Histones constitute the chief protein component of DNA. They help to maintain chromatin structure and regulate gene expression. The long double-stranded DNA molecule winds around histone octamers to form nucleosomes which serve the purpose of compacting DNA within the confines of the nuclear membrane. There are five major types of histones, namely H1/H5, H2, H3 and H4. H3.3 is a subtype of H3 histone and can be encoded either by the H3F3A or H3F3B genes independently. Amino acids such as lysine and arginine found in the histone tails are sites of post-translational modifications (PTMs) such as methylation and acetylation. These PTMs in histones are involved in the regulation of gene expression by chromatin remodelling and by controlling DNA methylation patterns. Mutations in histone genes can affect sites of PTMs causing changes in local and global DNA methylation status. These effects are directly linked to neoplastic transformation by altered gene expression. Recurrent H3.3 histone mutations are increasingly identified in several malignancies and developmental disorders. The following review attempts to shed light on the diseases associated with H3.3 histone mutations.
- molecular biology
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Histones are basic nuclear proteins that act as architectural scaffolds for the compaction of double-stranded DNA.1 The long double-stranded DNA molecule winds around the histones akin to a thread around beads so that it can be packed within the nucleus. The beaded structure formed by the wrapping of double-stranded DNA around the histone proteins is called a nucleosome (figure 1A). Hence, the primary function of histones seems to be involved in the formation of nucleosomes. Histone proteins are highly conserved among species and are the chief protein constituent of DNA.
There are five major histone families
Five major families of histone proteins, namely H1/H5 (linker histones), H2, H3 and H4 (core histones), have been recognised.2 A nucleosome is formed by winding approximately 146 base pairs of double-stranded DNA around a histone octamer. This histone octamer is formed by two molecules each of the four histones namely H2A, H2B, H3 and H4 which are termed the core histones (figure 1B). H1 is called the linker histone and it interacts with the double-stranded DNA between the two nucleosomes (internucleosomal DNA segment).3 Many histone variants exist except for H4.3 Histone variants are generated due to changes in the amino acid sequences along the N terminal end. H3 is a member of the core histone complex and seven variants of H3 histones have been identified in mammals (figure 2).2
Difference between canonical and non-canonical histones
Apart from the types mentioned earlier, histone proteins can be further categorised as canonical and non-canonical. Canonical histones are those that are produced and incorporated in the DNA only during the DNA synthesis phase and hence these are replication dependant. Non-canonical histones are constitutively expressed in the cells and aids in replication independent nucleosomal assembly.1 4 H3.1 and H3.2 are canonical H3 histones. H3.3, the centromere specific variant CENP-A and H3t (testis specific histone) are non-canonical histones. The newly characterised variant includes H3.Y.5 Further discussion will be restricted only to the H3.3 histone variant which is encoded by H3F3A and H3F3B genes.
Histone 3.3 variant
H3.3 protein is made up of 136 amino acids and is encoded by two independent genes, H3F3A and H3F3B, located in chromosome 1 (1q42.12) and 17 (17q25), respectively. Both the genes differ in their exonic and intronic sequences, but code for histone variant H3.3 that contains identical amino acid sequences.5 6 Both the genes appear to have a different set of promoters and regulators as well. Hence, a heterozygous mutation in either of the genes would be expected to affect nearly 25% of the total H3.3 protein in the cells depending on the differential expression of the genes.6 7 A unique feature of H3.3 histone genes is that they contain introns and the mRNA is polyadenylated, unlike most other histone genes which are polycistronic and intronless.5
H3.3 differs from the canonical histones H3.1 and H3.2 at the amino acid positions 31, 87, 89, 90 and 96 (figure 3).
Deposition of histones is aided by histone chaperon proteins
H3.3 histone is deposited predominantly at three sites within a chromosome:
Sites of active gene transcription requiring a high turnover of the nucleosomes.
Pericentromeric location—aids in proper separation of chromatin during cell division.
Peritelomeric location—helps to maintain telomere integrity.
H3.3 supports chromosomal heterochromatic structures, thus maintaining genome integrity during mammalian development.8 Deposition of H3.3 at these chromatin sites is mediated by specialised proteins called histone chaperon proteins. The two histone chaperon proteins for H3.3 include histone regulator A complex and ATRX/DAXX (alpha thalassemia mental retardation syndrome X linked/ death domain associated protein).4
Post-translational modifications in histones help in chromatin remodeling and regulation of gene expression
Histones are subjected to a wide variety of post-translational modifications (PTMs) which influence gene transcription. Acetylation, methylation, phosphorylation, ubiquitination are some of the common PTMs that occur in the amino acids comprising the histone proteins. These PTMs are maintained by code writers, erasers and code readers, which are proteins/enzymes that introduce, remove and recognise amino acid modifications, respectively. Lysine residues located along the N terminal frequently undergo PTMs. Acetylation or methylation of the lysine residues at H3K4, H3K36 and H3K79 lead to active chromatin marks while methylation of lysine residues at H3K27 and H3K9 lead to repressive chromatin marks.9 These PTMs are known as histone marks. These marks are recognised by specific protein complexes which act as chromatin remodelling complexes and help in the regulation of gene expression.
