Histone methylation is an important epigenetic mechanism that regulates chromatin states and gene expression. Different modification sites and degrees of methylation produce distinct transcriptional effects, and their dysregulation is closely associated with tumorigenesis, impaired cell differentiation, and disrupted genome stability.

Keywords: histone methylation; H3K4; H3K27; H3K79; EZH2; DOT1L; LSD1; PRMT5; epigenetics

1 Overview of Histone Methylation

1.1 Nucleosomes and Histone Modifications

In eukaryotic cells, DNA wraps around a histone octamer composed of H2A, H2B, H3, and H4 to form nucleosomes, which are further organized into chromatin with different degrees of compaction. Histones contain multiple lysine and arginine residues that can undergo post-translational modifications. Methylation, acetylation, phosphorylation, ubiquitination, and other modifications can alter the recruitment of chromatin regulatory proteins and nucleosome functions, thereby participating in processes such as gene expression, DNA replication, and damage repair.

1.2 Major Forms of Histone Methylation

(1) Lysine Methylation

Lysine residues can undergo monomethylation (me1), dimethylation (me2), or trimethylation (me3). Different methylation states have independent biological significance and do not simply represent progressively increasing levels of modification. For example, H3K4me1 is primarily associated with enhancer states, whereas H3K4me3 is predominantly enriched at active gene promoters.

(2) Arginine Methylation

Arginine residues can undergo monomethylation or symmetric or asymmetric dimethylation. This process is primarily catalyzed by protein arginine methyltransferases (PRMTs) and participates in transcriptional regulation, RNA processing, DNA damage repair, and protein interactions.

(3) Site-Dependent Transcriptional Effects

Histone methylation generally does not substantially alter the charge of lysine residues. Its functions are mainly achieved by establishing specific protein-recognition sites. Whether methylation produces transcriptional activation or repression therefore depends primarily on the modification site, degree of methylation, and corresponding reader proteins. Histone methylation cannot simply be equated with gene silencing.

2 Major Histone Methylation Marks

2.1 H3K4 Methylation

H3K4 methylation is generally associated with transcriptionally active states. H3K4me3 is primarily enriched near the transcription start sites of active genes and participates in the recruitment of transcription initiation complexes and chromatin regulatory proteins, whereas H3K4me1 is mainly distributed at enhancer regions. The SET1/KMT2 family comprises important catalytic enzymes for H3K4 methylation, while LSD1/KDM1A and the KDM5 family participate in removing H3K4 methylation marks at different methylation states.

2.2 H3K9 and H3K27 Methylation

(1) H3K9 Methylation

H3K9me2 and H3K9me3 are generally associated with transcriptional repression and heterochromatin formation. G9a/EHMT2 and GLP/EHMT1 primarily participate in the formation of H3K9me1/2, whereas SUV39H1/2 mainly catalyze H3K9me3. H3K9me3 can further recruit proteins such as HP1, promoting chromatin compaction and stable gene silencing.

(2) H3K27 Methylation

H3K27me3 is an important mark of Polycomb-mediated transcriptional repression. EZH2 or EZH1 within the PRC2 complex catalyzes H3K27 methylation, and the resulting H3K27me3 participates in maintaining the repressed states of genes associated with development and cell fate. KDM6A/UTX and KDM6B/JMJD3 participate in the removal of H3K27 methylation.

2.3 H3K36 and H3K79 Methylation

(1) H3K36 Methylation

H3K36me3 is mainly distributed across the gene bodies of actively transcribed genes and is associated with transcriptional elongation, RNA splicing, suppression of aberrant transcription initiation, and DNA repair. SETD2 is the major methyltransferase responsible for H3K36me3 formation in mammalian cells, whereas the NSD family is more extensively involved in regulating H3K36me1/2.

