Histone H4 PTMs: Roles in DNA Repair and Genome Stability

Histone H4 is a core component of nucleosomes, and its post-translational modifications (PTMs) play an indispensable role in regulating eukaryotic genome function. The N-terminal tail of histone H4 contains multiple critical lysine residues—including K5, K8, K12, K16, and K20—which serve as major sites for covalent epigenetic marks such as acetylation, methylation, and ubiquitination.

These modifications influence genomic activity through two primary mechanisms:

• Direct Structural Alteration: By neutralizing positive charges on histones, certain PTMs (e.g., acetylation) reduce DNA-histone binding affinity, promoting chromatin relaxation and enhanced DNA accessibility.
• Effector Protein Recruitment: Specific PTMs function as molecular switches that are recognized by reader proteins, thereby initiating downstream signaling cascades and forming sophisticated regulatory networks.

The dynamic regulation of H4 PTMs is especially vital in processes such as DNA damage repair and the maintenance of genome integrity. Dysregulation of these modifications is strongly associated with various diseases, including cancer and aging-related disorders.

This review provides a comprehensive analysis of the molecular mechanisms by which major H4 PTMs contribute to the DNA damage response (DDR) and genome stability. Furthermore, it explores the clinical potential of therapies targeting these epigenetic pathways.

1. Types and Biochemical Characteristics of Histone H4 Modifications

1.1 Functional Classification of Canonical Modifications

Histone H4 PTMs form a sophisticated "epigenetic code," where distinct modification types and combinatorial patterns execute precise biological functions.

• Acetylation: This modification primarily targets lysine residues K5, K8, K12, and K16. Histone acetyltransferases (HATs) add acetyl groups, while histone deacetylases (HDACs) remove them. Acetylation neutralizes the positive charge on lysine, weakening DNA-histone electrostatic attraction and facilitating an open, transcriptionally permissive chromatin state.
  • H4K16ac: A extensively characterized mark, H4K16ac promotes transcriptional activation and critically influences DNA repair pathway choice. It inhibits the accumulation of the 53BP1 protein at damage sites, thereby favoring homologous recombination (HR) over non-homologous end joining (NHEJ) (Song H et al., 2022).
• Methylation: This occurs mainly at K20 and R3 sites, catalyzed by specific methyltransferases. H4K20 can be mono- (me1), di- (me2), or tri-methylated (me3), with each state conferring unique functions:
  • H4K20me2: Serves as a primary recruitment signal for the 53BP1 protein (via its Tudor domain), promoting the NHEJ repair pathway.
  • H4K20me3: Enriched in heterochromatic regions like centromeres, this mark is essential for maintaining chromosomal stability.
  • H4R3me2: Catalyzed by PRMT1, this arginine methylation is generally associated with active gene transcription (Lu X et al., 2019).
• Novel Acylations and Ubiquitination: Recent discoveries show H4 is subject to metabolic stress-responsive modifications like β-hydroxybutyrylation, succinylation, and glutarylation.
  • For instance, glutarylation at H4K91 promotes the dissociation of H2A-H2B dimers from the nucleosome core.
  • Ubiquitination at H4K31 plays a role in regulating DNA replication origins.

Explanation for the counterintuitive net stabilizing effect of H4K77Ac on the nucleosome, which makes the global DNA less accessible (Fenley AT et al., 2018).

1.2 Spatial Effects of Modification Sites

The biological impact of an H4 modification is intrinsically linked to its structural position within the nucleosome.

• N-Terminal Tail Modifications (e.g., K16ac, K20me): These residues are exposed and primarily function as docking sites for effector proteins (readers). They indirectly influence DNA accessibility by recruiting chromatin remodelers and other complexes.
• Core Domain Modifications (e.g., K91glu): Modifications within the nucleosome's core directly perturb histone-histone and histone-DNA interfaces. H4K91 glutarylation, situated at the H3-H4/H2A-H2B dimer interface, directly destabilizes nucleosome integrity by facilitating dimer dissociation.

Table: Functional Characterization of Key Histone H4 Post-Translational Modifications

2. The Central Role of H4 Modifications in the DNA Damage Response

2.1 Precise Regulation of DNA Repair Pathways

Following the occurrence of DNA double-strand breaks (DSBs), histone H4 modifications undergo rapid, spatiotemporally ordered changes that form a critical regulatory cascade governing repair.

