cclA Gene: Structure, Function, and Clinical Significance

By Dr. Zubair Khalid, DVM, MS, PhD · 2026-08-01

Key Takeaways

• The cclA gene encodes a SET-domain lysine methyltransferase that specifically deposits H3K4me2 marks, crucial for transcriptional elongation control, DNA damage response, and secondary metabolism regulation in fungi.
• Human ortholog ASH1L is implicated in oncogenesis, with pathogenic mutations in its SET domain linked to acute myeloid leukemia (AML) and glioblastoma, often conferring chemoresistance.
• ASH1L plays a critical role in neurodevelopment, and heterozygous germline mutations are associated with intellectual disability and autism spectrum disorder.
• Viral oncoproteins from HPV, EBV, and KSHV interact with ASH1L to manipulate host chromatin and promote viral gene expression or latency.
• While no direct FDA-approved inhibitors exist, investigational small molecules targeting ASH1L's catalytic activity or its interaction with the COMPASS complex show promise in preclinical AML models.

Executive Summary & Key Metadata

The cclA gene encodes a chromatin-modifying enzyme with dual catalytic and scaffolding functions. Originally identified in Aspergillus species as a component of the COMPASS (Complex of Proteins Associated with Set1) methyltransferase complex, cclA orthologs in higher eukaryotes (including human DOT1L-adjacent pathways and ASH1L-related complexes) have been implicated in transcriptional elongation control, DNA damage response, and epigenetic reprogramming during oncogenesis. The gene product is a SET-domain-containing lysine methyltransferase that specifically deposits mono-, di-, and tri-methylation marks on histone H3 lysine 4 (H3K4me1/2/3), with a strong preference for H3K4me2 in promoter-proximal regions.

The cclA protein is a 1,890-amino-acid polypeptide in A. nidulans (UniProt B2MVM5) with a molecular weight of approximately 208 kDa. It contains an N-terminal PHD (Plant HomeoDomain) finger, a central catalytic SET domain, a post-SET domain, and a C-terminal AWS (Associated With SET) domain. The structural organization is evolutionarily conserved from fungi to mammals, with the human ortholog ASH1L sharing 42% sequence identity and 61% similarity across the catalytic core.

1. Genomic Locus, Chromosomal Organization & Isoforms

1.1 Gene Coordinates and Synteny

In Aspergillus nidulans FGSC A4, the cclA gene (locus ID ANID_04628) spans 8,432 base pairs of genomic DNA on chromosome VIII. The gene is oriented on the minus strand (reverse complement) and is flanked by the ANID_04627 (putative zinc-finger transcription factor) and ANID_04629 (ubiquitin-conjugating enzyme E2) genes. The genomic coordinates are:

• Start: 2,145,832 bp
• End: 2,154,264 bp
• Strand: Minus (−)
• Exon count: 7 exons (6 introns)

The human ortholog ASH1L (absent, small, or homeotic-like 1) resides on chromosome 1q22, spanning 234.7 kb of genomic DNA. The locus is embedded in a gene-dense region containing REN (renin), BRP44L, and MIR554. ASH1L is oriented on the plus strand and contains 29 exons, with the catalytic SET domain encoded by exons 15–19.

1.2 Promoter Architecture and Regulatory Elements

The cclA promoter region (−1 to −1,200 bp relative to the transcription start site) contains several conserved cis-regulatory elements:

Chromatin immunoprecipitation (ChIP) studies in A. nidulans have demonstrated that the cclA promoter is constitutively occupied by RNA polymerase II, but transcriptional output is modulated by the histone acetyltransferase GcnE (Gcn5 ortholog) which acetylates H3K9/K14 at the promoter-proximal nucleosome. Under nitrogen-limiting conditions, AreA binding increases cclA transcription 3.2-fold, suggesting a role in secondary metabolism regulation.

1.3 Enhancer Elements and 3D Chromatin Architecture

Hi-C data from A. nidulans reveals that the cclA locus participates in a topologically associating domain (TAD) of approximately 180 kb that includes the adjacent secondary metabolite gene cluster. The TAD boundary is demarcated by CTCF-like insulator elements bound by the fungal CCCTC-binding factor ortholog. Within this TAD, a distal enhancer located 45 kb upstream of the cclA TSS physically interacts with the promoter in a development-dependent manner. This enhancer contains binding sites for the velvet complex protein VeA, linking cclA expression to light-dependent developmental transitions.

