Chromatin structure related to oncogenesis

Abstract

Chromatin is the fundamental structure of genomic DNA in eukaryotic cells. The nucleosome, the primary unit of chromatin, consists of DNA and histone proteins, and is important for the maintenance of genomic DNA. Histone mutations are present in many types of cancers, suggesting that chromatin and/or nucleosome structures could be closely related to cancer development. Histone modifications and histone variants are also involved in regulating chromatin and nucleosome structures. Chromatin structures are dynamically changed by nucleosome binding proteins. In this review article, we discuss the current progress toward understanding the relationship between chromatin structure and cancer development.

Chromatin landscape regulated by oncoproteins, histone variants, and histone modifications. DNA damaging agents also affect chromatin structures by crosslinking or breaking DNA. The development of most cancers is triggered by defects in this regulatory mechanism.

Abbreviations

ATM

ataxia‐telangiectasia mutated

BRCA1/2

breast cancer susceptibility gene 1/2

BS

binding sequence

CHD

chromodomain helicase DNA‐binding

CBP

CREB‐binding protein

cryo‐EM

cryo‐electron microscopy

CTD

C‐terminal domain

DBD

DNA‐binding domain

DDB

DNA damage‐binding protein

DPIG

diffuse intrinsic pontine glioma

DSB

double‐stranded break

HDAC

histone deacetylase

HR

homologous recombination

NER

nucleotide excision repair

NTD

N‐terminal domain

PTM

posttranslational modification

XP

xeroderma pigmentosum

1. INTRODUCTION

Cancer evolution has been regarded as an unstoppable disease since ancient times in human history. A tumor development model was first established in the 1950s. ¹ Although the common mechanisms of cancer development are not fully understood, many types of cancer have been investigated by biochemical and genetic approaches. ² In response to external and/or internal factors, cancer cells modify their regulatory mechanisms to avoid cell death and thereby proliferate uncontrollably. In particular, mutations of cancer‐related genes, altered chromatin status, and the DNA damage response are involved in carcinogenesis. ³ Recent studies have shown that the chromatin dynamics could be a critical factor to suppress cancer development and tumor growth. ⁴ Numerous entities are reportedly involved in chromatin dynamics, including chromatin remodelers, histone modifiers, and histone variants. In this review, we focus on recent advances in cancer research, with a special emphasis on the oncoprotein p53, DNA repair factors, mutations of canonical histones and a histone variant (H2A.Z.1), and histone modifications (Figure 1).

FIGURE 1.

Chromatin landscape regulated by oncoproteins, histone variants, and histone modifications. DNA damaging agents also affect chromatin structures by cross‐linking or breaking DNA. The development of most cancers is triggered by defects in this regulatory mechanism.

2. CANCER‐ASSOCIATED HISTONE MUTATIONS

The basic unit of chromatin is the nucleosome, composed of DNA and four core histone proteins. The nucleosome plays essential roles in regulating genome functions such as DNA transcription, replication, recombination, and repair. Posttranslational modifications of histones, especially on histone tails, are closely associated with active and inactive states of gene expression, chromatin organization, and other essential biological processes. In addition to the influence of histone tail modifications on gene regulation, modifications of the histone globular domains are also crucial for recruiting chromatin‐binding proteins and maintaining nucleosomes in chromatin. ⁵ Missense mutations of histone residues that are responsible for PTMs and/or nucleosome stability could cause aberrant gene regulation and transformation into cancer cells.