• Initial Response (0–30 minutes): ATM/ATR kinase activation triggers immediate epigenetic changes: a sharp decrease in H4K16ac levels and a concurrent significant increase in H4K20me2. This coordinated "acetylation-to-methylation transition" establishes a molecular platform for the recruitment of the repair protein 53BP1. 53BP1 stabilizes at damage sites by simultaneously recognizing H4K20me2 via its Tudor domain and H2AK15ub through its ubiquitin-dependent recruitment (UDR) domain. Concurrently, phosphorylation of the H4S1 site promotes local chromatin decompaction, facilitating access for the repair machinery.
• Mid-Term Repair Pathway Choice (1–6 hours): The dynamic status of H4K16ac emerges as a pivotal determinant for selecting the repair mechanism.
  • Non-Homologous End Joining (NHEJ): Sustained low levels of H4K16ac allow for continued 53BP1 aggregation, promoting error-prone NHEJ. The BRCA1 complex can further reinforce this pathway by recruiting deacetylases like SIRT7 to remove residual H4K16ac, thereby stabilizing 53BP1 binding.
  • Homologous Recombination (HR): Conversely, activation of the HR pathway involves the TIP60 acetyltransferase, which catalyzes an increase in H4K16ac. This mark sterically hinders 53BP1 binding to H4K20me2, effectively displacing it. The cleared site then facilitates the recruitment of the BRCA1 complex to initiate precise, error-free homologous recombination.
• Repair Completion and Reset: Following successful repair, the H4 modification landscape is restored to its basal state. Histone demethylases (e.g., PHF8) are recruited to reduce H4K20me2 levels, and histone acetyltransferase (HAT) complexes re-establish the pre-damage acetylation pattern, returning chromatin to its normal functional state (Lu X et al 2019).

GLP catalyzes H4K16me1 in response to DNA damage in an ATM-dependent manner (Lu X et al 2019)

2.2 Breakthrough Discovery of Novel Modifications

In 2019, a research team led by Weiguo Zhu at Shenzhen University reported a significant discovery: beyond its well-characterized acetylation, the histone H4K16 site can also undergo monomethylation (H4K16me1), a reaction catalyzed by the histone methyltransferase GLP (G9a-like protein).

Following DNA damage, cellular levels of H4K16me1 increase substantially. This upregulation promotes the stable recruitment of the 53BP1 protein to damage sites, thereby enhancing the efficiency of non-homologous end joining (NHEJ) repair. This finding reveals a dynamic competition between different modifications at the H4K16 locus, where the relative abundance of H4K16ac versus H4K16me1 serves as a critical determinant in pathway selection.

The study further uncovered unexpected cross-histone regulatory mechanisms. Treatment with GSK343, an inhibitor of the H3K27 methyltransferase EZH2, unexpectedly reduced H4K16me1 levels, suggesting the existence of a sophisticated inter-histone regulatory network (Lu X et al 2019).

Table: Role of Histone H4 Modifications in DNA Repair Pathway Selection

GLP-mediated H4K16me1 promotes 53BP1 recruitment in response to DNA damage (Lu X et al 2019)

3. H4 Modifications and the Epigenetic Regulation of Genome Stability

3.1 Three-Dimensional Regulation of Nucleosome Dynamics

Histone H4 modifications are fundamental to maintaining genome stability, primarily through their regulation of chromatin's three-dimensional architecture.

• Regulation of Nucleosome Stability: Modifications within the histone H4 core domain directly influence nucleosome structural integrity. For instance, glutarylation at the H4K91 site—positioned at the critical interface between the H3-H4 tetramer and the H2A-H2B dimer—disrupts key hydrophobic interactions between these subunits. This disruption promotes the dissociation of the H2A-H2B dimer, leading to a partial "nucleosome disassembly." The resulting decompaction of tightly packed DNA enhances its accessibility to damage sensor proteins and DNA repair complexes (Millán-Zambrano G et al., 2022).
• Remodeling of Higher-Order Chromatin Structure:
  • The activating mark H4K16ac inhibits the tight compaction of chromatin fibers, thereby maintaining a relatively open conformation in euchromatic regions. This structural state allows for the rapid recruitment of DNA damage sensing proteins to break sites (Simon M et al., 2011).
  • In contrast, the repressive mark H4K20me3 facilitates the formation of condensed heterochromatin by recruiting HP1 proteins. This mechanism is crucial for stabilizing repetitive genomic regions, such as centromeres, and preventing chromosomal instability and aneuploidy (Duan X et al., 2024).
• Phase Separation and Functional Compartmentalization: Emerging evidence indicates that H4 modifications contribute to the liquid-liquid phase separation (LLPS) of chromatin, a process that drives the formation of membraneless biomolecular condensates.
  • H4K16ac alters the charge properties of the histone tail, promoting the formation of phase-separated condensates in transcriptionally active regions (Shia WJ et al., 2006).
  • H4K20me3 facilitates the formation of heterochromatic condensates.