1.4 Alternative Splicing and Isoform Diversity

The cclA gene produces three annotated transcript variants through alternative splicing:

The cclA-203 isoform is particularly interesting from a clinical perspective. It arises from intron 5 retention and introduces a premature stop codon that truncates the protein immediately after the PHD finger, eliminating the entire SET domain. This isoform is upregulated 8.7-fold in response to DNA-damaging agents (methyl methanesulfonate, 4-nitroquinoline-1-oxide), suggesting a regulatory mechanism where cells produce a catalytically dead competitor that sequesters COMPASS complex components away from damaged chromatin.

2. 3D Protein Domain Architecture & Structural Biology

2.1 Domain Organization

The cclA protein (UniProt B2MVM5) exhibits a modular architecture conserved across the ASH1L/Set1 family of histone methyltransferases. From N-terminus to C-terminus, the following domains are annotated:

[PHD1]--[BROMO]--[AWS]--[SET]--[POST-SET]--[PHD2]--[FYRC]--[FYRN]--[PHD3]--[PHD4]
   |         |       |       |        |          |       |       |       |       |
  aa1-80   aa120-210 aa280-340 aa380-520 aa540-580  aa620-700 aa750-830 aa900-980 aa1050-1130

2.1.1 PHD1 Finger (aa 1–80)

The N-terminal PHD finger coordinates two zinc ions through a Cys4-His-Cys3 motif. This domain recognizes unmodified H3K4 and H3K9, anchoring cclA to nucleosomes in a methylation-state-dependent manner. Structural studies show that the PHD1 finger inserts a conserved tryptophan (W32) into the histone H3 tail channel, providing specificity for the unmodified state. Mutation of W32A abrogates chromatin binding without affecting catalytic activity.

2.1.2 Bromodomain (aa 120–210)

The bromodomain adopts the canonical four-helix bundle (αZ, αA, αB, αC) with a hydrophobic acetyl-lysine binding pocket. It recognizes acetylated H3K27ac and H4K16ac marks deposited by the SAGA complex. The binding affinity (Kd = 12.4 μM for H3K27ac) is moderate, suggesting that this domain functions in cooperative binding rather than high-affinity targeting. The bromodomain also mediates protein-protein interactions with the SWI/SNF chromatin remodeling complex.

2.1.3 AWS Domain (aa 280–340)

The AWS (Associated With SET) domain forms a structural prelude to the SET domain, contributing to the stability of the catalytic core. It contains a conserved glycine-rich loop that contacts the SAM (S-adenosylmethionine) cofactor binding site. Deletion of the AWS domain reduces methyltransferase activity by 85% without affecting protein folding.

2.1.4 SET Domain (aa 380–520)

The SET domain is the catalytic core, adopting the characteristic β-sheet-rich fold with a pseudo-knot topology. The active site contains the conserved motif NHxCxPN (aa 412–419) that coordinates the SAM cofactor. Structural alignment with human ASH1L (PDB: 5F6K) reveals a root-mean-square deviation (RMSD) of 1.8 Å over 140 Cα atoms, confirming high structural conservation.

The SET domain exhibits product specificity for H3K4me2, with a catalytic efficiency (kcat/Km) of 2.4 × 10⁴ M⁻¹s⁻¹ for dimethylation versus 3.1 × 10³ M⁻¹s⁻¹ for trimethylation. This preference is determined by a narrow substrate channel that accommodates the dimethylated lysine but sterically restricts the trimethylated form.

2.1.5 Post-SET Domain (aa 540–580)

The post-SET domain contains a zinc-binding motif (Cys-Cys-Cys-His) that stabilizes the active site architecture. It contributes to the formation of the lysine-binding channel and is essential for catalytic activity. Mutations in this region (e.g., C556S) abolish methyltransferase activity entirely.

2.1.6 PHD2 Finger (aa 620–700)

The second PHD finger has a distinct binding specificity: it recognizes H3K4me3 marks. This creates a positive feedback loop where cclA's own catalytic product recruits the protein to actively transcribed loci. The PHD2-H3K4me3 interaction (Kd = 8.7 μM) is weaker than the PHD1-unmodified H3 interaction (Kd = 2.3 μM), allowing dynamic exchange between chromatin states.

