Epigenetics: CHIP- and ATAC-seq

Question 1

What is epigenetics

Answer 1

The study of how your behaviour and environment can cause changes that affect the way your genes work. Unlike genetic changes, epigenetic changes ARE reversible and do not change your DNA sequence. However, they can change how your body reads a DNA sequence

Question 2

What is important to open or close the accessibility of your DNA?

Answer 2

1. Histones. If DNA is tightly packed around a histone, TF cannot bind for protein production. This is regulated by the Histone tail
2. Acetylation and Methylation

Question 3

What does CHIP-seq and ATAC-seq stand for and how do they differ from each other?

Answer 3

CHIP-seq: Chromatin Immunoprecipitation. Analysis of histone modifications and protein-DNA interactions. It identify the binding sites of DNA-associated proteins. Specific for 1, so you already know what you want to analyse

ATAC-seq: Assay for Transposase-Accessible Chromatin. Checks for total chromatin accesibility. Provides a genome wide view of potentially active gene switches and TF-binding sites. It is more of a screening for open parts

Question 4

What is the principle of CHIP-seq

Answer 4

Primarily used to determine how TF/other chromatin-associated proteins influence phenotype-affecting mechanisms.

It start with crosslinking the TF’s to DNA. This can be with Formaldehyde for ex. Then there is the chromatin fragmentation. After that the immunoprecipitation where Ab’s bind to the proteins of interest. To get the DNA fragment, the only thing what need to be done is to purify the DNA and remove the crosslinks. Final, the purified DNA can be sequenced (by NGS for ex).

Question 5

What are the limitations for CHIP-seq

Answer 5

• As one is dependent of Ab’s, the research question is focussed and limited to the available Ab’s
• The resolution is limited to a resolution to the size of the DNA fragment generated following shearing, which makes it difficult to determine the exact bindingsite of a protein

Question 6

What technique can be used to get a better resolution for CHIP-seq

Answer 6

CHIP-exo! Exo is an enzyme what cleaves the DNA shorter so you will get smaller reads thus more specific.

Question 7

What is the principle of ATAC-seq

Answer 7

• It is a technique used to provide a genome-wide view of active genes and transcription factor binding sites. Moreover, ATAC –seq reveals genome-wide chromatin openness.
• It is an epigenetic discovery tool, and the technique is used to better understand chromatin accessibility, transcription factor binding, and gene regulation in cancer.
• Principle: Genomic DNA is exposed to Tn5, a highly active transposase(enzyme). Tn5 fragments DNA, inserts into open chromatin sites, and NGS adapters are loaded onto the transposase, which allows simultaneous fragmentation of chromatin and integration of those adapters into open chromatin regions. The library that is generated can be sequenced by NGS and the regions of the genome with open or accessible chromatin are analyzed using bioinformatics.

Question 8

What are the advantage and disadvantage of ATAC-seq

Answer 8

Advantages: It requires a low number of cells for input; Simple technique: 3 step-protocol; Cost-effective, Time efficient

Disadvantages: During sample processing, bound chromatin regions might open and be tagged by the transposase.