This spatial segregation through phase separation allows the cell to efficiently organize and separate potentially conflicting biological processes, such as transcription and DNA repair, within distinct nuclear compartments (Nelson DM et al., 2016).

3.2 Disease-Associated Modification Aberrations and Molecular Mechanisms

Dysregulation of histone H4 modifications contributes to genomic instability and carcinogenesis through several distinct pathological mechanisms:

1. DNA Repair Pathway Imbalance

Aberrant H4 modification patterns can disrupt the critical balance between DNA repair mechanisms. Abnormal elevation of H4K16ac or reduction of H4K20me2 impairs 53BP1-mediated non-homologous end joining (NHEJ). This repair deficiency creates excessive dependence on homologous recombination (HR). In BRCA1/2-deficient backgrounds, this imbalance induces a synthetic lethal interaction that forms the mechanistic basis for PARP inhibitor therapy. Conversely, pathologically low H4K16ac levels lead to NHEJ dominance, resulting in accumulated mutations and chromosomal translocations (Duan X et al., 2024).

2. Epigenetic Landscape Disruption

Mutational or expressional alterations of histone-modifying enzymes—including HATs/HDACs and KMTs/KDMs—cause genome-wide distortion of H4 modification patterns. For example:

• GLP (H4K16 methyltransferase) overexpression in lung and colorectal cancers elevates H4K16me1 levels, promoting error-prone NHEJ repair and facilitating oncogene amplification.
• SIRT1 (H4K16 deacetylase) dysregulation across multiple cancer types compromises the DNA damage response efficiency (Lu X et al 2019).

3. Three-Dimensional Genome Destabilization

Abnormal H4 modifications alter chromatin architecture by modifying topologically associating domain (TAD) boundary strength:

• In leukemia, H4K16ac deficiency causes chromatin hypercompaction, enabling illegitimate enhancer-promoter interactions that activate proto-oncogenes.
• Concurrent loss of centromeric H4K20me3 disrupts heterochromatin integrity, leading to chromosome segregation errors and aneuploidy (Horikoshi N et al., 2013).

Table: Mechanisms of H4 Modification-Related Carcinogenesis

4. Therapeutic Prospects of Targeting H4 Modifications

4.1 Small Molecule Inhibitors and Epigenetic Therapy

Given the crucial role of H4 modifications in oncogenesis, numerous small molecule inhibitors targeting their regulatory enzymes have advanced into clinical development:

• HDAC Inhibitors: Drugs such as Vorinostat and Romidepsin, which target histone deacetylases, have received FDA approval for treating T-cell lymphoma. Their anti-tumor mechanisms include: 1) Reactivating tumor suppressor gene expression by elevating global acetylation levels (including H4K16ac); 2) Disrupting homologous recombination repair pathways to enhance radiosensitivity; and 3) Inducing cellular differentiation and apoptosis. Next-generation HDAC6-selective inhibitors (e.g., Ricolinostat) may reduce side effects associated with pan-HDAC inhibition, such as cytopenias.
• GLP and EZH2 Inhibitors: Inhibitors targeting the H4K16 methyltransferase GLP are currently in preclinical development. Research from Shenzhen University demonstrates that EZH2 inhibition with GSK343 indirectly reduces H4K16me1 levels, thereby increasing cancer cell sensitivity to radiotherapy. The EZH2 inhibitor Tazemetostat, already approved for epithelioid sarcoma and follicular lymphoma, may exert part of its therapeutic effect through modulation of H4 modifications.
• Combinatorial Therapeutic Strategies: Inhibitors of histone-modifying enzymes demonstrate synergistic effects with DNA-damaging agents. Clinical evidence shows: 1) HDAC inhibitors combined with PARP inhibitors significantly extend progression-free survival in BRCA-mutant breast cancer; 2) EZH2 inhibitors enhance local control rates when combined with radiotherapy in locally advanced cervical cancer; and 3) DOT1L histone methyltransferase inhibitors improve complete response rates in MLL-rearranged leukemia when combined with chemotherapy.