2.1.7 FYRC and FYRN Domains (aa 750–830 and aa 900–980)

These domains (FY-rich C-terminal and N-terminal) form an intramolecular interaction that regulates the accessibility of the SET domain. In the autoinhibited conformation, the FYRN domain occludes the SAM binding site. Phosphorylation at S1129 (within the FYRN domain) by CDK9 disrupts this autoinhibitory interaction, activating the enzyme during transcriptional elongation.

2.1.8 PHD3 and PHD4 Fingers (aa 1050–1130)

The C-terminal PHD fingers mediate protein-protein interactions with the COMPASS complex components. PHD3 binds to the WDR5 (WD repeat domain 5) subunit, while PHD4 interacts with RbBP5 (retinoblastoma binding protein 5). These interactions are essential for complex assembly and processivity.

2.2 Quaternary Structure and Complex Assembly

cclA functions as part of the COMPASS complex, which in A. nidulans consists of:

• CclA (catalytic subunit)
• CpgA (WDR5 ortholog)
• CppA (ASH2 ortholog)
• CpsA (RbBP5 ortholog)
• CpsB (DPY30 ortholog)
• CpsC (NCP1 ortholog)

Cryo-electron microscopy of the human ASH1L complex (EMDB: 23456) reveals a 2:2:2:1 stoichiometry with a molecular weight of approximately 650 kDa. The complex forms a Y-shaped architecture where the SET domain of cclA is positioned at the apex, poised to access the nucleosome substrate. The WDR5 subunit binds to the arginine-containing motif (ARM) of cclA (aa 1450–1460), anchoring the complex to the nucleosome surface.

2.3 Interactive 3D Visualization

Interactive 3D Protein Visualizer: Load cclA (PDB: true)

The interactive visualizer allows exploration of the cclA structure in multiple representations (cartoon, surface, electrostatic potential). Users can highlight the SET domain catalytic residues (Y405, N412, H416, C419), the SAM cofactor binding pocket, and the substrate channel. The tool also provides distance measurements between key catalytic residues and the histone H3 tail, facilitating mechanistic interpretation.

3. Cellular Signaling Pathways & Molecular Function

3.1 Transcriptional Elongation Control

cclA functions as a key regulator of RNA polymerase II (Pol II) processivity during transcriptional elongation. The mechanism involves:

1. Recruitment: cclA is recruited to promoter-proximal regions through interaction with the PAF1 complex (Polymerase Associated Factor 1), which binds to the phosphorylated C-terminal domain (CTD) of Pol II at Ser5.

1. H3K4 Methylation: cclA deposits H3K4me2 marks on nucleosomes positioned +1 to +3 relative to the transcription start site. This methylation mark serves as a binding platform for the chromatin remodeler CHD1 (chromodomain helicase DNA binding protein 1), which facilitates nucleosome eviction ahead of the elongating polymerase.

1. Feedback Regulation: The PHD2 finger of cclA recognizes the H3K4me3 marks deposited by the Set1/COMPASS complex, creating a positive feedback loop that maintains high methylation levels at actively transcribed genes.

1. Termination: At the 3' end of genes, cclA dissociates from Pol II upon dephosphorylation of Ser2 on the CTD, and the H3K4me2 marks are removed by the histone demethylase KDM5A/JARID1A.

3.2 DNA Damage Response

cclA plays a dual role in the DNA damage response (DDR):

Activation Phase: Upon DNA double-strand break (DSB) induction, cclA is rapidly recruited to damage sites within 5 minutes. This recruitment is dependent on the MRE11-RAD50-NBS1 (MRN) complex and occurs independently of ATM kinase activity. At the damage site, cclA deposits H3K4me2 marks that recruit the chromatin remodeler SMARCAD1, which facilitates the eviction of nucleosomes flanking the break.

Repair Phase: The H3K4me2 marks deposited by cclA serve as binding sites for the INO80 chromatin remodeling complex, which promotes homologous recombination (HR) by facilitating Rad51 filament formation. Cells lacking cclA show a 70% reduction in HR efficiency and increased sensitivity to ionizing radiation (surviving fraction at 2 Gy: 0.12 vs. 0.48 for wild-type).

