Welcome back to the 4th and final installment in our epigenetics crash course series!
So far, we’ve introduced the field of epigenetics, and examined bisulfite as well as non-bisulfite approaches to studying nucleic acid methylation. In this last piece, we look at methods to study chromatin and histones.
Why Study Histones and Chromatin?
While studying nucleic acid methylation can tell us a lot about how individual genes and transcripts are switched on and off, studying histones and chromatin can provide a bigger picture about:
• How DNA interacts with the structural proteins and enzymes involved in epigenetics
• How these interactions regulate and impact critical processes such as embryonic development
• How aberrant processes can lead to neurological disorders, cancer and other diseases.
You will recall from Part 1 that chromatin is comprised of chromosomal DNA that is packaged very tightly into bead-like structures called nucleosomes, which are held in place by histones. Tightly packed chromatin is known as heterochromatin while loosely packed chromatin that is accessible to transcription machinery is known as euchromatin.
Figure 1: Illustrations of loosely packed euchromatin and tightly packed heterochromatin.
Image courtesy of Epigentek.
Genes within heterochromatic regions cannot be transcribed because they are not accessible to transcription machinery, so these appear silent. Conversely, genes within euchromatin are readily transcribed.
Packaging and unpackaging of chromatin is mediated by enzymes that modify chromatin structure, either directly (chromatin-modifying enzymes) or indirectly by influencing the function of histones (histone-modifying enzymes).
How are Histones and Chromatin Studied?
The function and impact of histone- and chromatin-modifying enzymes are deciphered using a variety of approaches that include:
• Microtitre plate assays that can quantitatively measure the activities of the modifying enzymes or the modifications themselves, e.g., histone modifications.
• immunohistochemistry (IHC)
• immunofluorescence (IF)
Histone Modification Analysis
So far, five distinct types of histones have been identified, and these fall into two categories; the core histones H2A, H2B, H3, and H4, and the linker histones H1 and its homologue H5. The core histones associate with DNA to form nucleosomes, while the linker histones mediate higher-order chromatin structures.
Post-translational modifications to histones, usually at their N-terminal tails, influence the expression of genes/whole regions by altering the chromatin structure. This is done directly or by recruiting the activity of other enzymes that can remodel chromatin, so that it transits between hetero- and euchromatic states.
The major histone modifications include methylation, phosphorylation, acetylation and ubiquitylation. Histones are typically modified at specific amino acid residues and there is considerable conservation across species (Figure 2).
Figure 2: Schematic of major histone tail post-translational modifications. In most species, histone H3 is primarily acetylated at lysines 9, 14, 18, 23, and 56, methylated at arginine 2 and lysines 4, 9, 27, 36, and 79, and phosphorylated at ser10, ser28, Thr3, and Thr11. Histone H4 is primarily acetylated at lysines 5, 8, 12 and 16, methylated at arginine 3 and lysine 20, and phosphorylated at serine 1. Image courtesy of Cusabio.
The most-studied histone-modifiying enzymes include histone acetyltransferases (HATs), histone deacetylaces (HDACs), histone methyltransferases (HMTs) and histone demethylases. These add or remove actyl or methyl groups, respectively, to histones at specific amino acid residues.
The activities of these enzymes can be monitored indirectly in ELISA-like assays that detect the presence of the corresponding histone marks using highly-specific antibodies, or directly in enzymatic activity assays that are designed to quantitatively detect the ability of a specific enzyme (present in the researcher’s sample) to modify a histone substrate supplied with the assay.
Nordic BioSite supplies a range of kits from EpiGentek to study all the major histone modifications and histone-modifying enzymes activity. Selected products from this offering include kits to study individual histone methylation and demethylation and kits to study individual histone acetylation & deacetylation. As well as this, there are two multiplex screening kits that allow one to simultaneously detect and quantify 21 different H3 or 10 different H4 modifications within a single sample.
Nordic BioSite also carries an extensive range of antibodies from multiple suppliers that can be used to detect single and combinations of histone modifications, which can be used in applications such as Western blotting, immunoprecipitation, and microscopic methods such as immunohistochemistry (IHC) and immunofluorescence (IF).