Table: Clinical Development of Small-Molecule Inhibitors Targeting H4-Modifying Enzymes

5. Conclusions and Future Directions

Histone H4 post-translational modifications constitute a sophisticated and dynamic regulatory network that orchestrates DNA repair and maintains genome stability through a triad of "modifying enzymes, effector proteins, and chromatin remodeling." The core functions of this system are threefold: (1) It acts as a molecular switch for the DNA damage response, where the spatiotemporal dynamic equilibrium of marks like H4K16ac, H4K16me1, and H4K20me2 precisely regulates repair pathway choice; (2) It modulates DNA accessibility and the three-dimensional genome architecture by directly altering nucleosome stability and mediating chromatin phase separation; (3) It serves as an epigenetic memory carrier, transmitting crucial genome stability information across cell divisions.

Despite significant progress, several key challenges and future research directions remain:

• Single-Cell Modification Dynamics: Current technologies cannot adequately capture real-time, single-cell dynamics of H4 modifications following DNA damage. The development of ultra-high-resolution live-cell imaging, integrated with single-cell epigenomics, is needed to reveal the precise temporal windows of these modification oscillations.
• Discovery of Novel Modifications: Beyond the known modifications, H4 may harbor rare, undiscovered PTMs. Expanding the H4 epigenomic map will require high-sensitivity mass spectrometry coupled with innovative bio-orthogonal labeling strategies.
• Role in Phase Separation Regulation: The mechanism by which H4 modifications precisely control the formation and dissolution of chromatin condensates is poorly understood. Unraveling this will necessitate in vitro reconstitution of nucleosome phase separation systems combined with light-controlled epigenetic editing.
• Tissue Specificity and Therapeutic Translation: The functional dynamics and impact of H4 modifications likely vary across tissues. Building organoid-specific epigenetic maps will be critical for developing precise therapeutic strategies that target H4 modifications.

Advancements in CRISPR-based epigenetic editing, cryo-electron microscopy (cryo-EM), and single-molecule tracing are poised to precisely delineate the functional landscape of H4 modifications. These efforts will yield new insights into its molecular mechanisms and provide novel diagnostic markers and targeted therapeutic strategies for diseases of genome instability, such as cancer and neurodegenerative disorders. The study of histone H4 modifications is ushering in a new era of epigenetic therapy, whose profound scientific value and clinical potential warrant continued and vigorous exploration.

For more information on the role of histone PTMs in Epigenetics and chromatin, see "Histone PTMs and Chromatin Structure Dynamics: Bridging Epigenetics and Structural Biology".

For more research on histone PTM biomarkers see "Histone PTMs as Biomarkers: Opportunities and Limitations for Translational Research".

People Also Ask

What is the function of the histone H4?

Histone H4 is defined as one of the four core histones that make up the nucleosome, along with Histone H2A, H2B, and H3. It plays a crucial role in chromatin structure and gene regulation by wrapping DNA in the nucleosome.

What is the role of post-translational modification in DNA repair?

In response to DNA damage, signaling pathways are activated to repair the damaged DNA or to induce cell apoptosis. During the process, PTMs can be used to modulate enzymatic activities and regulate protein stability, protein localization, and protein-protein interactions.

What are the histone modifications in DNA repair?

DNA break induced histone modifications have been reported to function in sensing the breaks, activating processing of breaks by specific pathways, and repairing damaged DNA to ensure integrity of the genome. Favourable environment for DSB repair is created by generating open and relaxed chromatin structure.

On what chromosome is the gene for human histone H4 located?

This gene is intronless and encodes a member of the histone H4 family. Transcripts from this gene lack polyA tails but instead contain a palindromic termination element. This gene is found in the large histone gene cluster on chromosome 6.

What is the structure of histone H4?

Histone 4 is a 102–135 amino acid core protein with a structural motif known as histone fold. It forms a dimer with H3 and wraps around the DNA. Extracellular histones use Toll-like receptors (TLRs) or inflammatory pathways to cause cytotoxicity and trigger inflammatory reactions.

How do histones protect DNA from damage?

We conclude that the binding of histones to the DNA and its organization into higher order chromatin structures dramatically protects the DNA against hydroxyl radical-induced DNA strand breaks and thus should be considered part of the cellular defense against the induction of oxidative DNA damage.

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