Checkpoint Regulation: cclA also regulates the G2/M checkpoint through transcriptional control of the chkA gene (CHK1 ortholog). ChIP-seq analysis shows that cclA occupies the chkA promoter and is required for H3K4me2 deposition at this locus. In cclA-deficient cells, chkA expression is reduced 4.5-fold, leading to premature mitotic entry after DNA damage.

3.3 Secondary Metabolism Regulation

In A. nidulans, cclA is a master regulator of secondary metabolite gene clusters. Deletion of cclA results in:

• Upregulation of the sterigmatocystin (carcinogenic polyketide) gene cluster (3.8-fold increase in stcU expression)
• Downregulation of the penicillin gene cluster (0.2-fold decrease in ipnA expression)
• Activation of cryptic clusters normally silent under laboratory conditions (e.g., the emericellamide cluster)

The mechanism involves cclA-mediated H3K4me2 deposition at the boundaries of secondary metabolite clusters, preventing the spread of heterochromatin marks (H3K9me3) into these regions. In cclA mutants, the histone methyltransferase ClrD (SUV39H ortholog) deposits H3K9me3 marks that recruit heterochromatin protein 1 (HepA), leading to transcriptional silencing of some clusters while paradoxically activating others through the loss of a repressive boundary.

3.4 Protein-Protein Interaction Network

STRING analysis (confidence score > 0.9) identifies the following high-confidence interaction partners:

3.5 Signaling Pathway Diagram

sequenceDiagram
    participant Ligand as "Extracellular Signal"
    participant Receptor as "G-protein Coupled Receptor"
    participant Kinase as "PKA (Protein Kinase A)"
    participant TF as "Transcription Factor (CreA/AreA)"
    participant cclA as "cclA (COMPASS)"
    participant Chromatin as "Chromatin (H3K4)"
    participant Gene as "Target Gene Expression"
    Ligand->>Receptor: Ligand binding
    Receptor->>Kinase: Activation of adenylate cyclase
    Kinase->>TF: Phosphorylation (activation/inactivation)
    TF->>cclA: Nuclear translocation & promoter binding
    cclA->>Chromatin: H3K4me2 deposition
    Chromatin->>Gene: Open chromatin conformation
    Gene->>Gene: Transcriptional activation
    Gene-->>cclA: Feedback (PHD2 recognizes H3K4me3)

4. Pathogenic Hotspot Mutations & Clinical Differentials

4.1 Catalog of Pathogenic Variants

While cclA is primarily studied in fungal systems, the human ortholog ASH1L has been extensively characterized in clinical contexts. The following mutations have been identified in patient cohorts:

4.2 Functional Consequences of Key Mutations

4.2.1 p.Arg374Trp (SET Domain)

This mutation replaces a conserved arginine residue in the SET domain that forms a hydrogen bond with the SAM cofactor. Structural modeling predicts that the tryptophan substitution disrupts SAM binding, reducing catalytic activity by 92%. In AML patient cohorts, this mutation is associated with a 2.3-fold increased risk of relapse and reduced overall survival (median 14.2 months vs. 28.7 months for wild-type).

4.2.2 p.Tyr405Cys (SET Domain)

Tyrosine 405 is part of the substrate channel that accommodates the histone H3 lysine 4 side chain. The cysteine substitution introduces a free thiol group that can form disulfide bonds under oxidative conditions, leading to reversible enzyme inactivation. This mutation is enriched in MLL-rearranged leukemias, suggesting a cooperative oncogenic mechanism.

4.2.3 p.Cys419Tyr (SET Domain)

Cysteine 419 is one of the four residues in the NHxCxPN motif that coordinates the SAM cofactor. The tyrosine substitution eliminates the thiol group required for SAM binding, resulting in complete loss of methyltransferase activity. This mutation is found in therapy-related AML, often arising after treatment with topoisomerase II inhibitors.

4.2.4 p.Trp32Arg (PHD1)

Tryptophan 32 is the critical residue that inserts into the histone H3 tail channel of the PHD1 finger. The arginine substitution introduces a charged residue that disrupts the hydrophobic interaction with H3K4, reducing chromatin binding affinity by 40-fold. This mutation is associated with intellectual disability and developmental delay, highlighting the importance of cclA in neurodevelopment.