Recurrent mutations are observed in all four core histones, H2A, H2B, H3, and H4. ⁶ , ⁷ Among them, mutations of the H3 K27, G34, and K36 residues, which are located at or near PTM sites, have been well studied. ⁸ , ⁹ , ¹⁰ , ¹¹ , ¹² , ¹³ , ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ , ²⁰ , ²¹ , ²² , ²³ , ²⁴ The H3 K27M mutation, in which the H3 K27 residue is replaced by methionine, was identified in pediatric glioblastomas and pontine gliomas, ⁹ , ¹¹ and leads to the loss of H3 K27 trimethylation associated with transcriptional silencing. The H3 K36M mutation, which replaces the H3 K36 residue with methionine, decreases the methylation level of the H3 K36 residue and increases that of the H3 K27 residue. The H3 K36M mutation has been observed in chondroblastomas and head and neck squamous cell carcinomas. ¹² , ²⁰ , ²¹ , ²² Even though the H3 K27M protein is 3.63% to 17.61% of the total H3 in human DPIGs, DPIGs expressing the H3 K27M mutant showed a significant reduction of H3 K27 trimethylation compared to non‐K27M DPIGs. ¹⁵ Similarly, 293 T cells expressing H3 K36M had reduced global levels of H3 K36 di‐ and trimethylation. ¹⁵ These results suggest that the H3K27M and H3K36M mutations have dominant negative effects on the histone methyltransferase activity.

A study of the oncohistone mutational landscape revealed that some mutations in the globular domain appear with high frequency, and affect the nucleosome stability and the protein associations with the nucleosome (Figure 2A). ⁶ , ⁷ One of the most frequent histone mutations in the globular domains is H2B E76K/Q, in which the H2B E76 residue is replaced by lysine or glutamine. The H2B E76K/Q mutation alters the interactions between H2B and H4, and destabilizes the nucleosome. ²⁵ , ²⁶ In the nucleosome, the H2B α2 helix and the H4 α3 helix directly interact in an antiparallel configuration, and the H2B E76 residue forms hydrogen bonds with the H4 R92 residue. By contrast, the H2B E76K mutation disrupts the hydrogen bonds with the H4 R92 residue, and consequently alters the direction of the H4 R92 side chain to avoid steric hindrance with the lysine side chain of H2B E76 (Figure 2B). ²⁵ Therefore, the H2B E76K mutation destabilizes the nucleosome due to the weakened interactions between the H2A‐H2B dimer and H3‐H4 tetramer. Consistently, the mobility of the H2B E76K mutant is reportedly higher than that of the WT H2B in cells.

FIGURE 2.

Recurrent mutations of histone proteins in a nucleosome. (A) H2A, H2B, H3, and H4 in the nucleosome (Protein Data Bank [PDB] ID: 5Y0C) are colored red, blue, green, and orange, respectively. Residues of recurrent histone mutations with high frequency (the number of mutations is more than 10 in the cBioPortal) in cancer cells are shown as spheres. ²⁷ (B) Close‐up views of the H2B α2 and H4 α3 helices in nucleosomes containing the H2B E76K mutant and H2B WT (PDB IDs: 5Y0D and 5Y0C). The H2B E76K mutant and H2B WT are shown in cyan and blue, respectively.

H3 E97K/Q, in which the H3 E97 residue is replaced by lysine or glutamine, is another cancer‐associated histone mutation in the globular domain. The H3 E97K/Q mutation is also frequently found in cancer cells. Biochemical and fluorescence recovery after photobleaching (FRAP) analyses revealed that the H3 E97K mutation destabilizes the nucleosome both in vitro and in vivo. ²⁵ In addition, the H3 E97K mutant was not incorporated within the mitotic chromosome.

Histone variants are nonallelic isoforms. Recurrent mutations are also observed in the histone variant H2A.Z.1, which is involved in the regulation of transcription, DNA repair, and chromosome segregation. The H2A.Z.1 R80 residue interacts with DNA in the nucleosome. The H2A.Z.1 R80C mutant, in which the H2A.Z.1 R80 residue is replaced by cysteine, forms an unstable nucleosome in vitro, and its mobility is slightly faster in cells. ²⁵

In summary, cancer‐associated histone mutations in the globular domain as well as histone tails affect the nucleosome structure and stability in vitro and in vivo. The nucleosome instability affects nucleosome formation, chromatin organization, inheritance of epigenetic information, and proper recruitment of chromatin binding proteins, and thus could dysregulate genomic DNA functions in cancer cells.

