Project description:We used microarray technology to investigate the transcriptional consequences of loss of Gli3 from the anterior mesenchyme of the developing mouse limb bud at both E11.5 and E12.5. Comparisons were made between RNA derived from the anterior margin of Gli3Xt/Xt limb buds and that derived from wild type littermates Keywords: genetic modification
Project description:Purpose: The response to Hedgehog signaling in the limb is driven by GLI bound enhancers and the majority of Hh targets in the developing limb bud are regulated solely by the activity of GLI-repressor. Currently we do not have a comprehensive understanding of how GLI bound enhancers respond Hedgehog signaling. The goal of this study is to identify how GLI bound enhancers are regulated by Hedgehog signaling and specifically by GLI-repressor. Methods: ChIP-seq was performed in Embryonic day 10.5 mouse limb buds from mice with endogenously FLAG tagged Gli3. Results: We identified 7282 GLI3 binding regions in the E10.5 limb bud.
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.
Project description:Profiling H3K4me1 & H3K27ac histone modifications across the ZRS and a selection of limb development genes within Mouse E11.5 distal limb bud tissue.
Project description:Sonic hedgehog (Shh) signals via Gli transcription factors to direct digit number and identity in the vertebrate limb. We have characterized the Gli-dependent cis-regulatory network through a combination of whole genome ChIP-on-chip and transcriptional profiling of the developing mouse limb. These analyses identified approximately 5,000 high quality Gli3 binding sites, including all known Gli-dependent enhancers. Discrete binding regions exhibit a higher-order clustering, highlighting the complexity of cis-regulatory interactions. Further, Gli3 binds inertly to previously identified neural-specific Gli enhancers, demonstrating the accessibility of their cis-regulatory elements. Intersection of DNA binding data with gene expression profiles predicted 205 putative limb target genes. The supplementary bed file contains all 5,274 high quality binding Gli3 binding sites reported in the paper.
Project description:Purpose: The response to Hedgehog signaling in the limb is driven by GLI bound enhancers and the majority of Hh targets in the developing limb bud are regulated solely by the activity of GLI-repressor. Currently we do not have a comprehensive understanding of how GLI bound enhancers respond Hedgehog signaling. The goal of this study is to identify how GLI bound enhancers are regulated by Hedgehog signaling and specifically by GLI-repressor. Methods: ChIP-seq was performed in Embryonic day 11.5 mouse forelimb and hidlimbs lfrom wildtype Swiss Webster mice. Results: We identified 15,347 HDAC1 binding sites in mouse E11.5 limb buds
Project description:Cohesin is implicated in establishing tissue-specific DNA loops that target enhancers to promoters, and also localizes to sites bound by the insulator protein CTCF, which blocks enhancer-promoter communication. However, cohesin-associated interactions have not been characterized on a genome-wide scale. Here we performed chromatin interaction analysis with paired-end tag sequencing (ChIA-PET) of the cohesin subunit SMC1A in developing mouse limb. We identified 2,264 SMC1A interactions, of which 1,491 (65%) involved sites co-occupied by CTCF. SMC1A participates in tissue- specific enhancer-promoter interactions and interactions that demarcate regions of correlated regulatory output. In contrast to previous studies, we also identified interactions between promoters and distal sites that are maintained in multiple tissues, but are poised in embryonic stem cells and resolve to tissue-specific activated or repressed chromatin states in the mouse embryo. Our results reveal the diversity of cohesin- associated interactions in the genome and highlight their role in establishing the regulatory architecture of development. Smc1a ChIA-PET, RNA-seq, chromatin state maps (H3K27ac, H3K27me3, H3K4m2), and CTCF and Smc1a binding in mouse embryonic limb bud (E11.5)
Project description:Understanding the origin of morphological diversity across vertebrates is central to evolutionary developmental biology. cis-regulatory elements (CRE) such as enhancers and promoters interpret precise spatiotemporal cues to control and coordinate gene expression. To get insights into both conserved and species-specific variations during early limb patterning and outgrowth, we leverage genome-wide comprehensive assessment of chromatin accessibility and transcriptional changes during mouse forelimb and chicken wing bud development. Our analysis reveals temporal modulation of chromatin accessibility and expression as well as their temporal relationship during the progression of mouse forelimb and chicken wing bud development. Transcription factor binding site enrichment analysis and putative TF occupancy as inferred by integrating TF binding motifs and chromatin accessibility information reveal temporal TF-DNA interactions during forelimb/wing bud patterning. Finally, the integration of accessibility, expression, and TF binding site information allowed to identify candidate gene targets of HAND2 and GLI3 that include conserved as well as species-specific transcriptional regulator-gene interactions.
Project description:Understanding the origin of morphological diversity across vertebrates is central to evolutionary developmental biology. cis-regulatory elements (CRE) such as enhancers and promoters interpret precise spatiotemporal cues to control and coordinate gene expression. To get insights into both conserved and species-specific variations during early limb patterning and outgrowth, we leverage genome-wide comprehensive assessment of chromatin accessibility and transcriptional changes during mouse forelimb and chicken wing bud development. Our analysis reveals temporal modulation of chromatin accessibility and expression as well as their temporal relationship during the progression of mouse forelimb and chicken wing bud development. Transcription factor binding site enrichment analysis and putative TF occupancy as inferred by integrating TF binding motifs and chromatin accessibility information reveal temporal TF-DNA interactions during forelimb/wing bud patterning. Finally, the integration of accessibility, expression, and TF binding site information allowed to identify candidate gene targets of HAND2 and GLI3 that include conserved as well as species-specific transcriptional regulator-gene interactions.