Learn about the most common histone modifications eg H3K4me3, H3K9me3, and H3K27me3 and their readers, writers, and erasers.
Chromatin architecture, nucleosomal positioning, and ultimately access to DNA for gene transcription, is largely controlled by histone proteins. Each nucleosome is made of two identical subunits, each of which contains four histones: H2A, H2B, H3, and H4. Meanwhile, the H1 protein acts as the linker histone to stabilize internucleosomal DNA and does not form part of the nucleosome itself.
Histone proteins undergo post-translational modification (PTM) in different ways, which impacts their interactions with DNA. Some modifications disrupt histone-DNA interactions, causing nucleosomes to unwind. In this open chromatin conformation, called euchromatin, DNA is accessible to binding of transcriptional machinery and subsequent gene activation. In contrast, modifications that strengthen histone-DNA interactions create a tightly packed chromatin structure called heterochromatin. In this compact form, transcriptional machinery cannot access DNA, resulting in gene silencing. In this way, modification of histones by chromatin remodeling complexes changes chromatin architecture and gene activation.
At least nine different types of histone modifications have been discovered. Acetylation, methylation, phosphorylation, and ubiquitylation are the most well-understood, while GlcNAcylation, citrullination, crotonylation, sumoylation, and isomerization are more recent discoveries that have yet to be thoroughly investigated. Each of these modifications are added or removed from histone amino acid residues by a specific set of enzymes.
Together, these histone modifications make up what is known as the histone code, which dictates the transcriptional state of the local genomic region. Examining histone modifications at a particular region, or across the genome, can reveal gene activation states, locations of promoters, enhancers, and other gene regulatory elements.
Acetylation is one of the most widely studied histone modifications since it was one of the first discovered to influence transcriptional regulation. Unmodified lysine residues are positively charged but acetylation results in neutralization of the charge on histones, which reduces the interaction of histones and negatively charged DNA. The charge neutralization results in a weaker histone: DNA interaction, allows transcription factor binding and significantly increases gene expression (Roth et al., 2001).
Histone acetylation is involved in cell cycle regulation, cell proliferation, and apoptosis and may play a vital role in regulating many other cellular processes, including cellular differentiation, DNA replication and repair, nuclear import and neuronal repression. An imbalance in the equilibrium of histone acetylation is associated with tumorigenesis and cancer progression.
Acetyl groups are added to lysine residues of histones H3 and H4 by histone acetyltransferases (HAT) and removed by deacetylases (HDAC). Histone acetylation is largely targeted to promoter regions, known as promoter-localized acetylation. For example, acetylation of K9 and K27 on histone H3 (H3K9ac and H3K27ac) is usually associated with enhancers and promoters of active genes. Low levels of global acetylation are also found throughout transcribed genes, whose function remains unclear.
Methylation is added to the lysine or arginine residues of histones H3 and H4, with different impacts on transcription. Arginine methylation promotes transcriptional activation (Greer et al., 2012) while lysine methylation is implicated in both transcriptional activation and repression depending on the methylation site. This flexibility may be explained by the fact that that methylation does not alter histone charge or directly impact histone-DNA interactions, unlike acetylation.
Lysines can be mono-, di-, or tri-methylated, providing further functional diversity to each site of methylation. For example, both mono- and tri-methylation on K4 of histone H3 (H3K4me1and H3K4me3) are activation markers, but with unique nuances: H3K4me1 typically marks transcriptional enhancers, while H3K4me3 marks gene promoters. Meanwhile, tri-methylation of K36 (H3K36me3) is an activation marker associated with transcribed regions in gene bodies.
In contrast, tri-methylation on K9 and K27 of histone H3 (H3K9me3 and H3K27me3) are repressive signals with unique functions: H3K27me3 is a temporary signal at promoter regions that controls development regulators in embryonic stem cells, including Hox and Sox genes. Meanwhile, H3K9me3 is a permanent signal for heterochromatin formation in gene-poor chromosomal regions with tandem repeat structures, such as satellite repeats, telomeres, and pericentromeres. It also marks retrotransposons and specific families of zinc finger genes (KRAB-ZFPs). Both marks are found on the inactive chromosome X, with H3K27me3 at intergenic and silenced coding regions and H3K9me3 predominantly in coding regions of active genes.
