF-induced signaling in greater detail, time-resolved measurements were carried out and analyzed on the transcriptomic as well as proteomic level. Daoy cells responded to the co-treatment by downregulating GLI1, PTCH, and HHIP on the transcript level which was also seen when Amphiregulin (AREG) was used instead of EGF. The finding that EGFR signaling silences proteins acting as negative regulators of HH signaling is firstly described here as a novel crosstalk mechanism. Furthermore, combined EGFR/HH signaling maintains high GLI1 protein levels contrasting its downregulation on the transcript level. On the other hand, high level synergism was observed with respect to a strong and significant upregulation of numerous canonical EGF-targets with putative tumor-promoting properties such as MMP7, VEGFA, and IL-8. In conclusion, synergistic effects between EGFR and HH signaling can selectively induce a switch from a canonical HH/GLI profile to a modulated specific target gene profile pointing to more wide-spread, yet context-dependent, interactions between HH/GLI and growth factor receptor signaling in human malignancies. To study interactions between HH- and EGF-induced signaling, time-resolved measurements were carried out over a period of 24 h at 14 different time points after stimulation by EGF with and without additional stimulation by SHH. Furthermore, as a control, cells without any stimulation by EGF and SHH (control) and cells in the presents of SHH were analyzed. Overall, three biological replicates of 60 different treatment/timepoints were analyzed yielding 180 different samples.
2014-07-31 | E-GEOD-46045 | biostudies-arrayexpress
Project description:The Hedgehog signaling pathway is essential for the maintenance and response of several types of stem cells. To study the transcriptional response of stem cells to HH signaling, we searched for proteins binding to GLI proteins, the transcriptional effectors of the HH pathway in mouse embryonic stem (ES) cells. We purified GLI protein complex from an ES cell line that contained a tamoxifen-inducible 3XFLAG-tagged GLI3 repressor allele by anti-FLAG immunoprecipitation and several novel GLI co-factors were identified in the complex by subsequent mass spectrometry analysis.
2016-02-04 | PXD002494 | Pride
Project description:Purpose: This study seeks to determine the role of Hedgehog signaling in larynx development. Methods: To determine how Hedgehog signaling directs larynx epithelial morphogenesis we used genomic approaches to identify a novel set of Hedgehog target genes within the early larynx. To this end, we performed bulk RNA-seq on 3-pooled E10.5 (32-35S) larynx tissues from wild-type (2 replicates) and Shh-/- (2 replicates) embryos. From this experiment we identified 1873 differentially expressed genes (p-value <0.05). Results: Hedgehog is essential for maintaining the identity of foregut-derived epithelial cells within the larynx vocal folds. In the absence of HH signaling these epithelial cells undergo specific transcriptional changes to initiate a partial epithelial-to-mesenchymal transition, migrate out of the epithelial layer and under go cell death. The layer is then replaced by a different population of cells.
2022-11-30 | GSE190281 | GEO
Project description:Aberrant activation of Hedgehog (HH) signaling has been identified as a key etiologic factor of many human malignancies. Signal strength, target gene specificity, and oncogenic activity of HH signaling profoundly depend on interactions with other pathways such as epidermal growth factor receptor-mediated signaling which has been shown to cooperate with HH/GLI in basal cell carcinoma and pancreatic cancer. We demonstrate that the human medulloblastoma cell line Daoy possesses a fully inducible endogenous HH pathway. Treatment of Daoy cells with Sonic Hedgehog or Smoothened agonist induced expression of GLI1 protein and prevented processing of GLI3 to its repressor form. To study interactions between HH- and EGF-induced signaling in greater detail, time-resolved measurements were carried out and analyzed on the transcriptomic as well as proteomic level. Daoy cells responded to the co-treatment by downregulating GLI1, PTCH, and HHIP on the transcript level which was also seen when Amphiregulin (AREG) was used instead of EGF. The finding that EGFR signaling silences proteins acting as negative regulators of HH signaling is firstly described here as a novel crosstalk mechanism. Furthermore, combined EGFR/HH signaling maintains high GLI1 protein levels contrasting its downregulation on the transcript level. On the other hand, high level synergism was observed with respect to a strong and significant upregulation of numerous canonical EGF-targets with putative tumor-promoting properties such as MMP7, VEGFA, and IL-8. In conclusion, synergistic effects between EGFR and HH signaling can selectively induce a switch from a canonical HH/GLI profile to a modulated specific target gene profile pointing to more wide-spread, yet context-dependent, interactions between HH/GLI and growth factor receptor signaling in human malignancies.
