Epigenetic gene regulation also is controlled by changes in histones that make up the nucleosome and histone modification. Canonical nucleosomes are octamers that consist of H2A, H2B, H3 and H4 proteins. However, there are several histone variants that can vary by a small number of amino acids or include large insertions (reviewed in Sarma and Reinberg, 2005). Often these histone variants are found at specific locations within the chromatin or are used to demarcate boundaries between heterochromatin and euchromatin regions.
The majority of histone-mediated regulation stems from histone modification, most often modification of the exposed amino termini of histones protruding from the nucleosome core. The predominant histone modifications include acetylation, methylation, phosphorylation, ubiquitination and sumoylation, with thousands of potential combinations of modifications within a single nucleosome. Of these, histone acetylation and methylation are the best understood, and some general trends have been observed. Trimethylation of histone H3, specifically the lysine at position 4 (H3K4me3), is a mark associated with transcriptionally active chromatin, whereas H3K27me3 leads to compact chromatin, which represses gene expression. The term “histone code” is used to describe how different combinations of histone modifications affect transcription levels.
Identification of proteins that read, write or erase these marks is critical to help unravel the complexities of epigenetic regulation. Chromatin immunoprecipitation (ChIP) is a powerful assay to identify proteins that bind to chromatin and map protein binding throughout the genome using techniques such as microarray analysis or high-throughput sequencing.
In ChIP analysis, protein:protein and protein:DNA complexes are crosslinked, immunoprecipitated using an antibody against the protein of interest and purified. The DNA sequence of interest then is amplified from the immunoprecipitated material using PCR. Alternatively, the immunoprecipitated DNA can be sequenced (ChIP-seq) or analyzed using microarrays (ChIP-chip) to identify target sequences.
One challenge of the traditional ChIP method is the availability of specific antibodies that recognize crosslinked epitopes. To overcome the need for suitable antibodies, Promega scientists developed the HaloCHIP™ System (Figure 3). This system takes advantage of the HaloTag® protein, which is a mutated hydrolase (Los et al. 2005; Los and Wood, 2007;
Los et al. 2008; Hartzell et al. 2009) that catalyzes a covalent attachment to a variety of ligands, including a resin-based ligand for immobilization. This tag can be fused to any protein; for ChIP, the DNA-binding protein of interest is fused to the HaloTag® protein by cloning the protein-coding region into a HaloTag® vector. The recombinant construct is transfected into cells for stable or transient expression, then cells are treated with formaldehyde to induce covalent protein:DNA and protein:protein crosslinks, lysed and sonicated to shear the DNA into smaller fragments. The crosslinked complexes are captured directly from the lysate through covalent binding of the HaloTag® moiety to the HaloLink™ Resin. Covalent binding allows more extensive and stringent washing than is possible with noncovalent interactions, resulting in reduced background and increased signal-to-noise ratio. Subsequent heating of the purified complexes reverses the crosslinks and releases captured DNA fragments, which can be purified and analyzed using PCR, sequencing or microarray analysis. For more information, see the
HaloCHIP™ System Technical Manual #TM075.
Histone Acetylation and Deacetylation
Acetylation of a lysine residue neutralizes a positive charge on a histone protein, reducing the electrostatic interaction with negatively charged DNA. This reduction in affinity leads to increased accessibility of the DNA to protein complexes, which can lead to increased gene expression. In addition, lysine acetylation can recruit nucleosome-remodeling complexes, such as Swi2/Snf2, via their
bromodomains to promote and maintain euchromatin structure (reviewed in Bernstein et al. 2007). However, the factors controlling gene expression are complex, and histone acetylation also can lead to reduced gene expression through indirect mechanisms.
Lysine acetylation occurs on the N-terminal tails of core histones and is controlled primarily by two enzyme families: histone acetyl transferases (HATs) and histone deactylases (HDACs). HATs use acetyl CoA as a coenzyme to transfer an acetyl group to the epsilon amino group of the lysine side chain. These enzymes are grouped into three families: GNAT, p300/CBP and MYST. HDACs reverse histone acetylation and promote gene silencing. HDACs are often components of large protein complexes and are recruited to sites of DNA methylation by methyl DNA-binding proteins. HDACs fall into four categories: Class I, which includes HDAC1, 2, 3 and 8; Class, II, which includes HDAC4, 5, 6, 7, 9 and 10; Class III, which includes the NAD+-dependent sirtuins (SIRTs); and Class IV, which includes HDAC11 (reviewed in Sun et al. 2012).
Misregulation of HATs and HDACs often is associated with development and progression of cancer and other diseases such as neurodegenerative disorders and cardiovascular diseases, making these enzymes attractive therapeutic drug targets. Many HDAC inhibitors promote cell cycle arrest at the G1/S phase, and studies have shown that tumor cells generally are more sensitive to HDAC inhibitors than normal cells (Johnstone, 2002). Also, HDAC inhibitors can restore the ability of animals to recall memory that had been lost in Alzheimer’s and Parkinson’s disease models, possibly by changing chromatin structure in neurons (Fischer et al. 2007).