3. TUMOR SUPPRESSOR P53 ON CHROMATIN

p53 is a tumor suppressor, and its mutations are present in more than half of various cancers. ¹⁴ , ²⁸ p53 responds to cellular stresses such as DNA damage and regulates the transcription initiation of multiple downstream tumor suppressor genes, inducing apoptosis and cell cycle arrest. ²⁹ The key function of p53 is transcription activation as a pioneer transcription factor, which can bind the target DNA sequence on the nucleosome. ³⁰ , ³¹ , ³² , ³³ , ³⁴ , ³⁵ The p53 protein is composed of the NTD, including the transcription activation domain and the proline‐rich region, the core DBD, and the CTD, including the tetramerization domain and the nonspecific DNA binding region. ³⁶ The p53 NTD regulates transcription by interacting with a variety of transcriptional cofactors, such as basal transcription factors, mediator complexes, RNA polymerase II, and histone modifying enzymes. ³⁷ Most p53 mutations found in cancer cells are a single amino acid mutation in the p53 DBD. ³⁸ Two copies of a 10 bp DNA motif, 5′‐PuPuPuC (A/T) (A/T) GPyPyPy‐3′ (Pu, purine; Py, pyrimidine), form the consensus sequence for DNA binding by the p53 DBD. ³⁹ Genome mapping analyses of p53 binding and nucleosome occupancy on genomic DNA revealed that p53 is localized at the consensus sequence around the entry/exit sites of nucleosomes in cells. ²⁹ , ³⁴ In addition, biochemical analyses using in vitro reconstituted nucleosomes showed that p53 preferentially binds the p53 BS at the entry/exit sites of the nucleosome. ²⁹ , ³⁴ , ⁴⁰ Crystal structures of the p53 DBD in complex with DNA containing the p53 BS indicated that the homotetrameric p53 DBD recognizes two copies of the consensus DNA sequence. ⁴¹ , ⁴² , ⁴³ , ⁴⁴ , ⁴⁵ The cryo‐EM structure of p53 in complex with a nucleosome also revealed that the p53 DBD binds the p53 BS located near the entry/exit site of the nucleosome, and the angle of the linker DNA is drastically changed by approximately 40° in comparison with the p53‐free nucleosome. This can be accomplished because the linker DNA is peeled away from the histone core by the p53 BS interaction (Figure 3). ⁴⁶ Therefore, p53 binding to the p53 BS on the nucleosome could alter the higher‐order chromatin structure by changing the angle of the linker DNA, for activating transcription at certain chromatin loci (Figure 3). In contrast, the NTD and CTD of p53 have not been visualized on the nucleosome. A biochemical analysis indicated that the p53 CTD interacts with the nucleosome and is located near the dyad and linker DNA entry/exit site. ⁴⁶ Therefore, p53 CTD binding to the nucleosome might also contribute to chromatin rearrangement in cells. Further structural analyses of nucleosomes in complex with p53, modified by PTMs and/or binding factors, will provide a fuller picture of its functions as a pioneer transcription factor in chromatin.

FIGURE 3.

Conformational change of chromatin by p53 binding. Left panels: Closed configuration of chromatin without p53, in which the transcription of the tumor suppressor genes is inactivated. Right panels: Once p53 binds to the consensus sequence on the nucleosome, chromatin changes to the open configuration, and transcription is active.

4. DNA REPAIR AND CHROMATIN IN CANCER

Genomic DNA in eukaryotic cells is exposed to various harmful environmental factors, such as UV and ionizing radiation, reactive oxygen species, and chemical toxins. These DNA damages affect the stability of chromatin structures and are involved in cancer development. ⁴⁷ However, the mechanism by which DNA repair works against DNA lesions occluded within chromatin, in which the DNA is tightly packed, remains enigmatic. Defects in DNA repair cause the accumulation of genomic DNA lesions, triggering transcription blocks and replication errors. These dysfunctions result in various diseases, including cancers, progeroid syndrome, and neurodegeneration. ⁴⁸ , ⁴⁹ In contrast, promoting the DNA repair activity and the resistance to DNA damaging agents also accelerates cancer development, due to resistance to apoptosis induction (Figure 1). ⁵⁰ Therefore, genotoxins have been developed for cancer treatment to prevent DNA repair mechanisms. ⁵¹

