KAT2A Human

What is KAT2A and what is its primary function in human cells?

KAT2A is a lysine acetyltransferase responsible primarily for histone H3 lysine 9 acetylation (H3K9ac), a modification associated with transcriptional activation . It functions as part of two distinct macromolecular complexes: Ada two-A-containing (ATAC) and Spt-Ada-Gcn5-Acetyltransferase (SAGA) . Its primary enzymatic role involves transferring acetyl groups from acetyl-CoA to specific lysine residues on histone proteins, particularly H3K9, which generally promotes gene activation by relaxing chromatin structure .

How is KAT2A expression distributed across different human tissues and cell types?

KAT2A expression varies considerably across different tissues and cancer types. Analysis of over 11,000 samples spanning 32 different human tumors showed that cholangiocarcinoma (CHOL) and testicular germ cell tumors (TGCT) exhibit the highest KAT2A expression, while kidney chromophobe (KICH) presents relatively low expression . In normal development, KAT2A is known to be important in bone and cartilage development , as well as in hematopoietic tissues where it regulates cell fate decisions .

What is the acyl-CoA specificity of KAT2A, and how can researchers distinguish between different acylation activities?

KAT2A demonstrates strong specificity for acetyl-CoA but shows limited activity with other acyl-CoA molecules. In vitro experiments have demonstrated that while KAT2A dramatically increases acetylation levels on multiple histone peptides, succinylated and malonylated peptides show similar abundance between samples with or without KAT2A . To effectively distinguish between these acylation activities, researchers should employ mass spectrometry analysis of reaction products when incubating histones with KAT2A and different acyl-CoA donors (acetyl-, succinyl-, or malonyl-CoA). Comparing peptide abundance ratios with appropriate statistical analysis (t-test with p-value thresholds <0.05) can conclusively determine significant activities .

How does integration of KAT2A into different complexes affect its HAT activity?

Integration of KAT2A into either ATAC or SAGA complex is required for its full histone acetyltransferase (HAT) activity . When examining KAT2A function, researchers must consider that its catalytic efficiency is significantly enhanced when it functions within these complexes rather than as an isolated enzyme. The SAGA complex comprises 19 subunits organized in 4 functionally distinct modules, including the HAT module containing KAT2A, TADA2B, TADA3, and CCDC101 (SGF29) . The structural arrangement within these complexes provides optimal positioning and substrate recognition for KAT2A to exert its enzymatic activity.

What are the structural and functional differences between ATAC and SAGA complexes containing KAT2A?

ATAC and SAGA are distinct macromolecular complexes that incorporate KAT2A but exhibit different chromatin specificities and regulate distinct sets of genes :

SAGA complex:

• Comprises 19 subunits organized in 4 functionally distinct modules

• Contains a HAT module (KAT2A, TADA2B, TADA3, CCDC101/SGF29)

• Includes an H2B deubiquitination (DUB) module centered on USP22

• Contains a core module with SPT20 (specific to SAGA) and 5 TBP-associated factors

• Includes a transcription factor interaction module (TRRAP)

• Has a core module with an octamer-like fold that facilitates TBP loading onto TATA promoters

• The enzymatic HAT and DUB modules connect flexibly to the core

ATAC complex:

• Exclusive to multicellular eukaryotes

• Initially linked to chromatin remodeling functions

• Controls ribosomal protein and translation-associated genes

• Participates in maintenance of biosynthetic molecular activity

Research has aligned ATAC with maintenance of biosynthetic activity, while SAGA participates in activation or maintenance of molecular programs underlying characteristics of individual cell types, contributing to cell identity preservation .

How can researchers experimentally distinguish between ATAC-dependent and SAGA-dependent KAT2A functions?

To experimentally distinguish between ATAC and SAGA functions:

• Perform selective knockdown studies of complex-specific components:

  • Target SAGA-specific components (e.g., SUPT20H/SPT20)

  • Target ATAC-specific components

  • Compare these with direct KAT2A knockdown

• Conduct ChIP-seq analysis to identify binding patterns of complex-specific components in comparison to KAT2A binding sites

• Perform transcriptomic analysis following selective knockdowns to identify:

  • ATAC-specific gene expression changes (enriched in biosynthetic pathways)

  • SAGA-specific gene expression changes (enriched in cell identity programs)

• Use pathway analysis to categorize affected genes (e.g., ATAC disruption uniquely affects ribosomal protein genes while SAGA disruption affects cell-type specific programs)

What role does KAT2A play in normal hematopoiesis, particularly in erythroid development?

KAT2A plays crucial, complex roles in hematopoiesis through its participation in both ATAC and SAGA complexes :

• Erythropoiesis-specific functions:

  • ATAC complex controls ribosomal protein genes that selectively affect early stages of erythropoiesis

  • Erythroid lineage is uniquely dependent on ribosomal assembly and protein synthesis rates

  • ATAC may affect the EPOR (erythropoietin receptor) locus, a candidate instructor of erythroid lineage commitment

  • SAGA becomes crucial in later stages of erythroid development

• Stage-specific dependencies:

  • Early erythropoiesis relies heavily on ATAC complex function

  • Post-commitment stages depend more on SAGA complex activity

  • USP22 (a SAGA component) requirements align with later stages of erythropoiesis

• Mechanistic impact:

  • KAT2A loss affects transcriptional stability

  • ATAC and SAGA control of gene transcription may be more specific than control of promoter H3K9 acetylation

  • Complex-specific enzymatic activities (H2B deubiquitination by USP22, H4 acetylation by KAT14) may contribute to transcriptional regulation specificity

How do KAT2A's functions in development differ between normal and malignant hematopoiesis?

