HAT1 Human

What is Histone Acetyltransferase 1 (HAT1) and what is its primary function?

Histone Acetyltransferase 1 (HAT1) is the founding member of the histone acetyltransferase superfamily that catalyzes the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to specific lysine residues on histone proteins . As a type B histone acetyltransferase, HAT1 predominantly acetylates free histones rather than those already incorporated into chromatin . Its primary function involves acetylating newly synthesized histone H4 at lysines 5 and 12 (K5 and K12) before incorporation into chromatin during DNA replication .

This post-translational modification pattern is highly conserved among all eukaryotes, suggesting fundamental importance in chromatin biology . The acetylation of newly synthesized histones is thought to facilitate their transport and assembly into nucleosomes, although this mark is typically removed approximately 20 minutes after incorporation into chromatin . Beyond its enzymatic role, HAT1 also functions as an important histone binding protein that contributes to various nuclear processes including chromatin assembly and DNA repair .

How is HAT1 classified and where is it localized within cells?

HAT1 belongs to the Gcn5-related N-acetyltransferase (GNAT) family and is classified as a type B histone acetyltransferase . This classification is based on several distinguishing characteristics: type B HATs primarily acetylate free histones (not incorporated into chromatin), while type A HATs modify histones within chromatin contexts . This functional distinction aligns with HAT1's role in modifying newly synthesized histones before their incorporation into nucleosomes.

• Cytoplasm: Where it was first identified and thought to exclusively function

• Nucleus: Where it forms specific protein complexes distinct from its cytoplasmic associations

• Mitochondria: Despite lacking a conventional mitochondrial localization signal, HAT1 has been detected in mitochondrial fractions

This multi-compartmental distribution reflects HAT1's diverse cellular functions beyond simply modifying new histones, including roles in DNA repair, chromatin assembly, and potentially mitochondrial gene regulation .

Which histone substrates does HAT1 specifically target?

HAT1 displays remarkable substrate specificity, primarily targeting lysine residues 5 and 12 (K5 and K12) on the amino-terminal tail of histone H4 . This highly specific acetylation pattern on newly synthesized histone H4 is evolutionarily conserved across eukaryotes, suggesting its fundamental importance in chromatin biology . The crystal structure of human HAT1 in complex with acetyl coenzyme A and histone H4 peptide reveals that the enzyme positions the substrate in a precise orientation that allows specific recognition of the sequence context around K5 and K12 .

While HAT1 is best known for modifying histone H4, evidence indicates potential interactions with other histones. Affinity purification studies using epitope-tagged versions of human histones H3.1 and H3.3 demonstrated that both variants co-purify with HAT1 and its associated proteins . Furthermore, HAT1 has been shown to facilitate the incorporation of H4K5/K12-acetylated H3.3 into double-strand break sites during DNA repair processes . This suggests that HAT1's substrate interactions extend beyond H4 to include histone H3 variants in specific functional contexts.

What is the structural basis for substrate specificity of human HAT1?

The structural basis for HAT1's remarkable substrate specificity has been elucidated through high-resolution crystallographic studies. A 1.9-Å resolution crystal structure of human HAT1 in complex with acetyl coenzyme A (AcCoA) and histone H4 peptide provided crucial insights into its molecular recognition mechanisms . This structure revealed that both the cofactor (AcCoA) and the side chain of lysine 12 of the histone H4 peptide are positioned in a canyon-like region located between the central and C-terminal domains of the enzyme .

The histone H4 peptide adopts a well-defined conformation when bound to HAT1 and establishes an extensive network of interactions with the enzyme . Particularly important are the invariant residues Glu64 and Trp199, which together govern the substrate-binding specificity of HAT1 . The precise positioning of these residues creates a molecular environment that selectively recognizes the unique sequence context surrounding lysines 5 and 12 of histone H4, explaining HAT1's highly selective acetylation pattern.

This structural information provides the molecular basis for understanding how HAT1 discriminates between different histone substrates and specific lysine residues, which is essential for its biological function in acetylating specific sites on newly synthesized histone H4.

How does HAT1 catalyze the acetylation reaction?

The catalytic mechanism of HAT1 involves several coordinated steps that facilitate the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to specific lysine residues on histone H4. Structure-guided enzyme kinetic studies have revealed important details about this process:

• Initial binding: The cofactor (AcCoA) and histone substrate bind in the canyon between the central and C-terminal domains of HAT1 .

