16.13: ChIP - Imunoprecipitação de cromatina

Chromatin immunoprecipitation, or ChIP, is an antibody-based technique used to identify sites on DNA that bind to transcription factors of interest or histone proteins. It also helps determine the type of histone modifications such as acetylation, phosphorylation, or methylation.

Types of ChIP

ChIP can be divided into two types – X-ChIP and N-ChIP. X-ChIP involves in vivo cross-linking of histones and regulatory proteins to DNA, fragmenting the DNA by sonication, and isolating the protein-DNA complexes by immunoprecipitating them with specific antibodies. On the other hand, N-ChIP does not involve cross-linking of DNA to proteins, and digestion is carried out using nucleases.

Downstream analysis of DNA  – protein interactions

After isolating the DNA of interest, techniques such as PCR, microarrays, or southern blot can be used for analysis. Alternatively, this DNA can be used for deep sequencing,  known as ChIP-Seq. ChIP can be modified using other methodologies for different analysis such as ChIA-PET, a technique that combines the principles of ChIP with chromosome conformation capture to detect long-range chromatin interactions mediated via a protein of interest; enChIP, a technique which employs the CRISPR/Cas9 system to target specific genomic regions and RIP-ChIP/RIP-Seq, a modification of ChIP used to analyze protein-RNA interactions.

Comparison between N-ChIP and X-ChIP

N-ChIP and X-ChIP have their own advantages and disadvantages. N-ChIP results in stronger antibody binding and efficient and highly specific immuno-precipitation. However, N-ChIP is suitable only in the case of tightly bound proteins such as histones, as transcription factors may get detached during processing. Additionally, not all of the nuclease-digested chromatin gets solubilized, resulting in the missing out of certain fractions of the sample.  X-ChIP is an excellent methodology for studying transcription factors that are not bound tightly to the DNA due to its cross-linking step. X-ChIP assay is also more sensitive than N-ChIP and requires lower amounts of samples as well as antibodies. Disadvantages of X-chip include possible difficulty in fragmentation due to excess cross-linking and false positives due to the cross-linking of transient-DNA protein interactions.