Studying Histone Modifications with Key Tools and Techniques

Introduction

Abcam can help users make their mark in epigenetic research, whether they are quantifying histone modifications or inhibiting or measuring the activity of erasers and writers. This article guides users on how to select reagents and techniques that are ideal for their experiments, right from sample preparation through to modification identification and characterization.

Getting started – sample preparation

Whether users are using tissues or cells, well-prepared starting material is needed to obtain accurate data. The following table sums up the different types of extraction methods, the type of application each method is best suited to, as well as the kits developed by Abcam to help users to achieve this.

 

 

Whole cell extraction

Nuclear extraction

Nuclear extraction (nucleic acid-free)

Histone extraction

Chromatin extraction

Applications

Enzyme activity assay

Enzyme activity assay

Protein detection

Histone detection

Chromatin IP

 

Protein detection

Protein detection

 

 

DNA-protein binding assays

 

 

 

 

 

Nuclear enzyme assays

Sample type and amount

Cells: 2-5 million

Cells: 2-5 million

Cells: 2-5 million

Cells: 2-5 million

Cells: 0.1-10 million

 

 

Tissue: variable

Tissue: variable

Tissue: 10 mg

Tissue: 50-200 mg

Assay time

≤ 45 minutes

≤ 60 minutes

≤ 60 minutes

≤ 60 minutes

≤ 60 minutes

Product code

ab113475

ab113474

ab113477

ab113476

ab117152

 

Quantifying universal levels of histone modifications

When studying histone post-translational modifications (PTMs), the first step is to observe the total changes in the PTMs levels across the whole genome. In techniques like western blot (WB), ELISA, immunocytochemistry (ICC), and immunohistochemistry (IHC), antibodies against specific protein modifications or proteins are available for use.

For instance, Abcam’s histone WB protocol can be utilized to evaluate the total level of histone PTMs in disease samples against the healthy ones. In this analysis, anti-histone H3 (ab1791) – a nuclear control antibody – will be used for result normalization.

Specific assays with fluorometric or colorimetric readouts can also be used to quantify histone PTMs, and these assays also provide a rapid way to scale up the experiments when there are large sample cohorts.

 

Detecting and quantifying histone modifications by ChIP

With Chromatin Immunoprecipitation (ChIP), users can identify the location of histone modifications within the genome. ChIP employs antibodies to separate a target protein or modification, along with any bound DNA. This can be subsequently used to discover the location of the target protein or modification inside the genome as well as its relative abundance at each site.

 

ChIP’ing histone modification is a robust tool for analyzing chromatin structure and regulation of gene expression. For instance, H3K9me3 marks satellite repeats and heterochromatin, H3K4me1 active enhancers, H3K27me3 promoters in gene-rich regions, and RNA pol II phospho S2 and S5 respectively correlate with initiation and elongation for transcription. With Abcam’s Ch IP kits, users can carry out reproducible, high-quality ChIP. The antibody is the most critical factor in a ChIP experiment.

 

ChIP-grade antibodies to key histone modifications and related proteins

Histone H3

H3K4me1

H3K27me3

H3K9me3

H3K27ac

H3K9ac

H3S10p

H3R2cit

H3K27M

H2AK119ub

γH2A.X

Histone H4

H4K16ac

RNA pol II phopsho S2

RNA pol II phopsho S5

RNA pol II

 

Quantifying the activity of writers and erasers

Enzymes called erasers and writers perform the addition and removal process of histone modifications. Enzyme activity assays can be used to determine the activity of these modifications. Applications include defining the histone modification pathways in the context of drug discovery and fundamental epigenetic mechanisms, where compounds like potential inhibitors can be screened against a series of assays. The following table shows some of the eraser and writer enzymes that play a role in specific histone PTMs.

 

Modification

Human recombinant proteins

H3K4 methylation

SETD7, NSD3, KMT2A, KMT2C, KMT2D, PRDM9, SETD1A, SETD1B, SMYD3

H3K4 demethylation

KDM1A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, PHF8, C14-orf169/NO66

H3K27 methylation

EZH1, EZH2, NSD2, NSD3, G9A, EHMT1

H3K27 demethylation

PHF8, KDM6A, KDM6B, KDM7A

Histone deacetylation

HDAC1 to 11

 

Inhibiting readers, writers, and erasers

Using tiny molecules to inhibit these regulatory enzymes can be useful for studying the biological functions of histone modifications. The function of histone modification binding proteins (also called ‘readers’) is inhibited by certain compounds. For instance, JQ1 uses acetylated lysines to inhibit the interaction of bromodomains in the BET protein family.

Inhibitors of readers, writers, and erasers essential tools for gaining a deeper insight into the epigenetic modification pathways and also crucial for validating ‘druggable’ targets in regard to pre-clinical researches in academia as well as the pharmaceutical sector.

 

Mass spectrometry for identifying novel histone modifications

Mass spectrometry (MS) is an important method for characterizing histone PTMs1, and is powerful enough to detect many novel modifications in single peptides using a somewhat modified version of the standard MS bottom-up technique2. Here, the samples are subjected to chemical derivatization in order to increase sequence coverage. A mass shift is promoted by PTMs that is evident in the MS spectra: for instance, +142 Da for acetyl groups and +14 Da for methyl groups.

Orbitraps are high-resolution analyzers that are often used to perform histone PTM analysis because of their ability to differentiate between PTMs with almost identical mass signatures (for instance, tri-methylation at 42.0470 Da and acetylation at 42.0106 Da)3.

Using additional liquid chromatography elution experiments and MS/MS fragmentation, the newly identified in vivo modifications were validated, often along with labeling of heavy isotopes and detection of antibodies.

 

References

  1. Karch, K. R., DeNizio, J. E., Black, B. E. & Garcia, B. A. Identification and interrogation of combinatorial histone modifications. Front. Genet. 4, 1–15 (2013).
  2. Arnaudo, A. R., Garcia, B.A. Proteomic characterisation of novel post-translational histone modifications. Epigenetics and Chromatin 6:24 (2013).
  3. Karch, K. R., Zee, B. M. & Garcia, B. A. High Resolution Is Not a Strict Requirement for Characterization and Quanti fi cation of Histone Post-Translational Modi fi cations. J. Proteome Res. (2014).

About Abcam

Abcam is a global life sciences company providing highly validated antibodies and other binders and assays to the research and clinical communities to help advance the understanding of biology and causes of disease.

Abcam’s mission is to serve life scientists to help them achieve their mission faster by listening to their needs, continuously innovating and improving and by giving them the tools, data and experience they want. Abcam’s ambition is to become the most influential life science company for researchers worldwide.

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Last updated: Jul 14, 2018 at 6:35 PM

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