(2) H3K79 Methylation

H3K79 is located within the globular core region of histone H3, and its methylation is primarily catalyzed by DOT1L. H3K79 methylation is associated with active transcription, transcriptional elongation, DNA damage responses, and hematopoietic regulation. DOT1L lacks a typical SET domain and possesses a unique catalytic structure among histone lysine methyltransferases.

2.4 H4K20 and Arginine Methylation

(1) H4K20 Methylation

H4K20 can exist in different methylation states, including me1, me2, and me3. H4K20me1 participates in regulating chromatin structure and the cell cycle, H4K20me2 participates in DNA damage responses and constitutes part of the chromatin environment required for 53BP1 recruitment, and H4K20me3 is more commonly associated with heterochromatin and transcriptional repression.

(2) Histone Arginine Methylation

The PRMT family catalyzes multiple histone arginine sites. Certain asymmetric dimethylation marks mediated by PRMT4/CARM1 are associated with transcriptional activation, whereas symmetric dimethylation marks such as H4R3me2s and H3R8me2s mediated by PRMT5 can participate in the formation of repressive chromatin and other transcriptional regulatory processes.

3 Writing, Erasing, and Reading Histone Methylation

3.1 Histone Methyltransferases

(1) Lysine Methyltransferases

Histone lysine methyltransferases (KMTs) primarily use S-adenosylmethionine (SAM) as a methyl donor to transfer methyl groups to specific lysine residues. Most KMTs, including EZH2, SETD2, G9a, and the KMT2 family, contain a catalytic SET domain, whereas DOT1L employs a distinct non-SET catalytic structure.

(2) Arginine Methyltransferases

The PRMT family catalyzes protein arginine methylation. Type I PRMTs primarily generate asymmetric dimethylarginine, whereas type II PRMTs primarily generate symmetric dimethylarginine. PRMT1, PRMT4/CARM1, PRMT5, and PRMT6 can all regulate transcription and cellular functions through histone and non-histone substrates.

3.2 Histone Demethylases

(1) The LSD Family

LSD1/KDM1A and LSD2/KDM1B belong to the family of FAD-dependent amine oxidases. Because of the limitations of their catalytic mechanism, LSD-family enzymes primarily remove mono- and dimethylated lysine states and cannot directly catalyze the demethylation of trimethylated lysines. Within different protein complexes, LSD1 can participate in the regulation of H3K4me1/2 or H3K9me1/2.

(2) The JmjC Family

Jumonji C (JmjC) domain-containing demethylases depend on Fe²⁺ and α-ketoglutarate (α-KG) to perform oxidative demethylation and can act on multiple modification states, including trimethylated lysines. The KDM5 and KDM6 families both belong to this class of demethylases.

3.3 Methylation Reader Proteins

Histone methylation must be converted into downstream chromatin effects through specific recognition domains. PHD fingers, chromodomains, Tudor domains, and MBT domains can selectively recognize different methylated residues. For example, HP1 recognizes H3K9me3 and promotes heterochromatin formation, whereas certain PHD domains recognize H3K4me3 and participate in recruiting active transcriptional complexes.

4 Functional Regulation by Histone Methylation

4.1 Promoter and Enhancer States

H3K4me3 and H3K4me1 constitute important epigenetic features of active promoters and enhancers, respectively. Different methylation marks can influence the recruitment of transcription factors, chromatin-remodeling complexes, and the transcriptional machinery, thereby regulating the activity of cis-regulatory elements.

4.2 Heterochromatin and Transcriptional Repression

H3K9me3 and H3K27me3 participate in the formation of different types of repressive chromatin. H3K9me3 primarily contributes to the maintenance of repetitive sequences and constitutive heterochromatin, whereas H3K27me3 mainly mediates the reversible repression of genes associated with development and cell fate.

4.3 Transcriptional Elongation and DNA Damage Responses

H3K36me3 and H3K79 methylation are closely associated with transcriptional elongation across active genes and also participate in maintaining genome stability. H3K36me3 can influence DNA repair processes such as mismatch repair and homologous recombination, whereas H4K20 methylation states participate in damage recognition and repair-factor recruitment following DNA double-strand breaks.