Chromatin Analysis by Immunoprecipitation
Chromatin immunoprecipitation or simply ChIP is the prevailing method
to investigate protein-DNA interactions. This is an enrichment method that is performed using a highly specific antibody that can bind to a target DNA-binding protein within a chromatin extract or a cell lysate. Prior to ChIP, the chromatin to be analysed is fragmented by digestion or sonication-based shearing, depending on whether a fixation step was included prior to cell lysis to crosslink the proteins to the chromatin.
Once the capture antibody has bound the target protein and any DNA sequences it is interacting with, the target is then precipitated from the rest of the sample. A cleanup step is usually performed on the bound DNA at this point to remove any reagents or material that could interfere with downstream applications.
The resulting enriched sample can then be used in a number of downstream applications, including PCR, bisulfite conversion, microarray or next-generation sequencing (NGS), to determine which regions of the gDNA were modified with the target methylation.
With ChIP, it is possible to study a particular protein-DNA interaction, several protein-DNA interactions, or interactions across the whole genome or a subset of genes. The capture antibody used in a ChIP experiment can be one that binds histones, histone modifications, transcription factors or cofactors, to provide information about chromatin states and gene transcription.
The remaining sections look at some of the most popular ChIP-based workflows for chromatin analysis.
As the name suggests, ChIP-Seq is a workflow that combines ChIP and NGS. ChIP-Seq is the method of choice for identifying genome-wide DNA-binding sites of transcriptions factors and other DNA-binding proteins.
ChIP-Seq generates large datasets and offers the benefit of resolving the specific genetic sequence of target protein binding sites that can be mapped on a genome-wide level.
In this workflow, real-time quantitative PCR (RT-qPCR) is combined with ChIP to analyse DNA-binding sites for selected histones and/or other DNA-binding proteins of interest.
ChIP-PCR is a good option when the researcher has a specific set of target loci in mind and wants to check whether or not the target protein is interacting with these loci. In this setup, the researcher designs a set of target-specific primers, which are used to amplify target loci from ChIP-enriched samples in a real-time PCR reaction.
For fast, sensitive and specific ChIP-PCR, Epigentek offers the EpiQuik™ Quantitative PCR Fast Kit, which employs a novel hot-start polymerase that reduces overall run time 70 minutes.
CUT&RUN and CUT&TAG
CUT&RUN is a recently developed method to study DNA-protein interactions. The name stands for Cleavage Under Target & Release Using Nuclease, and the procedure was developed as an improvement to conventional ChIP to release the captured target protein/DNA complexes from limited biological materials for mapping protein-DNA interactions.
In CUT&RUN, nuclei are isolated from cells and the target protein-DNA complex is bound with the ChIP-grade antibody of interest, just as in standard ChIP. A unique nucleic acid cleavage enzyme mix is then applied to fragment the chromatin. This cleaves and removes DNA sequences at both ends of the target protein/DNA complex without disturbing the DNA sequence bound by the target protein. The target protein-bound DNA is then purified and eluted, and the enriched DNA can be further analysed using ChIP-Seq, ChIP-PCR or another downstream application.
The removal of DNA sequences adjacent to the target-binding site has made it possible to perform ChIP with much less starting material and it has also greatly reduced background signals in downstream assays.
And as an extension to CUT&RUN, the cTIP (CUT & Tag In Place) procedure can be added to prepare a library for NGS. Epigentek’s CUT&RUN protocol followed by its CUT&TAG library preparation protocol provide a similar solution to ChIP-Seq while maintaining the benefits of CUT&RUN mentioned above (Figure 3).
Figure 3: Schematic depiction of CUT&RUN workflow. Image courtesy of Epigentek.
We’d Love to Hear From You
This concludes our Epigenetics Crash Course. Epigenetics is a huge area of research and the methods and tools used to study epigenetic modifications extend beyond the highlights that we have tried to condense in this series. If you would like to hear more about the methods covered by us so far or other methods that might better suit your research, please don’t hesitate to get in touch with us by sending an email to firstname.lastname@example.org.
Also, if there is a topic related to epigenetics that you would have liked to see covered in this series, please do write to us with your suggestion and we will take it into consideration for future blog posts!