• Substrate deprotonation: A critical step involves the deprotonation of the ε-amino group of the target lysine (K12) by active site residues . Three acidic residues—Glu187, Glu276, and Asp277—play crucial roles in this process, exhibiting a cumulative effect on the deprotonation step .

• Nucleophilic attack: Once deprotonated, the ε-amino group of lysine serves as a nucleophile that directly attacks the acetyl group of AcCoA .

• Acetyl transfer: The acetyl group is transferred from AcCoA to the lysine residue, resulting in acetylated histone H4 and free coenzyme A.

• Product release: The acetylated histone and coenzyme A are released from the enzyme.

This mechanistic understanding of HAT1's catalytic activity provides insights into the precise molecular events underlying its function in histone modification and helps explain how mutations in key catalytic residues might affect its activity in cellular contexts.

Which protein complexes does HAT1 form in human cells?

HAT1 operates within well-defined protein complexes rather than functioning in isolation. Unlike the large, multi-subunit complexes associated with type A histone acetyltransferases, HAT1-containing complexes have relatively simple compositions :

• Core HAT1 complex: The fundamental HAT1 complex consists of HAT1 and Rbap46/Rbap48 (mammalian homologs of yeast Hat2p) . These WD repeat proteins are components of several chromatin-related complexes and facilitate HAT1's interaction with histones and other proteins .

• NuB4-like complex: In humans, HAT1 associates with NASP (nuclear autoantigenic sperm protein), the homolog of yeast Hif1p, forming a complex similar to the yeast NuB4 complex . This was demonstrated through affinity purification studies showing that epitope-tagged human histones H3.1 and H3.3 co-purify with HAT1, Rbap46/48, and NASP .

• Interactions with replication and repair machinery: HAT1 has been found to interact with components of the DNA replication machinery, including the origin recognition complex (ORC) . These interactions suggest functional roles in coordinating histone modification with DNA replication and repair processes.

• Bromodomain protein interactions: HAT1-dependent acetylation of H4K5 and K12 appears to recruit bromodomain-containing proteins, including Brg1 (an ATP-dependent chromatin-remodeling enzyme), Baz1A, and Brd3 . These interactions may facilitate downstream chromatin remodeling events.

These protein-protein interactions highlight HAT1's integrated role within broader histone metabolism and chromatin assembly pathways, extending its function beyond simple enzymatic activity.

How does HAT1 contribute to DNA replication and chromatin assembly?

HAT1 plays multifaceted roles in DNA replication and the assembly of newly synthesized DNA into chromatin structures:

• Replication fork stability: HAT1 physically and transiently associates with DNA near replication sites . Loss of HAT1 has been observed to inhibit replication fork progression, increase fork stalling, destabilize stalled forks, and induce MRE11-dependent degradation of newly synthesized DNA . These findings indicate that HAT1 is crucial for maintaining replication fork stability.

• Histone modification for assembly: HAT1 acetylates newly synthesized histone H4 at lysines 5 and 12, a modification pattern highly conserved across eukaryotes . This acetylation is thought to facilitate the incorporation of newly synthesized histones into chromatin during replication-coupled assembly.

• Recruitment of chromatin assembly factors: HAT1-dependent acetylation of H4K5 and H4K12 recruits bromodomain proteins, including Brg1, Baz1A, and Brd3 . These proteins recognize acetylated lysine residues and may play roles in facilitating chromatin assembly through remodeling activities.

• Coordination with replication machinery: HAT1 interacts with components of the origin recognition complex (ORC) , potentially coordinating histone modification with the DNA replication process.

Interestingly, research has yielded some contradictory findings regarding the necessity of HAT1 for replication-coupled chromatin assembly. While some studies suggest HAT1 is dispensable (as HAT1-deficient DT40 cells showed no viability defects) , other research indicates important roles in replication fork stability and chromatin assembly . These apparent contradictions highlight the complexity of HAT1's functions and suggest potential redundancy in histone acetylation pathways.

What role does HAT1 play in DNA damage response and repair?

HAT1 has emerged as an important factor in the DNA damage response, particularly in the repair of double-strand breaks (DSBs):

• Recruitment to damage sites: Studies using inducible endonuclease systems followed by chromatin immunoprecipitation have demonstrated that HAT1 is recruited to double-strand breaks with kinetics similar to those of recombinational repair factors like Rad52 in yeast .

