Chromatin is decorated with many regulatory factors such as DNA methylation, histone post-translational modifications and chromatin remodelers in order to regulate gene expression in a differential manner (for details on this visit our Epigenetics 101 Article). The roles of different Histone PTMs got uncovered in the last decade as many groups especially in the context of the ENCODE Consortium have worked on characterizing the functional elements of the human and murine genome. With this short Article we want to mention the relevant resources, explain which Histone Marks will allow you to look at the elements you are interested in and at the end give you the opportunity to get a Promo-Code for certain Antibody Combinations to use for your CUT&Tag experiment.
In 2019 ENCODE published a unified encyclopedia of human functional DNA elements, an effort to combine all annotations into a single encyclopedia that catalogs all human regulatory elements. In this Paper they annotated 164 human cell types using 1615 data sets. This resource allows us now to profile the epigenetics landscape using the following Histone PTMs:
Marks representative for different chromatin states
| Chromatin state | Molecular markers | Chromatin regions |
|---|---|---|
| Constitutive heterochromatin | H3K9me3, HP1 | Permanently silenced regions, centromeres, telomeres, 10% of the genome |
| Facultative heterochromatin | H3K27me3, PRC | Polycomb-repressed chromatin, cell type-specific repression, 15% of the genome |
| Transcribed region | H3K36me3, H3K79me2 | Enriched in gene bodies |
| Promoter | H3K4me3, H3K9ac | Transcription factor binding sites, binding of RNA-PolII |
| Enhancer | H3K27ac, when active: H3K4me1, P300 | Binding of transcription factors and regulatory proteins like P300 and CTCF |
| Region with weak marks of regulation | H3K4me1 | Bivalent sites which are "poised" for transcription, both promoters and enhancers. |
| Regulatory element with marks of both activation and repression | H3K4me3 or H3K27ac, H3K27me3 |
CUT&TAG as novel tool to analyze chromatin states
The chromatin immunoprecipitation (ChIP) assay to this day was the gold standard technique to analyze the binding of transcription factors to DNA and the localization of histones and histone modifications throughout the genome. ChiP protocols involve a multi-step process where each step is of crucial importance to obtain interpretable and reproducible ChIP results that can be trusted. Furthermore, ChIP assays generally require a relatively high amount of starting material, which is difficult to obtain for some sample types.
While the ChIP assay is still the primary method to investigate protein-chromatin interactions, alternative strategies are being developed to overcome some of the limitations of ChIP assays. In particular, more and more scientists are starting to use Cleavage Under Targets and Tagmentation (CUT&Tag) technology to investigate genomic localization of some histone modifications and transcription factors, especially with low cell numbers and even single cells.
The CUT&Tag method is a variation of Active Motif’s patented TAM-ChIP™ technology. CUT&Tag is based on the main ChIP principles, antibody-based binding of the target protein or histone modification of interest, but instead of an immunoprecipitation step, antibody incubation is directly followed by the shearing of the chromatin and library preparation. CUT&Tag assays take advantage of a Tn5 transposase that is fused with protein A to direct the enzyme to the antibody bound to its target on chromatin. The Tn5 transposase is pre-loaded with sequencing adapters (generating the assembled pA-Tn5 adapter transposome) to carry out antibody-targeted tagmentation.
CUT&Tag is fast, does not require fixation or sonication and less sequencing is required. This makes CUT&Tag a perfect method to investigate Histone PTMs that bind strongly to DNA and where fixation is not needed. However, this is also its Achilles heel, when it comes to Transcription Factors. Since they often times bind very transiently to DNA, CUT&Tag might not be able to catch those interactions.
Products
Summary
Histone Marks mentioned above in the Pape of the ENCODE consortium are the ones listed below. For your convenience, we added the relevant catalog numbers to it and also a short description of the Mark. Bold Marks are already validated for CUT&Tag.
- H3K4me1 (39297) - primed enhancers
- H3K4me2 (39141) - TF binding sites, overlap with ATAC-seq
- H3K4me3 (39159) - active Promoters
- H3K9ac (39917) - active Promoters?
- H3K9me3 (39161, 39765) - repressive mark
- H3K27ac (91193, 39133) - active enhancers
- H3K27me3 (39135, 39155) - repressive mark
- H3K36me3 (61101) - active transcription
- H3K79me2 (39143) - active transcription
- H4K20me1 (39727) - transcriptional activation
- Pol2 (91151) - transcription
- p300 (61401) - enhancers
- CTCF (91285, 61311) - enhancers, promoters