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. 2014:96:e005.
doi: 10.1017/S0016672314000081.

The comprehensive transcriptional analysis in Caenorhabditis elegans by integrating ChIP-seq and gene expression data

Kan He  1 , Jiaofang Shao  2 , Zhongying Zhao  2 , Dahai Liu  1
Affiliations

The comprehensive transcriptional analysis in Caenorhabditis elegans by integrating ChIP-seq and gene expression data

Kan He et al. Genet Res (Camb). 2014.

Abstract

The fundamental step of learning transcriptional regulation mechanism is to identify the target genes regulated by transcription factors (TFs). Despite numerous target genes identified by chromatin immunoprecipitation followed by high-throughput sequencing technology (ChIP-seq) assays, it is not possible to infer function from binding alone in vivo. This is equally true in one of the best model systems, the nematode Caenorhabditis elegans (C. elegans), where regulation often occurs through diverse TF binding features of transcriptional networks identified in modENCODE. Here, we integrated ten ChIP-seq datasets with genome-wide expression data derived from tiling arrays, involved in six TFs (HLH-1, ELT-3, PQM-1, SKN-1, CEH-14 and LIN-11) with tissue-specific and four TFs (CEH-30, LIN-13, LIN-15B and MEP-1) with broad expression patterns. In common, TF bindings within 3 kb upstream of or within its target gene for these ten studies showed significantly elevated level of expression as opposed to that of non-target controls, indicated that these sites may be more likely to be functional through up-regulating its target genes. Intriguingly, expression of the target genes out of 5 kb upstream of their transcription start site also showed high levels, which was consistent with the results of following network component analysis. Our study has identified similar transcriptional regulation mechanisms of tissue-specific or broad expression TFs in C. elegans using ChIP-seq and gene expression data. It may also provide a novel insight into the mechanism of transcriptional regulation not only for simple organisms but also for more complex species.

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Figures

Fig. 1.
Fig. 1.
The workflow of gene expression and ChIP-seq data collection and integration analysis.
Fig. 2.
Fig. 2.
The target genes assignment and classification. The targets of each ChIP-seq study were assigned to unique gene and the target genes were classified into 15 classes based on their positions relative to the peak summits. Those with peak summit within the gene region were defined as the class of C0; those with peak summit located 0–5 kb upstream the TSS were defined as C1–5 for each 1 kb interval, respectively, whereas 0–5 kb downstream the TES as C11–15, respectively (a). For those peaks with no candidate genes in the 5-kb flanking region, the two nearest genes were found. The nearest gene upstream the peak was defined as C21, and the second nearest was C22. Similarly, the nearest gene downstream the peak was defined as C23, and the second nearest was C24 (b). If the same gene could be assigned to several classes due to the different peaks nearby, it was assigned into a class with the smallest number (c).
Fig. 3.
Fig. 3.
The proportion of target genes in each class of ten ChIP-seq studies. The bar charts show the proportion of target genes in each class of ten ChIP-seq studies, including the TFs of HLH-1, ELT-3, PQM-1, SKN-1, CEH-14 and LIN-11 with tissue-specific expression patterns as well as CEH-30, LIN-13, LIN-15B and MEP-1 with broad expression patterns. X-axis represents the different ChIP-seq studies; Y-axis represents the percentage (%) of target genes in each class (C0–C24).
Fig. 4.
Fig. 4.
The expression patterns of tissue specific TFs target genes. The Violin plots of normalized expression levels in specific tissues for different categories of corresponding TF target genes were performed for six TFs with tissue-specific expression pattern, including HLH-1 for body wall muscle (BWM), ELT-3 for hypodermis (Hyps), PQM-1 for intestine, SKN-1 for pharynx as well as CEH-14 and LIN-11 both for neurons, respectively. X-axis represents 15 different classes of target genes and reference of genome gene expression; Y-axis represents the normalized gene expression value. Significant levels by Mann–Whitney U-test (P < 10−3) are indicated in the plots, dark-red for significantly up-regulated and dark-green for significantly down-regulated.
Fig. 5.
Fig. 5.
The mean expression distribution and NCA results of each class in six tissue-specific studies. (a) It showed the mean expression distribution of each class in six tissue-specific studies, which is based on the integration analysis of ChIP-seq and gene expression data, including HLH-1 for body wall muscle (BWM), ELT-3 for hypodermis (Hyps), PQM-1 for intestine, SKN-1 for pharynx as well as CEH-14 and LIN-11 both for neurons, respectively. X-axis represents 15 different classes of target genes and reference of genome gene expression; Y-axis represents the mean level of normalized gene expression value. (b) It showed the transcription factor activity in 15 different classes for each of six TFs with tissue-specific expression pattern based on NCA method, including HLH-1 for BWM, ELT-3 for Hyps, PQM-1 for intestine, SKN-1 for pharynx as well as CEH-14 and LIN-11 both for neurons, respectively. X-axis represents 15 different classes of target genes and reference of genome gene expression; Y-axis represents the percentage of target genes with the corresponding A scores larger than 1 compared with the raw number of targets in ChIP-seq data.
Fig. 6.
Fig. 6.
The expression patterns of broad expressed TFs target genes. The Violin plots of normalized expression levels in whole animal for different categories of corresponding TF target genes were performed for four TFs with broad expression pattern, including CEH-30, LIN-13, LIN-15B and MEP-1, respectively. X-axis represents 15 different classes of target genes and reference of genome gene expression; Y-axis represents the normalized gene expression value. Significant levels by Mann–Whitney U-test (P < 10−3) are indicated in the plots, dark-red for significantly up-regulated and dark-green for significantly down-regulated.
Fig. 7.
Fig. 7.
The mean expression distribution and NCA results of each class in four broad expression TFs studies. (a) It showed the mean expression distribution of each class in four broad expression TFs studies, which is based on the integration analysis of ChIP-seq and gene expression data, including CEH-30, LIN-13, LIN-15B and MEP-1, respectively. X-axis represents 15 different classes of target genes and reference of genome gene expression; Y-axis represents the mean level of normalized gene expression value. (b) It showed the transcription factor activity in 15 different classes for each of four TFs with broad expression pattern based on NCA method, including CEH-30, LIN-13, LIN-15B and MEP-1, respectively. X-axis represents 15 different classes of target genes and reference of genome gene expression; Y-axis represents the percentage of target genes with the corresponding A scores larger than 1 compared with the raw number of targets in ChIP-seq data.
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