Alterations in the amino acid sequences due to mutations in the H3F3A/ H3F3B genes lead to changes in the PTMs in the H3.3 protein. This leads to global and local effects in DNA methylation status which acts as epigenetic causes of various diseases.10 11
Implications in disease
In general, mutations in the histone core affects nucleosomal stability while mutations in the tail region lead to changes in the dynamics of protein–protein interactions. Cancer causing histone mutations commonly affect the lysine residues in histone tails that are subjected frequently to PTMs. Mutations that affect the H3.3 gene can be either somatic or can be seen in germline configuration. Most of the cancer causing mutations observed so far are in somatic configuration. Germline H3.3 mutations are associated with neurological developmental disorders and not with malignancies12 (figure 4).
Role in brain cancers
Diffuse midline gliomas, H3 K27M mutant
Genome wide sequencing has identified recurrent somatic mutations in the H3F3A gene causing amino acid substitutions at two critical locations (K27M, G34R/V)(13,14).9 13 In addition to mutations in the H3.3 protein, mutations have also been identified to affect the ATRX and DAXX proteins which are histone chaperone proteins necessary for deposition of H3.3 histones within the nucleosomes in pericentromeric and telomeric regions.13 H3.3 K27M mutations are seen in diffuse midline gliomas affecting both paediatric and adult patients, while H3.3 G34R/V mutations are seen in diffuse supratentorial high-grade gliomas located within the cerebral hemispheres predominantly in older paediatric patients.4
Other mutations observed along with the K27M are loss of function mutations in p53, amplification or gain of function mutations in PDGFRa and FGFR1.1 The histone mutation K27M is heterozygous. A total of 70%–80% of paediatric diffuse midline gliomas have been found to harbour K27M mutations in the H3.3 variant, while in 30% of cases, similar K27M mutations have been reported in the H3.1 variant that is encoded by the HIST1H3B/C gene.10 Tumours resulting from K27M mutations in H3.3 (H3F3A) show poor response to radiotherapy with earlier relapses and metastasis in comparison with tumours resulting from mutations in H3.1 (encoded by the HIST1H3B/C gene).14 In the presence of an H3 K27M mutation, even lower-grade tumours are to be classified as WHO grade IV, due to the aggressive nature of these neoplasms.15
H3.3 K27M causes global DNA hypomethylation
H3.3 K27 is a well-recognised site of methylation during PTMs. Methylation of the lysine residue at the 27th position is a repressive chromatin mark. This mark is read by the EED domain of the polycomb repressive complex 2 (PRC2) protein. After recognition of the methylation mark in H3K27, the PRC2 complex causes methylation in the lysine residues of other H3.3 proteins in adjacent nucleosomes using a methyltransferase called EZH2, which is also a part of the PRC2 protein complex (figure 5A). H3 K27M mutation hinders this process and sequesters the active PRC2 complex thereby inhibiting its methyltransferase activity which leads to global DNA hypomethylation (figure 5B).16 This phenomenon is believed to contribute to gliomagenesis by altering the gene expression status. H3.3 K27M enhances neural progenitor cell proliferation and causes reprogramming of the cells to a more primitive state.
High-grade supratentorial paediatric gliomas
Glycine to arginine/valine substitution at the 34th position of H3.3 protein (G34R/V) has been observed in high grade gliomas that occur predominantly in the supratentorial regions in children and adolescents.1 The G34R/V mutation is associated with loss of function mutations in p53 and ATRX genes.13 H3.3 K27M and G34R/V mutations are mutually exclusive with each other, with IDH1/2 mutations, and also with analogous H3.1 mutations.4 H3.3 G34R/V mutant gliomas show a better prognosis in comparison with diffuse midline gliomas, K27M mutant type.13 It has been found that H3F3A G34 mutations cause upregulation of MYC-N, an important oncogene.11
Normally, there is a trimethylation PTM at the 36th lysine of H3.3, which is brought about by the action of SETD2 methyltransferase. ZMYND11 is a zinc finger protein that binds K36me3 and represses transcription by inhibiting RNA Polymerase II. In the presence of G34R/V, there is a reduction in the K36 trimethylation mark probably due to changes in the physical nature of the chromatin or other factors that hinder access of methyltransferases to K36 amino acid. Reduction in the K36me3 trimethylation mark has been found to enhance gene transcription due to local DNA hypomethylation which could lead to gliomagenesis.