Histone methylation is a stable mark propagated through multiple cell divisions, and for many years was thought to be irreversible. However, it was recently discovered to be an actively regulated and reversible process.
Histone phosphorylation is a critical intermediate step in chromosome condensation during cell division, transcriptional regulation, and DNA damage repair (Rossetto et al., 2012, Kschonsak et al., 2015). Unlike acetylation and methylation, histone phosphorylation establishes interactions between other histone modifications and serves as a platform for effector proteins, which leads to a downstream cascade of events.
Phosphorylation occurs on all core histones, with differential effects on each. Phosphorylation of histone H3 at serine 10 and 28, and histone H2A on T120, are involved in chromatin compaction and the regulation of chromatin structure and function during mitosis. These are important markers of cell cycle and cell growth that are conserved throughout eukaryotes. Phosphorylation of H2AX at S139 (resulting in γH2AX) serves as a recruiting point for DNA damage repair proteins (Lowndes et al., 2005, Pinto et al., 2010) and is one of the earliest events to occur after DNA double-strand breaks. H2B phosphorylation is not as well studies but is found to facilitate apoptosis-related chromatin condensation, DNA fragmentation, and cell death (Füllgrabe et al., 2010).
All histone core proteins can be ubiquitylated, but H2A and H2B are most commonly and are two of the most highly ubiquitylated proteins in the nucleus (Cao et al., 2012). Histone ubiquitylation plays a central role in the DNA damage response.
Monoubiquitylation of histones H2A, H2B, and H2AX is found at sites of DNA double-strand breaks. The most common forms are monoubiquitylated H2A on K119 and H2B on K123 (yeast)/K120 (vertebrates). Monoubiquitylated H2A is also associated with gene silencing, whereas H2B is also associated with transcription activation.
Poly-ubiquitylation is less common but is also important in DNA repair-- polyubiquitylation of H2A and H2AX on K63 provides a recognition site for DNA repair proteins, like RAP80.
Like other histone modifications, monoubiquitylation of H2A and H2B is reversible and is tightly regulated by histone ubiquitin ligases and deubiquitylating enzymes.
Table 1. A cheat sheet for the most common histone modifications and where to find them:
ChIP uses antibodies to isolate a protein or modification of interest, along with the DNA to which it is bound (figure 5). The DNA is then sequenced and mapped to the genome to identify the protein or modification’s location and abundance.
Figure 5: Histone modification ChIP. Antibodies bind directly to modified histone tails. Immunoprecipitation and DNA purification allow for the isolation and identification of the genomic regions that the modifications occupy.
Utilizing antibodies against specific histones and histone modifications in ChIP experiments can reveal the specific locations of:
If the function of a histone modification is known, ChIP can identify specific genes and regions with this histone modification signature and the corresponding function across the genome. These genes and regions can then be further examined for their role in the biological process of interest. Using ChIP against H3K4me1, for example, will reveal the locations and sequences of active enhancers throughout the genome, pointing to genes and genetic programs of interest.
Alternatively, if the function of the histone modification is not known, ChIP can identify sequences, genes, and locations with this signature, which can then be used to infer the function of the modification. This technique was pivotal in decoding much of the histone code and is still valuable in ascertaining the function of newly discovered modifications like ubiquitylation and other novel markings.
Histone modifications are dynamically added and removed from histone proteins by specific enzymes (table 2). The balance between these writers and erasers dictates which marks are present on histones, and at what levels, to ultimately control whether specific genetic programs and the cellular processes they orchestrate, are turned on or off.
Table 2. The major categories of histone writers and erasers:
For more details on the readers, writers, and erasers of histone modifications take a look at our epigenetic modification’s poster.
Identifying modification pathways and the specific writers and erasers at play can reveal:
For drug development efforts, compounds can easily be screened for their impact on writer and eraser activity.
In general, histone methyltransferase (HMT) assays are challenging to develop, and most have several drawbacks
