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SRX24003467: GSM8155806: CHiC_FL_Shox2trac_E135_DECOM; Mus musculus; Hi-C
1 ILLUMINA (Illumina NovaSeq 6000) run: 147.6M spots, 14.8G bases, 4.9Gb downloads

External Id: GSM8155806_r1
Submitted by: Department of Genetic Medicine and Development, University of Geneva
Study: Temporal constraints on enhancer usage shape the regulation of limb gene transcription
show Abstracthide Abstract
Repertoires of transcriptional enhancers orchestrate gene expression during embryonic development, thereby shaping the forms and functions of organs. Within these repertoires individual enhancers display spatially distinct or overlapping activities that collectively build up the expression domain of cognate genes. However, the temporal specificity of these enhancers - how their activities change over developmental time to dynamically influence gene expression - remains uncharacterized. Here, we observed that temporally restricted enhancer repertoires are embedded at numerous loci associated with mouse limb development. To monitor how such enhancer repertoires govern gene transcription in vivo across extensive developmental periods, we introduce the regulatory trajectory framework. This paradigm conceptually involves transcriptional initiation, marking the beginning of gene expression, followed by its maintenance over time, and ultimately decommissioning, leading to gene repression. To track and sort cells undergoing these distinct phases, we devised a transgenic recorder approach at the Shox2 model locus. Through this method, we discovered that cells maintaining Shox2 transcription in early and late limb development relies on distinct, temporally restricted enhancer repertoires. We demonstrate that eliminating early- or late-acting enhancers only transiently affects Shox2 expression indicating that these enhancer repertoires function independently. Additionally, we found that changes in the 3D topology of the locus associate with enhancer activities and that a rapid loss of enhancer-promoter contacts occurs during decommissioning. Finally, we show that the decommissioning of the Shox2 locus can be actively driven by Hoxd13, a gene which expression is known to antagonize Shox2. Overall, our work uncovers the dependency of developmental genes on enhancers with temporally restricted activities to generate complex expression patterns over time and shed light on the dynamics of enhancer-promoter interactions. Overall design: We devised a dual-reporter system to monitor Shox2 regulatory trajectories and sort cells undergoing these distinct phases. First, we inserted in mouse embryonic stem cells (mESCs), 1kb upstream of the Shox2 transcriptional start site, a regulatory sensor cassette constituted by a minimal ß-globin promoter, a mCherry reporter gene followed by a destabilized PEST sequence, a P2A self-cleavage sequence and a CRE recombinase gene. These cells cells were then retargeted to integrate, at the Rosa26 locus, a cassette with a splice acceptor followed by a floxed 3x SV40pA STOP signal and the EYFP gene open reading frame. We studied the transcriptional identity of these cells by scRNA-seq. We the profile the identity of sorted cells by RNA-seq and their regulatory profile by CHiC and ChIP-seq
Sample: CHiC_FL_Shox2trac_E135_DECOM
SAMN40556277 • SRS20799912 • All experiments • All runs
Organism: Mus musculus
Library:
Name: GSM8155806
Instrument: Illumina NovaSeq 6000
Strategy: Hi-C
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: Tissues were homogenized in typsin-EDTA solution, fixed with 2% PFA at room temperature 10 min. Then fixation was halted with 1M glycine and the cells were lysed in custom-made lysis buffer (10mM Tris pH7.5, 10mM NaCl, 5mM MgCl2, 1mM EGTA, Protease Inhibitor, #04693159001). Upon PBS wash, samples were frozen and stored at -80°C. Samples were then digested with DpnII, re-ligated, de-crosslinked and purified. The 3C-library was then sheared and adaptors were added to the sheared DNA and amplified according to Agilent instructions for Illumina sequencing. The library was hybridized to the custom-designed SureSelect beads and indexed for sequencing (50 to 100 bp paired-end) following Agilent instructions. Libraries were sequenced an Illumina HiSeq 4000 or Illumina NovaSeq 6000 sequencer.
Runs: 1 run, 147.6M spots, 14.8G bases, 4.9Gb
Run# of Spots# of BasesSizePublished
SRR28398779147,623,90614.8G4.9Gb2024-03-22

ID:
32309523

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