• Chromatin dynamics at break sites: HAT1 facilitates the incorporation of H4K5/K12-acetylated H3.3 (H3.3-H4K5/12ac) into DSB sites through HIRA-dependent histone turnover activity . This histone variant incorporation appears to create a chromatin environment conducive to repair processes.

• Repair factor recruitment: HAT1 promotes the recruitment of the repair factor Rad51 to DNA damage sites . This key recombination protein is essential for homologous recombination-mediated DNA repair.

• Promotion of homologous recombination: By facilitating appropriate chromatin environments at damage sites and supporting repair factor recruitment, HAT1 ultimately promotes homologous recombination-mediated DNA repair .

These findings significantly expand the functional repertoire of HAT1 beyond its classical role in acetylating newly synthesized histones. They highlight HAT1's importance in maintaining genome integrity by facilitating efficient DNA repair processes through chromatin-based mechanisms.

How does HAT1 influence nuclear structure and genome accessibility?

Recent research has revealed HAT1 as an important regulator of nuclear structure and genome accessibility:

• HAT1-dependent accessibility domains (HADs): Assays for transposase-accessible chromatin using sequencing (ATAC-seq) with mouse embryonic fibroblasts revealed that HAT1 loss reduces genome accessibility at 1,895 specific sites . These HAT1-dependent accessibility domains (HADs) have distinctive characteristics:

  • Located primarily in distal intergenic regions with low GC content and low gene enrichment

  • Range in size from 0.9 kb to 11 Mb

  • Display heterochromatin characteristics

• Relationship with nuclear lamina: HADs show substantial overlap (86%) with lamina-associated domains (LADs), particularly with constitutive LADs (85% overlap) . This indicates that HAT1 regulates the accessibility of chromatin domains associated with the nuclear lamina.

• Histone modification regulation: HAT1 functions as a global repressor of histone H3 lysine 9 methylation . The HADs correspond to regions where HAT1 regulates the abundance of H3K9 methylation, a mark traditionally associated with heterochromatin formation.

• Nuclear morphology: Loss of HAT1 results in increased nuclear size, similar to effects observed with decreased lamina expression or mutations that compromise lamina function . This suggests that HAT1 contributes to maintaining normal nuclear morphology.

These findings demonstrate that HAT1 plays crucial roles in regulating nuclear architecture and genome accessibility, extending its functions well beyond its classical role as an enzyme that acetylates newly synthesized histones.

What is the relationship between HAT1 and mitochondrial function?

Unexpected connections between HAT1 and mitochondrial functions have emerged from recent research:

• Mitochondrial localization: Despite lacking a conventional mitochondrial localization signal, HAT1 has been found to localize to mitochondria, as demonstrated by subcellular fractionation and mitochondrial purification studies .

• AMPK-mediated regulation: Adenosine monophosphate-activated protein kinase (AMPK) phosphorylates HAT1 in human umbilical vein endothelial cells (HUVECs) . This phosphorylation appears to activate HAT1 and leads to significant nucleosomal remodeling.

• Regulation of mitochondrial genes: The AMPK-mediated activation of HAT1 results in the upregulation of nuclear genes encoding proteins involved in mitochondrial biogenesis and function . These include:

  • Transcription factor A (Tfam)

  • Uncoupling proteins 2 and 3 (UCP2 and UCP3)

  • Peroxisome proliferator-activated receptor γ coactivator-1a (PGC-1a)

These findings suggest that HAT1 may serve as a critical link between cellular energy status (sensed by AMPK) and mitochondrial function, potentially through its ability to influence chromatin structure and gene expression patterns. This represents a novel aspect of HAT1 biology that extends its significance beyond histone metabolism to include roles in cellular energetics and mitochondrial homeostasis.

What experimental approaches are used to study HAT1 enzymatic activity?

Researchers employ several specialized approaches to study HAT1's enzymatic activity and catalytic properties:

• In vitro acetyltransferase assays: These typically include:

  • Purified recombinant HAT1 protein (alone or with binding partners)

  • Histone substrates (synthetic peptides or purified histone proteins)

  • Acetyl-CoA (often radiolabeled for detection)

  • Reaction conditions optimized for HAT1 activity

  • Quantification methods to measure acetyl transfer rates

• Structure-guided enzyme kinetic studies: These have been particularly valuable for understanding HAT1's catalytic mechanism. Researchers have used site-directed mutagenesis to alter specific active site residues (such as Glu187, Glu276, and Asp277) and measured the effects on catalytic activity . This approach identified residues crucial for deprotonating the ε-amino group of target lysines.

