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. 2024 Jun;11(24):e2305706.
doi: 10.1002/advs.202305706. Epub 2024 Apr 6.

Building Haplotype-Resolved 3D Genome Maps of Chicken Skeletal Muscle

Jing Li  1 , Yu Lin  1 , Diyan Li  2 , Mengnan He  3 , Hua Kui  1 , Jingyi Bai  1 , Ziyu Chen  1 , Yuwei Gou  1 , Jiaman Zhang  1 , Tao Wang  2 , Qianzi Tang  1 , Fanli Kong  4 , Long Jin  1 , Mingzhou Li  1
Affiliations

Building Haplotype-Resolved 3D Genome Maps of Chicken Skeletal Muscle

Jing Li et al. Adv Sci (Weinh). 2024 Jun.

Abstract

Haplotype-resolved 3D chromatin architecture related to allelic differences in avian skeletal muscle development has not been addressed so far, although chicken husbandry for meat consumption has been prevalent feature of cultures on every continent for more than thousands of years. Here, high-resolution Hi-C diploid maps (1.2-kb maximum resolution) are generated for skeletal muscle tissues in chicken across three developmental stages (embryonic day 15 to day 30 post-hatching). The sequence features governing spatial arrangement of chromosomes and characterize homolog pairing in the nucleus, are identified. Multi-scale characterization of chromatin reorganization between stages from myogenesis in the fetus to myofiber hypertrophy after hatching show concordant changes in transcriptional regulation by relevant signaling pathways. Further interrogation of parent-of-origin-specific chromatin conformation supported that genomic imprinting is absent in birds. This study also reveals promoter-enhancer interaction (PEI) differences between broiler and layer haplotypes in skeletal muscle development-related genes are related to genetic variation between breeds, however, only a minority of breed-specific variations likely contribute to phenotypic divergence in skeletal muscle potentially via allelic PEI rewiring. Beyond defining the haplotype-specific 3D chromatin architecture in chicken, this study provides a rich resource for investigating allelic regulatory divergence among chicken breeds.

