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Impact of Chromatin Immunoprecipitation Crosslinking Conditions on Selective Genomic Interactions by NF‐κB Family Members

ABSTRACT: The ability to capture protein: DNA interactions in a quantitative, high-resolution, and temporal manner in the cell context is crucial for elucidating the mechanisms underlying transcriptional regulation. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a widely used technique that enables the study of histone modifications and genome-wide interactions of transcription factors and chromatin regulators. Although ChIP-seq generates a vast amount of information, crosslinking conditions need to be carefully evaluated to minimize the capture of non-specific interactions. In this study, we conduct an in-depth analysis to evaluate the impact of crosslinking conditions on ChIP-seq results for two NF-kB family members, RelA and c-Rel. We titrate two commonly used chemical crosslinkers, formaldehyde and DSG. We demonstrate that while raising the crosslinking concentrations increases the total number of binding sites and the strength of the binding signal, high crosslinking conditions lead to an abundance of interactions detected at locations that lack anticipated motifs or annotations to potential NF-kB target genes. Our findings emphasize the importance of a comprehensive evaluation of ChIP-seq results and a cautious evaluation of chemical crosslinking conditions. Moreover, we compare ChIP-seq to CUT&TAG, a newer technique that does not require crosslinking. This comparison revealed strongly overlapping genomic interactions between the two methods. However, both methods display evidence of a large number of non-specific signals that exhibit little overlap between the two methods.

ORGANISM(S): Mus musculus

PROVIDER: GSE249834 | GEO | 2024/08/11

REPOSITORIES: GEO

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Project description:Protein-RNA interactions are critical for post-transcriptional regulatory processes that govern RNA metabolism. Several existing high throughput methods based to detect these interactions are reported for the biases associated to the inherent crosslinking approaches. In this study, we used Protein Occupancy Profile-Sequencing (POP-seq), a phase separation based method that does not require crosslinking, to examine the regulatory protein RNA interactions in cancer cell lines: K562, HepG2, A549, MCF7, Jurkat, and HEK293. POP-seq provides unbiased protein occupancy profiles in a transcriptome wide manner due to its crosslinking free approach. Our study demonstrates that POP-seq can enrich the protein occupied sites with over 60,000 peaks identified across multiple cancer cell lines. In our initial analysis, we compared POP-seq identified interactions with two protocols in presence and absence of UV crosslinking in K562 and HepG2 cells and results showed >70% overlap between the two approaches at the gene level, indicating that POP-seq can efficiently map the unbiased protein occupied sites in transcriptome wide manner. Using downstream bioinformatics analysis we found majority of POP-seq peaks on the exonic region of the transcript followed by introns and 3’ UTRs, while less than 10% abundance was recorded for repetitive elements like SINEs and LINEs. Among the various gene types, we found abundance of POP-seq peaks on protein coding genes followed by non-coding RNAs, that are typically highly expressed in cancer cell lines. We used CRISPR Cas 9 system to evaluate the functionality of POP-seq sites and observed that identified interactions could potentially impact the expression of nearby exons. Overall, this study provides the first comprehensive resource of transcriptome wide protein-RNA interaction maps in multiple cell line using a crosslinking free approach that further opens the opportunity to implement the method in primary tissues for detecting the regulatory interactions.

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Impact of Chromatin Immunoprecipitation Crosslinking Conditions on Selective Genomic Interactions by NF‐κB Family Members

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