4.4 Metabolic States and Histone Methylation

(1) SAM and SAH

SAM is the major methyl donor used by KMTs and PRMTs to catalyze methyl-transfer reactions, which produce S-adenosylhomocysteine (SAH). The levels and ratio of SAM and SAH can influence cellular methyl-transfer capacity, directly linking one-carbon metabolism to histone methylation.

(2) α-KG-Dependent Demethylation

JmjC-family demethylases require α-KG, Fe²⁺, and oxygen for catalysis. Cellular metabolic states, redox environments, and hypoxic conditions can therefore influence histone demethylation.

(3) 2-Hydroxyglutarate

Mutant IDH1/2 can aberrantly produce 2-hydroxyglutarate (2-HG). 2-HG competitively inhibits multiple classes of α-KG-dependent dioxygenases, including certain histone demethylases, thereby causing abnormal methylation accumulation and disrupting normal cellular differentiation programs.

5 Aberrant Histone Methylation and Tumors

5.1 EZH2–H3K27me3 Abnormalities

EZH2 is the core catalytic subunit of the PRC2 complex and promotes H3K27me3 formation. Gain-of-function EZH2 mutations occur in some germinal center-derived B-cell lymphomas and can cause aberrant H3K27 methylation and sustained repression of differentiation-related genes. Certain tumors with SWI/SNF complex deficiencies can also develop a specific dependence on EZH2 function.

5.2 KMT2A–DOT1L–H3K79 Abnormalities

Chromosomal rearrangements involving KMT2A can generate multiple fusion proteins. Some KMT2A fusion proteins aberrantly recruit DOT1L, continuously maintaining H3K79 methylation and abnormal transcription at leukemia-associated genes such as HOXA and MEIS1 and thereby establishing an important epigenetic dependency in KMT2A-rearranged leukemia.

5.3 LSD1 and Abnormal Differentiation States

LSD1 not only possesses histone demethylase activity but also serves as an important component of transcriptional complexes such as CoREST. In certain acute myeloid leukemias, LSD1 participates in maintaining abnormal transcriptional networks and myeloid differentiation blockade. LSD1 inhibition can alter differentiation-related gene programs and has therefore become an important research direction in epigenetic differentiation therapy.

5.4 Oncogenic Histone Mutations

(1) H3K27M

The H3K27M mutation can disrupt normal PRC2-mediated H3K27 methylation and cause genome-wide redistribution of H3K27me3, representing an important molecular feature of diffuse midline glioma. Its principal effect is to reshape the H3K27 methylation landscape rather than simply alter a single methylation level.

(2) H3K36M

H3K36M can interfere with the functions of the NSD family, SETD2, and other related methyltransferases, resulting in reduced H3K36 methylation and secondary changes in H3K27 methylation distribution. This demonstrates that changes in histone sequences themselves can drive extensive epigenetic reprogramming.

6 Research Progress in Targeting Histone Methylation

6.1 Tazemetostat and EZH2 Inhibition

(1) EZH2-Targeting Mechanism

Tazemetostat is a SAM-competitive EZH2 inhibitor that suppresses EZH2 methyltransferase activity and reduces H3K27me3. Its clinical investigation has demonstrated that aberrant histone methyltransferases can establish tumor dependencies with practical pharmacological intervention value, providing important clinical proof of concept for histone methylation-targeted therapy.

(2) Clinical Research and Regulatory Changes

Tazemetostat received accelerated approval in the United States in 2020 for certain patients with epithelioid sarcoma and follicular lymphoma. Because subsequent studies identified safety signals related to secondary hematological malignancies, the relevant U.S. accelerated approvals were withdrawn on June 22, 2026. Therefore, Tazemetostat is currently more appropriately regarded as a clinical validation case for EZH2 druggability and should no longer be described as a currently FDA-approved therapeutic drug.