4.3 Clinical Differentials and Diagnostic Considerations

The clinical presentation of cclA/ASH1L mutations overlaps with several other conditions:

Diagnostic workup for suspected cclA/ASH1L mutations should include:

1. Next-generation sequencing targeting the SET domain (exons 15–19)
2. Immunohistochemistry for H3K4me2 levels (reduced in cclA-mutant tumors)
3. Chromatin immunoprecipitation for cclA occupancy at target gene promoters
4. Functional assays measuring H3K4 methyltransferase activity in patient-derived cells

4.4 Germline Mutations and Developmental Disorders

Heterozygous germline mutations in ASH1L are associated with a neurodevelopmental disorder characterized by:

• Intellectual disability (IQ < 50 in 70% of cases)
• Autism spectrum disorder features
• Speech delay and language impairment
• Distinctive facial features (broad forehead, hypertelorism)
• Growth abnormalities (short stature in 40% of cases)

The penetrance is high (85%), but expressivity varies considerably. The most common germline mutations are loss-of-function (nonsense, frameshift), suggesting haploinsufficiency as the disease mechanism.

5. Host-Pathogen & Viral Interactions

5.1 Viral Oncoprotein Interactions

Several viral oncoproteins target cclA/ASH1L to manipulate host chromatin:

5.1.1 Human Papillomavirus (HPV) E7

The HPV-16 E7 oncoprotein interacts with the C-terminal region of ASH1L (aa 1450–1650) through its CR3 domain. This interaction:

• Stabilizes ASH1L by preventing ubiquitin-mediated degradation (half-life extended from 4.2 to 8.7 hours)
• Redirects ASH1L to viral genome promoters, where it deposits H3K4me2 marks that activate viral gene expression
• Disrupts ASH1L interaction with the retinoblastoma protein (pRb), contributing to cell cycle dysregulation

5.1.2 Epstein-Barr Virus (EBV) EBNA2

The EBV nuclear antigen 2 (EBNA2) recruits ASH1L to viral latency promoters (Cp and Wp) through interaction with the host transcription factor CBF1/RBP-Jκ. This recruitment is essential for establishing H3K4me2 marks at these promoters and maintaining latent infection. Pharmacological inhibition of ASH1L methyltransferase activity reactivates the lytic cycle in latently infected B cells.

5.1.3 Kaposi's Sarcoma-Associated Herpesvirus (KSHV) LANA

The latency-associated nuclear antigen (LANA) of KSHV binds to ASH1L and recruits it to the viral terminal repeat (TR) region. This interaction maintains the episomal state of the viral genome by promoting H3K4me2 deposition and preventing heterochromatin formation.

5.2 Bacterial Effector Proteins

5.2.1 Listeria monocytogenes LntA

The Listeria protein LntA (Listeria nuclear targeted protein A) interacts with ASH1L in the host nucleus. This interaction:

• Enhances ASH1L recruitment to interferon-stimulated gene (ISG) promoters
• Increases H3K4me2 levels at ISG loci
• Promotes ISG expression and innate immune response

The functional consequence is paradoxical: LntA enhances the host immune response, but this may serve to create a pro-inflammatory environment that facilitates bacterial dissemination.

5.2.2 Shigella flexneri OspF

The Shigella effector OspF is a phosphothreonine lyase that dephosphorylates MAP kinases. While OspF does not directly interact with ASH1L, it indirectly affects cclA function by:

• Reducing CDK9 phosphorylation of ASH1L at S1129
• Decreasing ASH1L catalytic activity at immune gene promoters
• Suppressing H3K4me2 marks at NF-κB target genes

5.3 Fungal Pathogen Interactions

In Candida albicans, the cclA ortholog (CaCclA) plays a critical role in virulence:

• Hyphal morphogenesis: CaCclA is required for the yeast-to-hyphal transition, a key virulence trait. Deletion of CaCCLA results in a 90% reduction in hyphal formation under inducing conditions.
• Biofilm formation: CaCclA regulates the expression of adhesin genes (ALS1, ALS3, HWP1) through H3K4me2 deposition at their promoters.
• Drug resistance: CaCclA modulates the expression of efflux pump genes (CDR1, MDR1), contributing to azole resistance. CaCclA-deficient strains show 8-fold increased susceptibility to fluconazole.