2014-07-31 | GSE46045 | GEO
Project description:Recent fate mapping studies concluded that EMT is not required for metastasis of carcinomas. Here we challenge this conclusion by showing that it failed to account for possible crosstalk between EMT and non-EMT cells that promotes dissemination of non-EMT cells. In breast cancer models, EMT cells induce increased metastasis of weakly metastatic, non-EMT tumor cells in a paracrine manner in part by non-cell autonomous activation of the GLI transcription factor. Treatment with GANT61, a GLI1/2 inhibitor, but not with IPI-926, a Smoothened inhibitor, blocks this effect, and inhibits growth in PDX models. In human breast tumors, the EMT-TFs strongly correlate with activated Hedgehog/GLI signaling, but not with the Hh ligands. Our findings indicate that EMT contributes to metastasis in part via non-cell autonomous effects that activate the Hh pathway. While all Hh inhibitors may act against tumors with canonical Hh/GLI signaling, only GLI inhibitors would act against EMT-induced GLI activation. In recent years, immunohistochemical analyses and multiplex high-throughput single cell sequencing of human tumor cells have shown that tumors are composed of diverse cell subpopulations containing different driver mutations, gene and protein expression profiles, growth rates, and responses to chemotherapeutics. Such heterogeneity is exacerbated by cellular plasticity, where some cells may undergo oncogenic epithelial-to-mesenchymal transition (EMT), resulting in loss of cell-cell adhesion and polarity, reduced epithelial and elevated mesenchymal protein expression, increased migration and invasion, and enhanced dissemination from the primary tumor. Because metastases in patients appear epithelial, the reverse process, mesenchymal-to-epithelial transition (MET), may occur to allow tumor cell colonization in secondary metastatic sites, establishing cellular plasticity as an important aspect of tumor progression. However, the role of EMT in carcinoma metastasis is controversial. Recent lineage tracing studies argue against the requirement of EMT for metastasis, as reporter-tagged cells that underwent a previous EMT were not found at the secondary site. However, these studies did not address the potential cooperation between EMT and non-EMT cells during the metastatic process, as EMT cancer cells may enable non-EMT cells to gain access to the secondary site, leading to macrometastatic growth. Thus, metastasis can be influenced by intratumoral heterogeneity: where a small proportion of primary tumor cells that have undergone an EMT may influence neighboring, non-EMT tumor cells. Twist1, Snail1, and Six1 are EMT-inducing transcription factors (EMT-TFs) that have all been associated with breast cancer metastasis. All three EMT-TFs regulate critical developmental processes such as cell survival, migration and invasion; in part by influencing EMT. In addition, they are downregulated post-embryogenesis, but re-expressed in various cancers where they cell autonomously induce EMT, resulting in increased percentages of tumor initiating cells (TICs) and enhanced metastasis. In carcinomas, Twist1 and Snail1 transcriptionally repress E-Cadherin (E- Cad) and upregulate mesenchymal genes. Similarly, Six1 overexpression induces EMT by regulating E-Cad localization and altering other EMT markers. During development and cancer, EMT-TFs act in concert with several signaling networks including TGFβ, Wnt, and Hedghog (Hh). The Hedgehog (Hh) signaling pathway is a prominent regulator of embryonic development, where Hh ligands function as morphogens to control numerous developmental processes. Interestingly, in Drosophila eye development, hh is a direct target of sine oculis (the homolog of Six1) and Six1 regulates Hh/GLI signaling during lung development and in fibroblasts. Additionally, Twist1 and Hh/GLI signaling are intimately linked during development, and recently Twist1 and Snail1 were associated with the Hh pathway in TICs. In mammals, canonical activation of Hh/GLI signaling involves binding of one the Hh ligands such as(either, Desert (DHH), Indian (IHH), or Sonic Hedgehog (SHH) to Patched-1 (PTCH1) or Patched-2 (PTCH2) receptors, relieving the inhibitory activity of PTCH on Smoothened (SMO). When inhibition is relieved, levels of the transcriptional activator forms of one or more GLI transcription factors (GLI1, 2, or 3) increase in the nucleus, resulting in activation of Hh target genes 12. Non-canonical activation of the GLI TFs can occur in a Hh- or SMO-independent manner via secreted factors like TGF-β19. Importantly, autocrine and paracrine Hh-mediated crosstalk between tumor cells and the tumor microenvironment 20 results in increased proliferation, stem cell self-renewal and metastasis in various cancers 21. In basal cell carcinoma (BCC) and medulloblastoma, activated Hh signaling is often due to mutations in pathway components such as PTCH and SMO, while in other tumor types including breast, mutations are not observed at high frequency. Instead there is evidence for Hh ligand-dependent pathway hyperactivity. Because numerous studies link Hh signaling to progression in multiple tumor types, derivatives of cyclopamine (e.g. GDC-0449 and IPI926), a naturally occurring plant-derived steroidal alkaloid which targets SMO, are in clinical trials for select patients with BCC and medulloblastoma with promising efficacy. However, these inhibitors have not shown promise in breast cancer despite evidence for activation of this pathway. Herein, we demonstrate that EMT-TFs Twist1, Snail1 and Six1 influence neighboring carcinoma cells in a non-cell autonomous manner, by increasing EMT features and aggressive properties of cells not expressing these TFs. Six1 is a key mediator of the non-cell autonomous effects downstream of Twist1 and Snail1, and can induce metastasis of non-Six1, non-EMT cells. All three EMT-TFs function non-cell autonomously by activation of GLI-mediated transcription in non-EMT cells, but employ different mechanisms of pathway activation, some of which are Hh ligand and SMO-independent. We find that pharmacological inhibition of GLI, but not SMO, in the non-EMT cells efficiently inhibits the non-cell autonomous phenotypes imparted by all three EMT-TFs. Importantly, we demonstrate that in selected patient derived breast cancer xenograft (PDX) models endogenously expressing high levels of these EMT-TFs (but not Hh ligands), GANT61 (a GLI1/2 inhibitor) inhibits tumor growth whereas IPI926 (a SMO inhibitor) did not. Our data suggest that upstream SMO inhibitors may not prove efficacious in tumors where a proportion of cells activate GLI-TF via EMT-TFs, and instead argue that inhibitors directly targeting GLI may be more effective.
2017-04-13 | GSE97726 | GEO