6.2 Pinometostat and DOT1L Inhibition

(1) Intervention in H3K79 Methylation

Pinometostat (EPZ-5676) is a selective DOT1L inhibitor that competes for the SAM-binding site of DOT1L and reduces H3K79 methylation, thereby suppressing aberrant transcriptional programs involving HOXA, MEIS1, and other genes in KMT2A-rearranged leukemia.

(2) Clinical Research

Early clinical studies demonstrated that DOT1L inhibition can produce clear pharmacodynamic changes in H3K79 methylation in patients, although its overall single-agent antitumor activity was limited. These findings validated the clinical tractability of DOT1L while suggesting that remodeling epigenetic states may require combination with other treatments to produce more substantial antitumor effects.

6.3 LSD1 Inhibition Strategies

(1) Regulation of Differentiation Programs

LSD1 inhibition can alter H3K4 methylation and the functions of associated transcriptional complexes and reverse abnormal differentiation blockade in certain myeloid malignancies. The core of this strategy is to remodel abnormal transcriptional and differentiation states rather than simply induce direct cytotoxicity.

(2) Combination Therapy

Studies combining LSD1 inhibitors with hypomethylating agents, retinoids, and other antitumor therapies continue to advance. Key development priorities include identifying tumor subtypes with well-defined LSD1 dependency, optimizing the therapeutic window, and establishing epigenetic biomarkers that can predict treatment efficacy.

6.4 PRMT5 and MTAP-Deletion Dependency

(1) MTAP Deletion Creates PRMT5 Vulnerability

MTAP deletion can cause the accumulation of methylthioadenosine (MTA), which partially inhibits PRMT5 activity and sensitizes MTAP-deficient tumors to further reductions in PRMT5 function, thereby creating a genotype-selective PRMT5 dependency.

(2) MTA-Cooperative PRMT5 Inhibition

MTA-cooperative PRMT5 inhibitors exploit MTA accumulation in MTAP-deficient tumors to create a selective pharmacological window. PRMT5 regulates histones, splicing proteins, and multiple non-histone substrates, and its antitumor effects therefore involve several processes, including arginine methylation, RNA processing, and transcriptional regulation.

7 Products

7.1 Products for Histone Lysine Methyltransferase Research

7.2 Products for Arginine Methyltransferase and Histone Demethylase Research

7.3 Products for Methylation and Demethylation Metabolism Research

Histone methylation shapes chromatin states through the coordinated processes of methyl-group transfer, demethylation, and specific recognition. Research on regulatory nodes such as EZH2, DOT1L, LSD1, and PRMT5 demonstrates that aberrant methylation programs are pharmacologically tractable and have become an important direction in epigenetic precision therapy.

References

[1] Hyun K, Jeon J, Park K, et al. Writing, erasing and reading histone lysine methylations. Exp Mol Med. 2017;49(4):e324.

[2] Husmann D, Gozani O. Histone lysine methyltransferases in biology and disease. Nat Struct Mol Biol. 2019;26(10):880–889.

[3] Morschhauser F, Tilly H, Chaidos A, et al. Tazemetostat for patients with relapsed or refractory follicular lymphoma: an open-label, single-arm, multicentre, phase 2 trial. Lancet Oncol. 2020;21(11):1433–1442.

[4] Stein EM, Garcia-Manero G, Rizzieri DA, et al. The DOT1L inhibitor pinometostat reduces H3K79 methylation and has modest clinical activity in adult acute leukemia. Blood. 2018;131(24):2661–2669.

For more related articles, please see below:

[1] Epigenetic Modifiers

[2] If the DNA Sequence Remains Unchanged, Why Does Gene Expression Change? Core Mechanisms and Research Methods of Epigenetic Regulation

[3] Noncanonical Functions of Epigenetic Enzymes and Regulation of Mitochondrial Metabolism

[4] Regulatory Mechanisms of 5mC DNA Methylation