• Crystallographic analyses: The 1.9-Å resolution crystal structure of human HAT1 in complex with acetyl coenzyme A and histone H4 peptide provided critical insights into substrate recognition and catalysis mechanisms . Such structures allow visualization of the molecular interactions within the active site and substrate binding pocket.

• Substrate specificity assays: Comparative analyses using different histone peptides or full-length histones can determine HAT1's substrate preferences and identify sequence elements that influence recognition.

• Inhibitor studies: Testing small molecules that interfere with HAT1 activity can provide insights into catalytic mechanisms and potential regulatory points.

These biochemical and structural approaches have collectively established our current understanding of HAT1's catalytic mechanism, substrate specificity, and the structural basis of its function.

How can one measure the acetylation status of histones modified by HAT1?

Measuring histone acetylation, particularly HAT1-mediated modifications at H4K5 and H4K12, requires specialized approaches:

• Antibody-based detection methods:

  • Western blotting with site-specific antibodies recognizing acetylated H4K5 and H4K12

  • Immunofluorescence microscopy to visualize modified histones in cellular contexts

  • Chromatin immunoprecipitation (ChIP) to identify genomic regions containing HAT1-modified histones

• Mass spectrometry approaches:

  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for quantitative analysis of histone modifications

  • Top-down proteomics to analyze intact histones and identify combinatorial modification patterns

  • Targeted multiple reaction monitoring for specific acetylation sites

• Fractionation-based analyses:

  • Separation of soluble (non-nucleosomal) and chromatin-bound histone pools

  • Cell cycle synchronization with hydroxyurea treatment to accumulate soluble histones for analysis

  • Subcellular fractionation to examine compartment-specific histone modification patterns

• Pulse-chase experiments:

  • Metabolic labeling with isotopically tagged acetate to track newly acetylated histones

  • Time-course analyses to monitor modification dynamics during specific processes

These methodological approaches have revealed important insights about HAT1 function. For example, HAT1 deletion significantly reduces but does not eliminate H4K5 and H4K12 acetylation on soluble histone H4, suggesting HAT1 is not the sole enzyme capable of these modifications . Additionally, these approaches have shown that HAT1 deletion affects acetylation of soluble histone H4 but not bulk chromatin-associated H4 , supporting its role in modifying newly synthesized histones before chromatin incorporation.

What strategies are effective for studying HAT1 loss-of-function?

Various approaches have been employed to study HAT1 loss-of-function, each with particular advantages for investigating different aspects of HAT1 biology:

• Genetic deletion approaches:

  • Complete HAT1 knockout cell lines (e.g., DT40 cells)

  • Conditional knockout models to study temporal aspects of HAT1 requirement

  • CRISPR-Cas9-mediated gene editing for precise HAT1 manipulation

• RNA interference techniques:

  • siRNA knockdown of HAT1 in mammalian cells

  • shRNA-mediated stable suppression of HAT1 expression

  • Inducible systems for temporal control of knockdown

• Catalytic inactivation strategies:

  • Mutations in key catalytic residues (Glu187, Glu276, Asp277)

  • Expression of dominant-negative HAT1 variants

  • Selective inhibitors of HAT1 enzymatic activity

• Functional readouts to assess HAT1 loss effects:

  • Histone acetylation analysis in different cellular fractions

  • DNA replication fork stability assessment

  • Cell cycle progression and proliferation monitoring

  • Chromatin accessibility measurement via ATAC-seq

  • Nuclear size and morphology examination

  • Mitochondrial gene expression quantification

These approaches have revealed key insights into HAT1 function, such as its roles in replication fork stability, DNA repair, and nuclear structure regulation. They have also exposed the complexity of HAT1 biology, including the finding that it is not the sole enzyme responsible for H4K5 and H4K12 acetylation, as these modifications are reduced but not eliminated in HAT1-deficient cells .

Is HAT1 the sole enzyme responsible for H4K5 and H4K12 acetylation?

