Epigenetic Mapping of Cis-Regulatory Elements in the Developing Mouse and Pig Forelimbs.

Cis-regulatory elements (CREs) orchestrate the spatio-temporal regulation of key transcriptional programs. These elements - including promoters, enhancers, and insulators - play a crucial role in embryonic development and in morphological evolution. We performed ChIP-seq for histone modifications (H3K4me3, H3K27ac, and H3K4me1) from equivalent stages of mouse (E11.5) and pig (day 23; D23) forelimb development to catalog the limb bud regulome in both species. The overlap of these epigenomic signatures with the pattern of open chromatin during limb development previously described for both species allowed us to classify these putative regulatory regions into different chromatin states. Our studies offer a valuable resource in the evo-devo field for exploring mechanisms underlying the morphological evolution of the tetrapod limb. Mouse (E11.5) or pig (D23) embryonic forelimbs were collected from littermates to generate biological replicates. Nuclei homogenization was performed mechanically, and chromatin was crosslinked with 1% formaldehyde. Material from each biological replicate was split in half to generate technical replicates for ChIP-seq. Due to technical limitations, only one biological replicate (with two technical replicates) was generated for the pig H3K4me1 mark. All libraries were generated by ChIPmentation (Schmidl et al., 2015; PMID: 26280331), using 1 μg of the following antibodies: anti-H3K4me3 (Abcam ab8580), anti-H3K27ac (Abcam ab4729), and anti-H3K4me1 (Abcam ab8895). All pig libraries were sequenced 50bp paired-end, except for H3K4me1. This sample, together with all mouse libraries, was sequenced using 100bp paired-end reads, and subsequently cropped to 50bp. Reads were aligned to the corresponding assemblies: mouse GRCm38/mm10 or pig SusScrofa11.1/susScr11. BigWigs were normalized to RPKM. Peak calling was performed using a broad IDR <0.01.