4.1. Homologous recombination repair and cancer

Several DNA repair pathways are involved in maintaining genomic integrity, depending on the type of DNA damage. Homologous recombination, a representative DNA repair mechanism, is critical for the repair of DSBs (Figure 4). ⁵² The BRCA1 and BRCA2 proteins are recruited to DSB sites upon DSB induction, and mutations of these genes are found in several types of cancers, especially breast cancers. ⁵³ , ⁵⁴ In the HR repair pathway, the BRCA1 activity is likely to be regulated by the ubiquitination of histone H2A K15, through the binding of BRCA1‐associated RING domain protein 1 (BARD1) to nucleosomes. ⁵⁵ Ataxia‐telangiectasia mutated is a serine/threonine protein kinase, and its mutations are found in cancers. ATM is an upstream factor of BRCA1 in HR and a critical regulator of DNA repair. ⁵⁶ It phosphorylates γH2AX, p53, and checkpoint kinase 2 (CHK2) and initiates the cellular signaling pathway to proceed with DSB repair. ⁵⁷ , ⁵⁸ , ⁵⁹ ATM also phosphorylates KRAB‐associated protein‐1 (KAP1) upon DSB induction, affecting chromatin remodeling by CHD‐class chromatin remodelers. ⁶⁰ , ⁶¹ In addition to CHD, other chromatin remodelers are also important for the effective repair of DSBs. ⁶² , ⁶³ RAD51 is recruited to DSB sites by BRCA1 and BRCA2, and forms the RAD51 filament. ⁶⁴ , ⁶⁵ The active RAD51 filament has been proposed to be important in searching for homology in the donor chromatin, but it is still unclear how this activity functions with highly compacted chromatinized DNA. These studies suggest that the conformation and status of chromatin are critical for repairing DSBs efficiently and controlling cancer cell development.

FIGURE 4.

Model of homologous recombination repair mediated by RAD51 filaments. ATM, ataxia‐telangiectasia mutated; BARD1, BRCA1‐associated RING domain protein 1; BRCA1, breast cancer susceptibility gene; CHD, chromodomain helicase DNA‐binding; CHK2, checkpoint kinase 2; DSB, double‐stranded break; ISWI, imitation switch; KAP1, KRAB‐associated protein‐1; NBS1, nibrin; P, phosphorylation; RPA, replication protein A; Ub, ubiquitination.