To facilitate screening of potential HDAC inhibitors, Promega offers the HDAC-Glo™ I/II Assays and Screening Systems and SIRT-Glo™ Assays Systems. The HDAC-Glo™ I/II and SIRT-Glo™ Assays are single-reagent-addition, homogeneous, luminescent assays that measure relative activities of HDAC class I and II enzymes and sirtuins, respectively. The HDAC-Glo™ I/II Assays use an acetylated, live-cell-permeant, luminogenic peptide substrate that is deacetylated by HDAC activities from cells, extracts or purified enzyme sources (Figure 4). The SIRT-Glo™ Assay uses a similar substrate to detect SIRT activities from purified enzyme sources (Figure 5). Deacetylation of the peptide substrate is measured using a coupled enzymatic system in which a protease in the Developer Reagent cleaves the deacetylated peptide from aminoluciferin, which is quantified in a luciferase-based reaction. The HDAC-mediated luminescent signal is proportional to enzyme activity and persistent, allowing batch processing of multiwell plates in high-throughput screening.
Figure 4. The single-reagent-addition HDAC-Glo™ I/II Assay.
HDAC activity deacetylates the luminogenic HDAC-Glo™ I/II Substrate, making the peptide sensitive to specific proteolytic cleavage that is mediated by the HDAC-Glo™ I/II Developer Reagent and liberates aminoluciferin. Free aminoluciferin then can be measured using Ultra-Glo™ firefly luciferase to produce stable, persistent light emission. Boc represents an amino-terminal blocking group that protects the substrate from nonspecific cleavage. XXLysine is an HDAC I/II-optimized consensus sequence derived from histone 4 (Smith et al. 2000).
Additional Resources for the HDAC-Glo™ I/II Assays and Screening Systems
Technical Bulletins and Manuals
HDAC-Glo™ I/II Assay and Screening System Technical Manual
Histone methylation occurs at lysine residues, which can be mono-, di- or trimethylated, and arginine residues, which can be mono- or dimethylated. Histone methylation is catalyzed by protein lysine methyltransferases (PKMTs) and protein arginine methyltransferases (PRMTs) but can be reversed by protein demethylases. To date, researchers have identified >30 demethylating enzymes, >50
protein lysine methyltransferases and >10 protein arginine methyltransferases, suggesting that protein methylation is a dynamic and complex process (Janzen et al. 2010). Histone methylation has different effects on transcriptional activity, depending on the number of methyl groups and position of the amino acid being modified. In general, the H3K9me1 mark is activating, whereas
H3K9me2 and H3K9me3 are repressive; H3K4me3 and H3K36me3 are associated with active chromatin, whereas H3K9me3, H3K27me3, H3K36me2 and H4K20me1 often are found in transcriptionally repressed heterochromatin.
The downstream effects of histone methylation are largely determined by proteins that bind to the modified histones. For example, H3K9me3 acts as a binding site for heterochromatin protein 1 (HP1), which then can recruit histone methyltransferases, histone deacetylases and other proteins that affect chromatin structure. H3K4me3 recruits proteins that promote euchromatin, whereas H3K9me1, H3K9me2 and H3K27me3 interact with proteins that promote heterochromatin. Two such groups of proteins are the polycomb group (PcG) proteins and their antagonists, the trithorax (trxG) group proteins, which were first identified as regulators of hox gene expression in Drosophila (Schwartz and Pirrotta, 2008). More recent studies have shown that related proteins exist in mammals and plants. PcG proteins repress transcription; trxG proteins activate transcription. Some PcG and trxG proteins possess histone methyltransferase activity and can modify histones directly, while others bind to and interpret histone modifications.
In embryonic stem (ES) cells, CpG islands that are regulated by PcG proteins often are “bivalent” in that they retain the permissive H3K36me2-depleted and H3K4me3-enriched environment but also exhibit H3K27me3. Genes with bivalent promoters often are actively silenced in ES cells but lose the repressive H3K27me3 mark while retaining the activating H3K4me3 mark later during differentiation.
Histones can be phosphorylated on serine, threonine and tyrosine residues. Many of the serine and threonine phosphorylation events play a role in DNA repair or DNA condensation, segregation and decondensation during mitosis, but some are involved in epigenetic regulation of transcription, including H3T3ph, H3T6ph, H3T11ph, H2.AS1ph, H3S10ph and H4S41ph (reviewed in Pérez-Cadahía et al. 2010). H3S10ph is one of the best characterized of these histone modifications. In addition to its DNA-restructuring responsibilities during mitosis, H3S10ph seems important for chromatin decondensation associated with transcriptional activation of target genes. H3S10ph recruits chromatin-modifying enzymes and chromatin-remodeling complexes and prevents binding of HP1 to neighboring H3K9me3 marks at the onset of mitosis. H3S10ph, as well as H3T3ph and H3T11ph, can block binding of DNMT3a to H3, reducing methylation of nearby chromatin (Zhang et al. 2010).