The question of whether HAT1 exclusively controls H4K5 and H4K12 acetylation represents an important unresolved issue in chromatin biology. Current evidence points to a more complex reality:

• Partial acetylation reduction in HAT1-deficient cells: When the HAT1 gene is deleted, there is a significant but incomplete loss of H4K5 and H4K12 acetylation on soluble histone H4 . Similar results in S. cerevisiae showed that HAT1 deletion substantially decreased but did not eliminate these acetylation marks on accumulated cytosolic histones .

• System-specific effects: siRNA knockdown of HAT1 in mammalian cells shows more subtle effects on cytosolic histone H4 lysine 12 acetylation compared to genetic deletion in yeast . This may reflect incomplete knockdown efficiency or system-specific differences in compensatory mechanisms.

• Evidence for redundant enzymes: While HAT1 clearly contributes to H4K5 and H4K12 acetylation of newly synthesized histones, the persistence of these modifications in HAT1-deficient cells strongly suggests that other acetyltransferases can perform these modifications .

• Methodological challenges: Definitive identification of all enzymes capable of acetylating H4K5 and H4K12 would require pulse-labeling techniques in cells genetically deleted for HAT1 combined with systematic analysis of other potential acetyltransferases .

Identifying these potentially redundant acetyltransferases remains an important research goal that could significantly advance our understanding of histone modification pathways and the biological significance of H4K5 and H4K12 acetylation.

How do HAT1-mediated modifications coordinate with other histone marks?

Understanding how HAT1-mediated acetylation coordinates with other histone modifications represents a frontier in chromatin biology research:

• Temporal coordination during histone deposition:

  • HAT1-mediated acetylation occurs on newly synthesized histones

  • These marks are typically removed approximately 20 minutes after chromatin incorporation

  • This transient nature suggests "hand-off" mechanisms where pre-deposition acetylation marks are replaced by other modifications after assembly

• Regulatory relationships with repressive marks:

  • HAT1 functions as a global repressor of H3K9 methylation, a mark associated with heterochromatin

  • HAT1-dependent accessibility domains (HADs) correspond to regions where HAT1 regulates H3K9 methylation levels

  • This suggests that HAT1-mediated acetylation may influence the establishment or maintenance of repressive modifications in specific genomic regions

• Recruitment of chromatin regulators:

  • HAT1-dependent acetylation of H4K5 and H4K12 recruits bromodomain proteins like Brg1, Baz1A, and Brd3

  • These proteins could facilitate additional modification or remodeling activities

  • Such interactions potentially form the basis of a "histone modification code" directing downstream processes

• Context-specific modification patterns:

  • During DNA repair, HAT1 facilitates the incorporation of H4K5/K12-acetylated H3.3 into double-strand break sites

  • This suggests coordination between histone variant incorporation and specific modification patterns in response to DNA damage

Understanding these complex interrelationships requires integrated approaches combining genetics, biochemistry, proteomics, and high-resolution imaging techniques to map the spatiotemporal dynamics of histone modifications in different cellular contexts.

What are the implications of HAT1 research for understanding human disease?

HAT1's involvement in fundamental cellular processes suggests potential connections to human disease states, though this remains a developing research area:

• Cancer biology implications:

  • HAT1 is required for EGF-dependent proliferation of telomerase-immortalized human mammary epithelial cells

  • It plays essential roles in cell cycle progression into S phase

  • These proliferation-related functions suggest that dysregulated HAT1 activity might contribute to abnormal cell growth in cancer contexts

• Genomic instability disorders:

  • HAT1 facilitates DNA repair factor recruitment and promotes homologous recombination

  • Defects in these processes could potentially lead to genomic instability, a hallmark of cancer and various genetic disorders

  • Understanding HAT1's role in maintaining genome integrity may provide insights into disease mechanisms

• Nuclear structure abnormalities:

  • HAT1 loss increases nuclear size , reminiscent of nuclear irregularities in various cancers

  • HAT1's regulation of nuclear lamina-associated domains suggests potential implications for laminopathies, diverse genetic disorders affecting nuclear envelope proteins

• Mitochondrial and metabolic disorders:

  • HAT1's involvement in regulating mitochondrial gene expression suggests potential connections to mitochondrial dysfunction disorders

  • The AMPK-HAT1 pathway upregulates genes involved in mitochondrial biogenesis and function , pointing to possible roles in metabolic diseases

Future research directions should include examining HAT1 expression and activity in disease tissues, investigating correlations between HAT1 genetic variations and disease susceptibility, and exploring targeted approaches to modulate HAT1 function in disease contexts.