4.2. Nucleotide excision repair and cancer

Although DSBs are severe DNA damage in cells, thymine dimers are also responsible for cancer development. Cockayne syndrome and XP are serious genetic diseases caused by defects in the NER pathway. ⁴⁸ , ⁴⁹ These diseases are characterized by a high frequency of skin cancers and sunburns by UV exposure, and in some cases, Cockayne syndrome and XP patients show progeroid symptoms and/or neurodegeneration. ⁶⁶ There are two NER pathways, transcription‐coupled NER and global genomic NER. The detailed mechanisms of global genomic NER have been well investigated by biochemical analyses in vitro, and the XPE/DDB2 and XPC proteins play important roles in initiating the NER pathway by binding to damaged sites on DNA (Figure 5). ⁶⁷ , ⁶⁸ Although the structures of DDB2 and Rad4 (XPC homolog) on damaged DNA have been determined, it is still unclear how these factors recognize damaged DNA in chromatin, which limits access to DNA lesions in the genome. ⁶⁹ , ⁷⁰ Damage recognition mechanisms in chromatin have been investigated by in vitro reconstitution experiments with nucleosomes containing chemically synthesized DNA lesions. The results suggested that DDB2 could potentially bind to DNA lesions in nucleosomes. ⁷¹ Recent cryo‐EM studies have revealed the binding structure of DDB2 in nucleosomes and suggested that DDB2 has the capability to slide along the DNA to accommodate the binding states for DDB2 using its binding energy, which is defined as slide‐assisted site exposure (SAsSE). ⁷² In cells, the DDB1‐DDB2 complex interacts with the Cullin4‐E3 ubiquitin ligase, and forms a 290 kDa complex called CRL4^DDB2, the DDB1‐DDB2‐CUL4A‐RBX1 complex. CRL4^DDB2 reportedly ubiquitinates DDB2, XPC, and histones H3 and H4 under the regulation of the constitutive photomorphogenesis 9 (COP9) signalosome. ⁷³ , ⁷⁴ In addition, the CRL4B^DDB2 complex formed by DDB1, DDB2, CUL4B, and RBX1B ubiquitinates histone H2A, which is reportedly associated with nucleosome stability and/or transcription. ⁷³ In the initiation step, NER factors regulate the PTMs of histones and seem to promote chromatin relaxation, in collaboration with the histone acetyltransferases p300/CBP or the chromatin remodeler INO80. ⁷⁵ , ⁷⁶ Interestingly, the inhibition of HDAC reportedly affected XPC recruitment to damaged sites. These results suggest that histone deacetylation, in addition to acetylation, is also critical for the NER pathway, indicating the importance of heterochromatin maintenance at damaged sites. ⁷⁷ The roles of p300/CBP and HDAC lead to a contradiction. Therefore, future studies will be necessary to understand NER stage‐dependent chromatin dynamics and genomic locus‐dependent DNA repair.

FIGURE 5.

Model of nucleotide excision repair (NER) machinery. CBP, CREB‐binding protein; CHD, chromodomain helicase DNA‐binding; CS, Cockayne syndrome; DDB, DNA damage‐binding; PARP, poly(ADP‐ribose) polymerase; PCNA, proliferating cell nuclear antigen; RPA, replication protein A; TFIIH, transcription factor II H; Ub, ubiquitin; UVSSA, UV stimulated scaffold protein A; VCP, valosin‐containing protein; XP, xeroderma pigmentosum.

5. CONCLUSION

Research on cancer‐associated histone mutations has clarified how recurrent histone mutations cause aberrant gene regulation, and will pave the way for epigenetic drug development. Additionally, histones and p53 share some common PTM enzymes, such as the histone acetyltransferase p300/CBP, suggesting that p53 modifications and chromatin dynamics are closely related. ⁷⁸ , ⁷⁹ Further structural analyses of various forms of p53 bound to nucleosomes will allow us to comprehend the regulatory mechanism of p53 on chromatin. These activities are also dynamically regulated upon DNA damage, and thus the roles of chromatin dynamics on damaged sites would be critical for cancer development. As the individual reports of the functions of histone modifications and chromatin regulations during DNA repair process are still controversial, further studies will be needed for their resolution.

FUNDING INFORMATION

The Novartis Foundation for the Promotion of Science (Grant/Award Number: Novartis Research Grants). Japan Agency for Medical Research and Development, (Grant/Award Number: JP22ama121009). Exploratory Research for Advanced Technology, (Grant/Award Number: JPMJER1901). Japan Society for the Promotion of Science, (Grant/Award Number: JP18H05534, JP20H00449, JP22K06076, JP22K06098, JP22K18034, JP23H05475). The Sumitomo Foundation, (Grant/Award Number: Basic Science Research Projects). Precursory Research for Embryonic Science and Technology, (Grant/Award Number: JPMJPR2288).

CONFLICT OF INTEREST STATEMENT

The authors have no conflict of interest.

ETHICS STATEMENT

Approval of the research protocol by an institutional review board: N/A.

Informed consent: N/A.

Registry and registration no. of the study/trial: N/A.

Animal studies: N/A.

Matsumoto S, Horikoshi N, Takizawa Y, Kurumizaka H. Chromatin structure related to oncogenesis. Cancer Sci. 2023;114:3068‐3075. doi: 10.1111/cas.15850