Analysis workflow and quality control of H3K4me3 and H3K27ac peaks in the fat-tailed dunnart (Sminthopsis crassicaudata).

(A) Drawing of a D0 dunnart pouch young with dissected orofacial tissue shown in gray. Short-read alignment and peak calling workflow and numbers of reproducible peaks identified for H3K27ac (orange), H3K4me3 (blue) for craniofacial tissue. (B) Log10 distance to the nearest TSS for putative enhancer (orange) and promoters (blue). (C) Log10 of peak intensity and peak length are represented as boxplots and violin plots for putative enhancers (orange) and promoters (blue). Peak intensities correspond to average fold enrichment values over total input DNA across biological replicates. (D) Dunnart pouch young on the day of birth. Scale bar = 1mm. (E) Adult female dunnart carrying four young.

Predicted functional enrichment for dunnart peaks.

(A) 304 significantly enriched GO terms clustered based on similarity of the terms. The function of the terms in each group are summarized by word clouds of the keywords. Rows marked by P were driven by genes linked to putative promoters, rows marked by E were driven by genes linked to putative enhancers. (B) Enriched TF motifs for transcription factor families (HOMER). PWM logos for preferred binding motifs of TFs are shown. The letter size indicates the probability of a TF binding given the nucleotide composition.

Genes linked to craniofacial enhancers and promoters in the dunnart are reproducibly expressed and involved in embryonic vasculature, muscle, skin and sensory system development.

(A) The majority of nearest genes assigned to candidate enhancers and promoters were reproducibly expressed in dunnart face tissue, and (B) reproducibly expressed genes in dunnart were associated with both a promoter and enhancer region. (C) Biological processes enriched for genes medium to highly expressed (>10TPM) and linked to both a promoter and enhancer region (FDR-corrected, p < 0.01).

Analysis workflow and features of H3K4me3 and H3K27ac enhancers and promoters in dunnart and mouse.

(A) Alignment filtering and peak calling workflow and (B) number of reproducible predicted regulatory elements identified in the dunnart and mouse embryonic stages. Log10 of distance to the nearest TSS for putative (C) enhancers and (D) promoters.

Genes near enhancers highly expressed only in dunnart are involved in development of the skin, muscle and mechanosensory systems.

Gene set intersections across mouse (E10.5-E15.5) and dunnart (P0) for (A) genes near promoters and (B) genes near enhancers. (C) Gene ontology term enrichment for top 500 highly expressed genes in dunnart. (D) Top 50 highly expressed genes (TPM) in dunnart compared with mouse embryonic stages. Scale bar in E. (E) Expression levels (TPM) for keratin genes across dunnart and mouse.

Dunnart expressed genes are associated with two gene clusters with distinct temporal expression patterns in the mouse.

(A) Genes in cluster 2 and cluster 3 plotted with their z-scaled temporal expression (logCPM). Color-coding represents membership value (degree to which data points of a gene belong to the cluster). Gene Ontology enrichment for biological processes enriched in (B) cluster 2 and (C) cluster 3 (FDR-corrected p<0.01).