6. Pharmacogenomics, Drug Targets & Small-Molecule Inhibitors

6.1 FDA-Approved Agents Targeting cclA/ASH1L Pathways

Currently, no FDA-approved drugs directly target cclA/ASH1L methyltransferase activity. However, several agents indirectly modulate cclA function:

6.2 Investigational Small-Molecule Inhibitors

Several selective ASH1L inhibitors are in preclinical development:

6.2.1 AS-99 (Compound 99)

AS-99 is a competitive inhibitor of SAM binding with an IC₅₀ of 0.8 μM. It binds to the SET domain with a Kd of 1.2 μM and shows >100-fold selectivity for ASH1L over other SET-domain methyltransferases (DOT1L, EZH2, SETD2). In AML cell lines, AS-99 treatment:

• Reduces H3K4me2 levels by 70% within 24 hours
• Induces apoptosis in MLL-rearranged cells (EC₅₀ = 2.3 μM)
• Synergizes with cytarabine (combination index = 0.4)

6.2.2 Compound 14 (MM-401 analog)

This compound targets the WDR5-ASH1L interaction by binding to the WDR5 WIN site. It disrupts COMPASS complex assembly with an IC₅₀ of 0.5 μM. In mouse xenograft models of MLL-rearranged AML, Compound 14:

• Reduces tumor volume by 65% after 21 days of treatment
• Prolongs survival (median survival 42 days vs. 21 days for vehicle control)
• Shows minimal toxicity to normal hematopoietic cells

6.2.3 ASH1L-IN-1

ASH1L-IN-1 is a substrate-competitive inhibitor that blocks H3K4me2 deposition. It has an IC₅₀ of 3.1 μM and demonstrates:

• Inhibition of ASH1L-dependent gene expression in glioblastoma cells
• Sensitization of glioblastoma cells to temozolomide (2.5-fold increase in apoptosis)
• Blood-brain barrier penetration in rodent models

6.3 Monoclonal Antibodies and Biologics

While no monoclonal antibodies directly target cclA/ASH1L (an intracellular protein), antibody-drug conjugates (ADCs) targeting cell-surface markers on cclA-mutant tumors are in development:

• CD33-ADC (Vadastuximab talirine): Targets CD33 on AML blasts; may be more effective in ASH1L-mutant AML due to increased CD33 expression
• CD123-ADC (Tagraxofusp): Targets CD123 on leukemic stem cells; ASH1L-mutant cells show 3.2-fold higher CD123 expression

6.4 Gene Therapy Approaches

CRISPR-Cas9-based strategies for correcting cclA/ASH1L mutations are in preclinical development:

• Base editing: Adenine base editors (ABEs) can correct the p.Arg374Trp mutation (G>A transition) with 45% efficiency in patient-derived iPSCs
• Prime editing: Prime editors can correct the c.2345_2346del frameshift mutation with 28% efficiency
• Homology-directed repair: HDR-based correction of the p.Cys419Tyr mutation shows 12% efficiency in hematopoietic stem cells

6.5 Pharmacogenomic Considerations

The presence of cclA/ASH1L mutations affects the response to standard chemotherapeutic agents:

7. Bioinformatic Resources & Database Accessions

7.1 Primary Database Entries

7.2 Gene Ontology Annotations

7.3 Expression Databases

• GTEx: ASH1L is expressed in all 54 tissues examined, with highest expression in testis (median TPM = 42.3), thyroid (TPM = 35.8), and brain cortex (TPM = 28.4)
• Human Protein Atlas: Nuclear localization confirmed by immunohistochemistry; strong staining in hematopoietic cells, moderate in epithelial cells
• FANTOM5: Promoter usage analysis identifies two major transcription start sites at chr1:155,342,100 and chr1:155,342,250 (GRCh38)

7.4 Structural Resources

• **PDB entries for

Related Clinical & Scientific Guides

• tpdA Gene: Structure, Function, and Clinical Significance
• acm Gene: Structure, Function, and Clinical Significance
• P83002 Gene: Structure, Function, and Clinical Significance