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. 2014;15 Suppl 1(Suppl 1):S1.
doi: 10.1186/1471-2164-15-S1-S1. Epub 2014 Jan 24.

An Improved ChIP-seq Peak Detection System for Simultaneously Identifying Post-Translational Modified Transcription Factors by Combinatorial Fusion, Using SUMOylation as an Example

Free PMC article

An Improved ChIP-seq Peak Detection System for Simultaneously Identifying Post-Translational Modified Transcription Factors by Combinatorial Fusion, Using SUMOylation as an Example

Chia-Yang Cheng et al. BMC Genomics. .
Free PMC article

Abstract

Background: Post-translational modification (PTM) of transcriptional factors and chromatin remodelling proteins is recognized as a major mechanism by which transcriptional regulation occurs. Chromatin immunoprecipitation (ChIP) in combination with high-throughput sequencing (ChIP-seq) is being applied as a gold standard when studying the genome-wide binding sites of transcription factor (TFs). This has greatly improved our understanding of protein-DNA interactions on a genomic-wide scale. However, current ChIP-seq peak calling tools are not sufficiently sensitive and are unable to simultaneously identify post-translational modified TFs based on ChIP-seq analysis; this is largely due to the wide-spread presence of multiple modified TFs. Using SUMO-1 modification as an example; we describe here an improved approach that allows the simultaneous identification of the particular genomic binding regions of all TFs with SUMO-1 modification.

Results: Traditional peak calling methods are inadequate when identifying multiple TF binding sites that involve long genomic regions and therefore we designed a ChIP-seq processing pipeline for the detection of peaks via a combinatorial fusion method. Then, we annotate the peaks with known transcription factor binding sites (TFBS) using the Transfac Matrix Database (v7.0), which predicts potential SUMOylated TFs. Next, the peak calling result was further analyzed based on the promoter proximity, TFBS annotation, a literature review, and was validated by ChIP-real-time quantitative PCR (qPCR) and ChIP-reChIP real-time qPCR. The results show clearly that SUMOylated TFs are able to be pinpointed using our pipeline.

Conclusion: A methodology is presented that analyzes SUMO-1 ChIP-seq patterns and predicts related TFs. Our analysis uses three peak calling tools. The fusion of these different tools increases the precision of the peak calling results. TFBS annotation method is able to predict potential SUMOylated TFs. Here, we offer a new approach that enhances ChIP-seq data analysis and allows the identification of multiple SUMOylated TF binding sites simultaneously, which can then be utilized for other functional PTM binding site prediction in future.

Figures

Figure 1
Figure 1
Overview of experimental design. The experimental design of the SUMO-1 ChIP-seq. DNA crosslinking with either SUMO-TF or SUMO-cofactor are identified using SUMO-1 antibody. Following size selection, all the resulting ChIP-DNA fragments were sequenced using an Illumina® Genome AnalyzerIIx.
Figure 2
Figure 2
Promoter region of NOSIP as explored by different peak detection methods with TFBS annotation.
Figure 3
Figure 3
Confirmation of ChIP-seq data for ELK1-binding sites with SUMO-1 enrichment in BCBL-1 cells using ChIP-qPCR. Confirmation of data derived from ChIP-seq for ELK1 binding sites with SUMO-1 enrichment in BCBL-1 cells. The ELK1 binding sites within the SUMO-1 peak of the promoters of (A) TARS2, (B) NDUFB7 and (C) ADAMTS10 genes were amplified using qPCR. (D) SNRPE, (E) INO80B and (F) LYSMD1 genes identified in our SUMO-1 ChIP-seq result and GSM608163 ChIP-seq data were analyzed by qPCR with their specific primer pairs. All reactions were run in triplicate and normalized against the input. Nonspecific IgG was used as the control ChIP antibody.
Figure 4
Figure 4
Colocalization of ELK1 and SUMO-1 in the promoters of TARS2 and NDUFB7 genes. Sequential chromatin immunoprecipitation (ChIP-reChIP) assay using control IgG and anti-SUMO-1 antibody for the first ChIP and anti-ELK-1 antibody for the reChIP was performed in formaldehyde-fixed chromatin derived fromTREx-F3H3-K-Rta BCBL-1 cells. Quantification of first ChIP and reChIP DNA recovered from (A) TARS2, (B) NDUFB7 and (C) SNRPE, (D) INO80B and (E) LYSMD1 by real-time qPCR using the promoter-specific primers.
Figure 5
Figure 5
Regulation of ELK-1 activity by SUMO-1 modification. (A) TREx-F3H3-K-Rta-shSUMO-1 BCBL-1 cells were treated with Dox for 48 hours. TCLs were analyzed by immunoblotting using anti-SUMO-1 antibody. (B to H) Two ELK-1 targeted genes, TARS2 (B) and NDUFB7 (C), showing SUMO-1 enrichment at the promoter region identified in our study and three genes, SNRPE (D), INO80B (E) and LYSMD1 (F), that have high quality ELK-1 binding sites identified in HeLa cells overlapping with our SUMO-1 enriched regions were chosen. Two genes, MCL-1 (G) and IRF-3 (H), with ELK-1 binding site at the promoter region showing no SUMO-1 enrichment were chosen as control. RNA samples derived from TREx-F3H3-K-Rta BCBL-1 and TREx-F3H3-K-Rta shSUMO-1 BCBL-1 cells before and after 48 hours of Dox induction were subjected to reverse transcription (RT) reaction. Following the RT reaction, the ELK-1 target genes were amplified by qPCR using gene-specific primer sets. All reactions were run in triplicate and normalized against GAPDH.
Figure 6
Figure 6
Potential SUMO-1 TF verified result. The percentage of literature verified SUMO-1 TFs predicted by the C, T, M*C*T, C*T, C+T and M+C+T methods, from top1 to top 35, plotted on a curve.
Figure 7
Figure 7
Peak calling by different software. The Venn diagram showing the overlaps among the peaks called by MACS, T-PIC and CisGenome, together with the numbers of peak presented. The numbers for the union and intersection of the peaks, and the mapped genes as obtained by the software can also be found in Table 4.
Figure 8
Figure 8
Potential SUMO-1 TF verified result by the combinational methods of M, C, T and B. The percentage of literature verified SUMO-1 TFs predicted by the combinational methods of M, C, T and B from top1 to top 35, plotted on a curve.
Figure 9
Figure 9
Diagram of the SUMO-1 ChIP-seq analysis workflow. Scheme used for the modified high-resolution ChIP-seq method and its validation. The literature was used to verify 17 of the top 19 SUMO-1-TF candidates. The SUMO-1-TF candidates were predicted by the following steps: (1) filtering poor and repeat reads out, and aligning reads to the human genome (hg19); (2) calling peaks using three tools MACS, T-PIC and CisGenome; (3) combining three peak sets; (4) annotating peaks using TFBS; (5) scoring and ranking SUMO-1 TF candidates; and finally (6) verifying SUMO-1 TF candidates via the literature.

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