Role in bone and cartilage tumours
Recurrent H3.3 mutations are also seen in bone and cartilage tumours, mainly in giant cell tumours, chondroblastomas and osteosarcomas. Chondroblastoma, which is a locally aggressive tumour seen in childhood and early adulthood is associated with K36M mutations commonly in H3F3B and occasionally in H3F3A.17 Presence of K36M mutations results in inhibition of methyltransferase enzymes such as MMSET and SETD2 by the mutant H3.3 K36M mutant proteins. Inhibition of methyltransferases leads to global reductions in H3K36me2/me3 methylation marks causing alterations in methylation landscape in tumour cells. These changes contribute to altered gene expression in cancer associated genes and lead to tumour formation.7 Point mutations in H3F3A such as G34W and G34L are highly prevalent among giant cell tumours of bone. Of importance is the fact, that these H3.3 somatic mutations have been detected only among the mononuclear stromal cells and not among the osteoclast-like multinucleate giant cells emphasising the neoplastic nature of the mononuclear stromal cells in these neoplasms.17
The G34R/V mutation in H3.3 variant documented among the supratentorial high grade gliomas in children has also been reported in a small fraction of osteosarcomas as well. Thus, among different subsets of bone and cartilage forming tumours, a very high frequency of H3.3 mutations is seen in chondroblastoma and giant cell tumours. Low prevalence of H3.3 mutations is noted in osteosarcoma, conventional chondrosarcoma and clear cell chondrosarcomas.17
Mutations affecting the K27, often associated with childhood brain tumours, are not seen among the bone and cartilage tumours. Likewise, K36 mutations which are commonly seen in skeletal tumours are not seen among brain tumours. Thus, there appears to be a close relationship between each tumour type and the specific H3.3 mutation.17
Role in non neoplastic conditions: neurodevelopmental disorders
Apart from the role of histone mutations in neoplastic conditions, a study by Bryant et al has demonstrated the presence of denovo heterozygous germline missense mutations affecting H3.3 proteins in neurodegenerative conditions and congenital anomalies.12 These missense mutations, identified primarily by exome or genome sequencing, were found to affect either H3F3A or H3F3B genes. The mutations are distributed throughout the coding regions without any hotspots. Importantly, these germline mutations were not associated with malignancies and appear to have a completely different pathogenic mechanism in comparison with cancer causing mutations as discussed earlier. These mutations can affect histone binding in the minor groove of DNA, may disrupt intermonomer contacts within the histone core, or may alter the chaperone binding sites leading to generalised loosening of chromatin structure (figure 6). The location of mutation does not correspond to the severity of phenotype. Most of these germline H3.3 mutations were found to be denovo among the 49 patients studied in the cohort.12 This study highlights the important role of H3.3 histones in neural development and function.
Mutations in H3F3A/ H3F3B genes that encode the H3.3 histone variant appear to play an important role in the formation of a subset of high grade glial tumours and primary skeletal tumours. Since these mutations appear to play a pivotal role in the causation of these neoplasms, these histones can be labelled as ‘oncohistones’. H3F3A/ H3F3B gene mutations can be somatic or germline in nature. Somatic mutations in the H3.3 protein lead to changes in global DNA methylation status which affects transcription and alterations in gene expression. While the somatic mutations are associated with the formation of malignancies, the germline mutations have been identified predominantly in patients with developmental disorders which highlights the important role played by histones in normal neural and embryological development. Understanding the role of H3.3 mutations will be instrumental in the development of potential targeted treatments.
Take home messages
H3.3 is a subtype of H3 histones and can be encoded either by H3F3A or H3F3B genes.
PTMs of histones play an important role in maintainence of chromatin structure and in regulation of gene expression.
Somatic H3.3 histone mutations have been implicated in diffuse midline gliomas, supratentorial paediatric gliomas, bone tumours such as giant cell tumours and chondroblastomas.
Neurodevelopmental disorders have been associated with germline H3.3 histone mutations.
Patient consent for publication
Handling editor Runjan Chetty.
Contributors VCK: contributed to the main writing work in the article and designed the illustrations. RP: provided technical assistance, proof reading of the article and contribution of ideas.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
Provenance and peer review Not commissioned; internally peer reviewed.
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