Keywords: Hi‐C; chicken; homolog; sequence variations; skeletal muscle development.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Experimental design and construction of diploid 3D genome maps for chicken skeletal muscle tissue. A) The experimental design of this study aimed to elucidate the regulatory roles of chromatin conformation in gene transcription regulation, as depicted in the schematic representation. B) Allele assignment of in situ Hi‐C contacts in F1 hybrid chickens. A total of approximately 3.54 billion valid Hi‐C contacts were generated for each of the 12 hybrid chicken samples. C,D) Estimation of resolution for haplotype‐resolved intra‐chromosomal (C) and inter‐chromosomal (D) Hi‐C maps. The resolution of the Hi‐C maps refers to the smallest bin size where at least 80% of bins contain a minimum of 1000 reads, ensuring reliable discernment of local features. The data are presented as the mean ± SD of the 24 haplotypes. The mean numbers of contacts at resolutions of 5 kb, 20 kb, and 100 kb are displayed. E) 3D genome structure of a hybrid chicken skeletal muscle sample (BL2‐1) at the E15 stage, constructed using normalized intra‐chromosomal (at 20 kb resolution) and inter‐chromosomal (at 500 kb resolution) contact matrices with miniMDS and the PYMOL program. Upper: an overall view from three different angles. Lower: the structures of autosome pairs. The haplotypes of three pairs of autosomes were marked as examples. Bottom: the lengths of autosomes are indicated with the bar plot.
Figure 2
Figure 2
Characteristics of homolog pairing in hybrid chicken samples. A) The close spatial localization of homologous chromosome pairs is highlighted by diagonal lines (black arrows) alongside intra‐chromosomal interaction squares that display relatively intense chromatin interactions between homologs on the Hi‐C map. This figure presents data from a sample (BL1‐1) at the E15 stage. B) The identification of tightly paired regions in the skeletal muscle tissue of hybrid chickens at the three developmental stages is based on the distribution of the homolog pairing score (HPS). Tightly‐paired loci are defined as those with an HPS above Q3 + 1.5 × IQR. C) The Venn plot illustrates the overlap of tightly paired genomic bins (at 20‐kb resolution) identified at the three developmental stages. Approximately 56.89–62.62% of these bins are shared across all three stages. D–I) Analysis of sequence features in the tightly paired regions. These regions exhibit significantly higher GC contents (D), an increased presence of CpG islands (E), more centromere sequences (F), more telomere sequences (G), and a greater abundance of CTCF (H) and YY1 (I) binding sites compared to other genomic regions. The P values were calculated using the Wilcoxon rank‐sum test in (D,E) and Fisher's exact test in (F–I). **0.001< p <0.01; *** p <0.001.
Figure 3
Figure 3
Characteristics of differential chromatin architectures identified between neighboring developmental stages in chicken skeletal muscle tissue. A) Expression differences are observed between neighboring stages for genes located in stage‐restricted, more active compartments, including A/B switched and A/B variable compartments. B–D) Examples of genes with switched compartments and differential expression across skeletal muscle development are EYA2 (B), CDK1 (C), and FGF4 (D), all of which play important roles in myogenesis and skeletal muscle growth. E) Comparison of differential expression levels for genes near shifted and conserved TAD boundaries between developmental stages. A total of 122 genes were located at shifted boundaries and had allelic expression data. An equal number of genes were randomly selected from conserved boundaries for 100,000 iterations. F,G) Stage‐specific TAD boundaries adjacent to genes responsible for cell differentiation and muscle development. An E15‐specific TAD boundary loss overlaps with CD82 (F), while a D30‐specific TAD boundary shift affects CMBL and SBK2 (G). These genes exhibit corresponding changes in expression across development. The figure includes Hi‐C maps (at 20‐kb resolution), TAD boundaries, local boundary scores (LBS), RNA‐seq signals in the corresponding region, as well as structures and transcripts per million (TPM) values of the putatively affected genes. Black dashed boxes indicate TAD boundary changes during development. H) Expression differences are observed for genes with significantly differential RPS between neighboring stages. I–K) Rewiring of PEIs is observed for three representative genes with differential RPS between neighboring stages: MYF5 and MYF6 (genes enriched in the “embryonic skeletal muscle system morphogenesis” gene ontology term and sharing the promoter bin; I), KCNJ2 (gene enriched in the “cGMP‐PKG signaling pathway” and “actin filament‐based process”; J), and CAVIN4 (gene enriched in “muscle structure development”; K). The figure presents Hi‐C maps (upper left), heatmaps of interaction intensity differences (upper right), gene expression levels (lower left), and 3D structural models (lower right) of the corresponding genomic regions. Promoters (green squares), enhancers (grey squares), and PEIs (connecting lines) are displayed alongside the Hi‐C maps. The significance of the differences in (A) and (H) was evaluated using the Wilcoxon rank‐sum test.
Figure 4
Figure 4
Comparison of absolute values of A‐B index differences (A), local boundary score (LBS) differences (B), and regulatory potential score (RPS) differences (C) between the 106 chicken orthologs of empirical imprinted genes and randomly selected genes (n = 106) at E15, D1, and D30 stages. Mat: maternal haplotypes; Pat: paternal haplotypes. The P values were calculated using a Wilcoxon rank‐sum test.
Figure 5
Figure 5
Characteristics of differential RPS genes between parental breeds in the hybrid chickens. A) Volcano plots illustrating genes with statistically different RPS (|ΔRPS| > 0.3 and P < 0.05, paired Student's t‐test) between parental breeds at E15 (left), D1 (middle), and D30 (right) developmental stages. B) Venn plots displaying genes with higher RPS in either broiler (left) or layer haplotypes (right) at the three developmental stages. Gene numbers are labeled on the plots. C) Results of functional enrichment analysis conducted for genes with elevated RPS in broiler haplotypes across the three developmental stages. The top twenty enriched terms are shown. D,E) PEI rewiring of two representative genes with differential RPS between breeds: SMAD1 (related to ‘response to growth factor’; D) and ROCK2 (related to ‘Rho protein signal transduction’; E). The figures display the Hi‐C maps (upper left), heatmaps of interaction intensity differences (upper right), gene expression levels (lower left), and 3D structural models (lower right) of the corresponding genomic regions. Promoter regions are denoted by green squares, enhancers by grey squares, and PEIs by connecting lines beside the Hi‐C maps.
Figure 6
Figure 6
Effects of highly divergent sequences (indicated by high‐F ST variants) in enhancer regions between breeds on PEI wiring in the hybrid chickens. A) Genes with enhancers containing high‐F ST variants exhibit greater changes in RPS. (A) Comparison of RPS differences for genes with enhancers carrying high‐F ST variants versus those lacking them. B) Comparison of enrichment in differential RPS genes for genes with enhancers carrying high‐F ST variants versus those lacking them. C)Genes with enhancers containing high‐F ST variants have a significantly greater number of enhancers compared to those lacking them. D) Genes with enhancers containing high‐F ST variants have a lower number and percentage of genes interacting with a single enhancer compared to those lacking high‐F ST variants. E) No significant expression differences between breeds were observed for genes with enhancers containing high‐F ST variants compared to those lacking them at the three developmental stages. F) NRAP, a differential RPS gene, with enhancers enriched for high‐F ST variants, displaying divergent expression between parental breeds. The figure showcases the comparison of Hi‐C maps (upper left), interaction intensity differences (upper right), RPS (middle left), gene expression levels (middle right), and PEI intensities and sequence divergence in the enhancer regions (measured by F ST of variants calculated using the genotypes of 24 purebred chickens; lower) between broilers and layers. Promoters (green square), enhancers (grey squares), and PEIs (connecting lines) are displayed beside the Hi‐C maps. The P values were calculated using the Wilcoxon rank‐sum test in (A,C,E) and Fisher's exact test in (B,D,F).
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