ChIPseq Technique 2024 PDF

Technique: ChIPseq DNA is associated with proteins Half of chromatin mass comes from protein Many associated proteins are histone proteins that form nucleosomes Also non-histone proteins that bind the DNA for various reasons (replication, repair, recombination, transcription, etc)...

Technique: ChIPseq DNA is associated with proteins Half of chromatin mass comes from protein Many associated proteins are histone proteins that form nucleosomes Also non-histone proteins that bind the DNA for various reasons (replication, repair, recombination, transcription, etc) Major interest in learning about the DNA binding sites of these proteins or being able to separate out DNA fragments that are associated with a specific protein  ChIP ChIP – chromatin immunoprecipitation Allows the determination of protein-binding sites on DNA Separate out DNA fragments that are bound to a particular protein for other downstream applications Fragments can be analyzed with microarrays (ChIP-ChIP) or next- generation sequencing (ChIPseq) ChIP-chip Cells fixed with formaldehyde to crosslink DNA to associated proteins, cells lysed and DNA sheared Proteins and associated DNA are immunoprecipitated with antibody for protein of interest Crosslinking is reversed and proteins are separated from DNA DNA is amplified, labeled, and loaded onto microarray Fluorescence signals indicate enriched DNA regions where proteins were bound ChIP-chip (Two-color microarray) Cross-linked chromatin is immunoprecipitated to separate out DNA fragments where a specific protein is bound Cross-linking is reversed, DNA separated from protein, fluorescently labelled red Cross-linked chromatin (no immunoprecipitation) is used as comparison Cross-linking is reversed, purified, and fluorescently labelled green Hybridized on microarray Red indicates DNA sequences that are enriched for protein binding https://www.abcam.com/index.html?pageconfig=resource&rid=10738  ChIP-chip can identify enhancer regions Identification of TFs Nanog, Sox2, and Oct4 binding locations Contribute to the renewing and differentiating properties of embryonic stem cells Over 100 potential enhancer regions are regulated by all 3 All 3 bind to an enhancer of Hox1b, which affects development in Drosophila Nanog (G), Sox2 (R), and Oct4 ChIP-chip limitations Not possible to determine the exact protein binding site sequence (because larger fragments ~200-300bp are identified with ChIP) Only proteins that have antibodies can be studied Need to have a suitable microarray with good probe design Limited in how much of the genome can be represented ChIPseq Chromatin immunoprecipitation sequencing (next generation) Sequencing of genomic DNA fragments that are the binding locations of certain proteins in an unbiased way Transcription factors Chromatin modifying enzymes Modified histones Transcriptional machinery ChIPseq Workflow ChIPseq Workflow Determine the Find genes with similar chromatin state by histone marks and examining histone identify their functions tail modifications in – potentially similar these locations To DNA that was not immunoprecipita ted Identify genome Large letters = locations where frequently associated protein is bound with protein (consensus Applications – Histone Marks Normalized read distribution of sharp histone marks Two histone marks in each track (both associated with transcription activation) See locations of binding compared to gene regions Nakato & Sakata 202  Applications-histone modification differences between males and females Female Males s ^Stars indicate significant overlaps in clustered genes between M and F (Jaccard Index) Colors show distribution of histone marks in clustered Palmateer et al., 2021 ChIPseq benefits and limitations Can determine exact binding site and sequence Not dependent on microarray design Can investigate the entire genome Antibodies have to work well for the protein of interest Does require some knowledge about the reference genome Correct bioinformatics