Several kinases are involved in phosphorylation of H3S10, including IkB kinase a (IKKa) (Yamamoto et al. 2003; Anest et al. 2003), proviral integration site for Moloney murine
leukemia virus 1 (PIM1) (Zippo et al. 2007) and ribosomal S6 kinase 2 (RSK2) (Sassone-Corsi et al. 1999). Addition of the H3S10ph mark to H3K9me3 is catalyzed by Aurora B kinase (Sabbattini et al. 2007), which also modulates chromosome structure during mitosis and mediates chromosome alignment and attachment to microtubules of the mitotic spindle.
Histones contain many highly conserved tyrosine residues, many of which can be phosphorylated. Phosphorylation of H3Y99 is critical for polyubiquitination and subsequent proteolysis of excess histones, which can increase a cell’s sensitivity to DNA-damaging agents, cause genomic instability and induce apoptosis. Another tyrosine residue, H3Y41, is important in chromatin structure and oncogenesis. In human hematopoietic cell lines, phosphorylation of H3Y41 by Janus kinase 2 (JAK2) destabilizes binding of HP1a to histone H3 (Dawson et al. 2009), leading to a more open chromatin structure around certain gene promoters such as leukemia oncogene LMO2, which can trigger oncogenesis in hematopoietic cells. Overexpression or aberrant activation of JAK2
activity leads to higher levels of H3Y41, loss of HP1a binding and higher expression of LMO2.
Promega offers a number of kinase enzyme systems to monitor the activity or identify inhibitors of different kinases involved in histone phosphorylation, including IKKa, PIM1, RSK2 and several cyclin-dependent kinases (CDKs) such as CDK1, CDK2 and CDK5. These luminescent assays convert ADP produced by these kinases to ATP, which is then converted to light by Ultra-Glo™ Luciferase. The resulting luminescent signal positively correlates with ADP amount and kinase activity. An example protocol is provided below. For a list of available Kinase Enzyme Systems, refer to the Human Kinome chart.
Additional Resources for Histone Phosphorylation
Technical Bulletins and Manuals
TM313 ADP-Glo™ Kinase Assay Technical Manual
Kinase Enzyme Systems
Conjugation of ubiquitin, a 76-amino acid protein, to lysine residues of histone proteins can affect transcription activity as well as nucleosome stability and, as a result, gene accessibility. The consequences of histone ubiquitination depend on the histone substrate and degree of ubiquitination (reviewed by Weake and Workman, 2008). Mono-ubiquitination of histone H2A (H2Aub1) is often considered a repressive mark, while H2B mono-ubiquitination can play a role in both transcriptional activation and silencing. In addition, there is evidence of cross-talk between histone ubiquitination and other forms of histone modification. For example, ubiquitinated H2B has been identified as a docking site for the COMPASS protein complex (Chandrasekharan et al. 2010), which includes the histone methyltransferase responsible for H3K4 methylation. Also, H2Aub, but not H2A, specifically represses di- and trimethylation of H3K4, and ubiquitin-specific protease 21 (USP21) relieves this repression (Nakagawa et al. 2008).
Ubiquitination of histones can be reversed by cleaving the peptide bond between ubiquitin and the ubiquitinated protein. Several deubiquitinases (DUBs) have been reported to deubiquitylate histones 2A, 2A.Z and 2B, including USP3, USP10, USP21, USP22 and Bap1. Histone deubiquitination has been associated with both transcription activation (Nakagawa et al. 2008; Draker et al. 2011; Gutiérrez et al. 2012) and repression (van der Knaap et al. 2005; van der Knaap et al. 2010).
Sumoylation as a Mechanism of Epigenetic Regulation
Another post-translational modification that plays an important role in epigenetic regulation is sumoylation, the addition of the small ubiquitin-related modifier SUMO (reviewed in Ouyang and Gill, 2009). This modification can stabilize proteins, alter subcellular localization, affect enzyme activity and mediate interactions with other proteins. Many transcription factors and cofactors can be sumoylated, which is generally indicative of transcription repression. In Drosophila, the sumoylated form of Sp3 recruits the polycomb protein Sfmbt (Steilow et al. 2008a) and HP1α, β and γ (Steilow et al. 2008b; Seeler et al. 2001) to repress transcription.
Many histone-modifying enzymes, nucleosome-remodeling complexes and their associated enzyme cofactors contain one or more SUMO interaction motifs (SIMs). This motif allows these proteins to interact with sumoylated transcription factors and cofactors, which can direct these enzymes to specific promoters. Two such groups of histone-modifying enzymes recruited by SUMO are histone deacetylases, which decrease histone acetylation at the target promoter, and histone demethylases such as lysine-specific demethylase 1, which catalyzes the removal of methyl groups from H3K4.