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ChIP-Seq

Overview

Chromatin Immunoprecipitation Sequencing (ChIP-Seq) provides genome-wide profiling of DNA targets for histone modification, transcription factors, and other DNA-associated proteins. It combines the selectivity of chromatin immuno-precipitation (ChIP) to recover specific protein-DNA complexes, with the power of next-generation sequencing (NGS) for high-throughput sequencing of the recovered DNA. Additionally, because the protein-DNA complexes are recovered from living cells, binding sites can be compared in different cell types and tissues, or under different conditions. At Novogene, we provide high-quality sequencing and comprehensive bioinformatics solutions for your ChIP-Seq projects.

Applications

  • Applications range from transcriptional regulation to developmental pathways to disease mechanisms and beyond.

Advantages

  • Cost-effective: Rapid and efficient genome-wide profiling of multiple samples, using only 1/100 of the amount of DNA required for ChIP-chip.
  • Unsurpassed data quality: We guarantee that ≥ 80% of bases have a sequencing quality score ≥ Q30, exceeding Illumina’s official guarantee of ≥ 75%.
  • Comprehensive analysis: Expert bioinformatics analyses utilizing industry standard MACS2 software and latest programs for motif prediction, peak annotation, functional analysis and data visualization.
  • Professional bioinformatics: A bioinformatics analysis team composed entirely of Ph.D. scientists for Chip-Seq data analysis.

Sample Requirements

Sample Type Required Amount Fragment size Purity
Enriched DNA Sample
≥ 50 ng (Concentration ≥ 2ng/μL)
100 bp~500bp
OD260/280=1.8-2.0

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Sequencing Parameter and Analysis

Platform Illumina Novaseq 6000
Read length Pair-end 150
Recommended Sequencing Depth ≥ 20 million read pairs per sample for the species with reference genome
Standard Data Analysis
  • Data quality control
  • Mapping onto reference genome
  • Peak calling
  • Motif prediction
  • Peak annotation and functional analysis of peak-associated genes
  • Summary of differential peaks and functional analysis of differential peak related genes
  • Visualization of ChIP-seq data
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    Note: Sequencing depths and bioinformatic analysis requests can be customized based on the project needs. Please contact us for more information.

    Project Workflow

    E6 Protein Expressed by High-Risk HPV Activates Super-Enhancers of the EGFR and c-MET Oncogenes by Destabilizing the Histone Demethylase KDM5C

    Background:

    Cervical cancer is one of the most common gynecological tumors, which seriously threatens women’s health. Infection by the high-risk (HR) types of HPV, HPV-16 and HPV-18, is the major cause of anogenital carcinomas in women and men, as well as a fraction of head and neck cancer. As a noncanonical function, HR HPV E6 plays an important role in regulating certain oncogene expression, such as EGFR and c-MET. However, the molecular mechanisms underlying the upregulation of these two proto-oncogenes are unknown. An emerging role in proto-oncogene activation is the abnormal epigenetic modifications. Previous studies have shown that HR HPV E6 interacts with both histone methyltransferases and acetyltransferases. Moreover, the generation and activation of super-enhancers can be a persistent regulatory element that drives the uncontrolled proliferation in cancer cells. A number of super-enhancers have been identified in various types of cancer, but it has not been reported in cervical carcinomas. It was demonstrated that the presence of HPV16 E6 is sufficient to upregulate the EGFR and c-MET super-enhancers, further elevating the expression of these two protooncogenes.

    Sampling & Sequencing Strategy:

    2.1. Samples:
    CaSki-pHAGE cells, CaSki-KDM5C cells

    2.2. Library preparation:
    ChIP-seq library and RNA-seq library

    2.3. Sequencing:
    Illumina HiSeq

    2.4. Bioinformatics analysis:
    ChIP-seq standard analysis and gene expression analysis

    Results:

    1) Whole genome ChIP-seq revealed existence of the EGFR and c-MET super-enhancers in the human cervical cancer cell line
    The existence of possible super-enhancers was screened by ChIP-seq analysis of H3K27Ac in the CaSki cells, as well as the CaSki-vector control and CaSki-KDM5C cells. It was found that there the two protooncogenes, EGFR and c-MET, each contained a super-enhancer. The EGFR super-enhancer is located in the first intron while the c-MET super-enhancer resides within the c-MET gene 50 region through intron 2 (Figure 1).

    Figure 1. KDM5C regulates super-enhancers activity and gene expression of the EGFR and c-MET.

    2) KDM5C regulates cervical cancer cell EGFR and c-MET expression by modulating their super-enhancer H3K4 methylation dynamics
    ChIP-qPCR of KDM5C was performed and displayed a direct correlation between the restoration of KDM5C and its enrichment. KDM5C restoration led to specific increased H3K4me1 in global super-enhancers rather than in adjacent regions, confirming KDM5C as a specific enhancer regulator (Figure 2).

    Figure 2. KDM5C were enriched at super-enhancer or other regions of EGFR and c-MET.

    Conclusion:

    This study generated a carcinogenic model of HPV infection: HPV16 E6 binds to KDM5C and form an E6‒E6AP‒KDM5C complex, thereby degrading KDM5C in a polyubiquitin-dependent manner. As a result, the super-enhancers of key protooncogenes, EGFR and c-MET, become highly upregulated, increasing their expressions and promoting tumor cell growth. This finding has provided novel insights into virus-induced cancer.

    Reference: Chen X, Loo J X, Shi X, et al. E6 Protein Expressed by High-Risk HPV Activates Super-Enhancers of the EGFR and c-MET Oncogenes by Destabilizing the Histone Demethylase KDM5C[J]. Cancer Research, 2018, 78(6): 1418-1430.


    Figure 1 Distribution of MAPQ


    Figure 2 Plots of strand cross correlation


    Figure 3 Distance distribution of peaks to TSS


    Figure 4 Motif analysis


    Figure 5 Peak distribution in functional gene region


    Figure 6 GO enrichment


    Figure 7 KEGG enrichment scatter