In normal hematopoiesis:

• KAT2A regulates proliferation and activation of T-cell subsets and maturation of invariant natural killer T (iNKT) cells

• It restricts terminal differentiation of granulocytic cells

• It does not play a central role in hematopoietic stem cells

• ATAC complex affects early erythropoiesis through ribosomal protein gene regulation

• SAGA complex stabilizes or ensures correct progression of cell type-specific programs

In malignant hematopoiesis:

• KAT2A is a requirement in acute myeloid leukemia (AML) cell lines and patient samples

• Loss of Kat2a results in transcriptional instability of general metabolic regulation programs

• This leads to probabilistic loss of functional leukemia stem-like cells

• ATAC complex selectively affects ribosomal protein genes that sustain AML self-renewal

• ATAC may control other self-renewal associated genes, including HOXA signature associated with KMT2A/MLL rearrangements

• SAGA complex appears to contribute to stabilization of cell identity programs in leukemia

These differential roles explain why KAT2A is a dependency in leukemia but not in normal hematopoietic stem cells .

How does KAT2A expression correlate with cancer prognosis in different human tumors?

KAT2A expression significantly correlates with prognosis in specific cancer types:

These findings suggest KAT2A expression has context-dependent prognostic significance, potentially reflecting its differential roles in various tumor types.

What experimental approaches can researchers use to investigate KAT2A's role in tumorigenesis?

Researchers investigating KAT2A's role in tumorigenesis should consider these methodological approaches:

• Expression analysis:

  • Compare KAT2A expression between tumor samples and matched normal tissues using transcriptomics

  • Analyze expression across large datasets (e.g., TCGA) to identify cancer-specific patterns

  • Use immunohistochemistry to validate protein expression in tissue samples

• Functional studies:

  • Employ shRNA or CRISPR-mediated knockdown/knockout of KAT2A in cancer cell lines

  • Analyze phenotypic changes in proliferation, apoptosis, migration, and invasion

  • Perform rescue experiments with wild-type or catalytically inactive KAT2A

• Complex-specific approaches:

  • Selectively target ATAC or SAGA complex components to distinguish their contributions

  • Investigate how complex-specific functions impact cancer hallmarks

• Survival analysis:

  • Correlate KAT2A expression with patient outcomes in specific cancer types

  • Stratify patients based on KAT2A expression levels and associated molecular features

• Mechanistic investigations:

  • Perform ChIP-seq to identify KAT2A binding sites in cancer genomes

  • Conduct RNA-seq following KAT2A manipulation to identify transcriptional targets

  • Investigate post-translational modifications of KAT2A in cancer contexts

What are the optimal methods for assessing KAT2A acetyltransferase activity in vitro and in cellular contexts?

For comprehensive assessment of KAT2A acetyltransferase activity, researchers should consider:

In vitro assays:

• Recombinant enzyme activity assays:

  • Use purified KAT2A catalytic domain (e.g., residues 497-662)

  • Incubate with histone substrates (e.g., calf thymus histone) and acetyl-CoA

  • Include appropriate controls (samples without KAT2A)

  • Perform reactions at 37°C for 30 minutes

  • Run each reaction in triplicate for statistical validity

• Mass spectrometry analysis:

  • Digest reaction products with trypsin

  • Analyze peptides by LC-MS/MS

  • Aim for at least 50% sequence coverage of each histone

  • Compare peptide abundance between samples with/without KAT2A

  • Apply appropriate statistical analysis (t-test with p-value thresholds)

Cellular context assays:

• Knockdown experiments:

  • Use shRNA targeting KAT2A (select constructs giving strongest knockdown)

  • Validate knockdown efficiency by Western blot

  • Extract histones using appropriate lysis buffers with protease inhibitors

  • Analyze changes in global histone acetylation levels

• Complex-specific activity:

  • Perform selective knockdown of complex components

  • Distinguish between ATAC and SAGA-dependent activities

  • Compare effects on H3K9ac levels at specific genomic loci

How can researchers effectively investigate the relationship between KAT2A and other epigenetic modifiers in transcriptional regulation networks?

To investigate relationships between KAT2A and other epigenetic modifiers:

• Multi-omics integration approaches:

  • Combine ChIP-seq for KAT2A, H3K9ac, and other relevant histone marks

  • Integrate with RNA-seq data to correlate binding patterns with transcriptional outcomes

  • Include ATAC-seq to assess chromatin accessibility changes

  • Apply machine learning algorithms to identify coordinated regulation patterns

• Protein-protein interaction studies:

  • Perform co-immunoprecipitation to identify direct KAT2A interactors

  • Use proximity labeling approaches (BioID, APEX) to map the broader KAT2A interactome

  • Validate interactions with known epigenetic modifiers

  • Map interactions to specific complex components (ATAC vs. SAGA)

• Perturbation experiments:

  • Conduct combinatorial knockdowns/knockouts of KAT2A with other epigenetic regulators

  • Analyze synergistic or antagonistic effects on gene expression

  • Assess changes in histone modification landscapes

• Single-cell approaches:

  • Apply single-cell RNA-seq following KAT2A perturbation to capture cellular heterogeneity

  • Investigate how KAT2A contributes to transcriptional variability

  • This is particularly relevant given KAT2A's role in sustaining rather than initiating gene transcription and stabilizing promoter activity

• Structural biology approaches:

  • Analyze how KAT2A integrates with different complex structures

  • Investigate how these structures facilitate interactions with other epigenetic modifiers

  • Consider the flexible connection of enzymatic modules to complex cores