Genome-wide open chromatin regions and their effects on the regulation of silk protein genes in Bombyx mori

Abstract

Nucleosome-depleted open chromatin regions (OCRs) often harbor transcription factor (TF) binding sites that are associated with active DNA regulatory elements. To investigate the regulation of silk-protein genes, DNA molecules isolated from the silk glands of third-day fifth-instar silkworm larvae and embryo-derived (BmE) cells were subjected to formal dehyde-assisted isolation of regulatory elements (FAIRE) and high-throughput sequencing. In total, 68,000 OCRs were identified, and a number of TF-binding motifs were predicted. In particular, OCRs located near silk-protein genes contained potential binding sites for functional TFs. Moreover, many TFs were found to bind to clusters of OCRs upstream of silk-protein genes, and to regulate the expression of these genes. The expression of silk protein genes may be related not only to regulating TFs (such as fkh, Bmdimm, and Bmsage), but also to developmental and hormone-induced TFs (such as zen, eve, Br, and eip74ef). Elucidation of genome-wide OCRs and their regulatory motifs in silk protein genes will provide valuable data and clues for characterizing the mechanisms of transcriptional control of silk protein genes.

Figure 1

Quality control analysis of the next-generation sequencing data. (a) Analysis workflow. (b) Coverage of reads by formaldehyde-assisted isolation of regulatory elements (FAIRE). One color represents one sample. Mean is the mean coverage per bp, i.e., 20% of the sampled bp has up to 11 overlapping reads. A tiny fraction of bp had >30 overlapping reads. (c) Principal component analysis (PCA) plot for the FAIRE replicates of Bombyx mori embryo-derived (BmE) cells and silk glands. PC1 (89.0%) and PC2 (9.4%) are the top two principal components. (d) The plot of FAIRE signal at Ubx peaks. (e) Browser representation of the slit locus. Silk gland FAIRE, from top to bottom: silk gland FAIRE peaks, BmE FAIRE peaks, and Ubx chip peaks.

Figure 2

The distribution and function of OCRs. (a) Profile and heatmap of open chromatin regions (OCRs) of Bombyx mori embryo-derived (BmE) cells and silk glands. The distribution profile of OCRs within+/−2 kb of transcriptional start sites (TSS). Heatmap of TSS+/−2 kb region OCRs were clustered using k-means = 4. The number of OCRs in every cluster was marked in the picture both silk gland and BmE cell. (b) The genomic location of OCRs. Orange bar represents silk gland OCRs; blue bar represents BmE OCRs. (c) OCRs and regulated genes of BmE cells and silk glands. Up-regulated and down-regulated genes are considered those whose expression level was significantly higher or lower in the silk gland than in BmE cells, respectively. OCRs located within 2 kb of a gene were considered that adjacent gene’s OCR. Orange bar represents genes; blue bar represents OCRs.

Figure 3

Network graphic for four transcription factors (TFs) and their adjacent genes. Hexagons represent known motifs. The points that form the purple circles represent genes regulated by the corresponding TF. Lines are used to indicate genes that might be regulated by the corresponding TFs. Genes located within the big circle located in the middle might be regulated by two or more TFs. Genes within the peripheral small circle might be regulated by only one TF.

Figure 4

The transcriptional regulation mode of Bmsage and Fibrohexamerin gene. Formaldehyde-assisted isolation of regulatory elements (FAIRE; 0–30) and RNA (Fibrohexamerin 0–50,000, Bmsage 0–200) signals for 10-kb upstream regions from (a) Bmsage and (b) Fibrohexamerin. Pink peaks are silk gland FAIRE-seq and RNA-seq peaks. Blue peaks are BmE FAIRE-seq and RNA-seq peaks. Other colored shadows show locations of known transcription factor binding sites, and each color corresponds to a particular transcription factor.