SIX1 Human

What is the functional role of SIX1 in human development?

SIX1 (sine oculis homeobox 1) is a developmentally regulated homeoprotein that functions as a crucial transcriptional regulator during embryonic development. Its primary role is in the development of cranial placodes, which are specialized regions of embryonic ectoderm that contribute to sensory structures in the head, particularly the inner ear.

Research has established SIX1 as a master regulator of cranial placode development, with loss-of-function studies demonstrating significant consequences: reduced expression of several placode genes and defects in otic development, including loss of hair cells and defective patterning of the sensory epithelium . SIX1 critically contributes to the development of both the auditory and vestibular structures, affecting the volume of otic cartilaginous capsules, otoliths, lumen, and sensory patches containing hair cells.

The importance of SIX1 extends beyond ear development to other embryonic processes, including neural crest cell migration and craniofacial formation, making it essential for proper organogenesis in multiple systems.

What are the key functional domains of the SIX1 protein?

The SIX1 protein contains two critical functional domains that are highly conserved across species:

• Protein-Protein Interaction Domain (SD): This domain enables interactions with cofactors such as EYA1 and EYA2, which are essential for SIX1's transcriptional activity. The SD domain serves as a molecular scaffold for assembling transcriptional complexes.

• DNA-Binding Homeodomain (HD): This domain binds to specific DNA sequences in the promoters of target genes, allowing SIX1 to regulate their expression. The homeodomain is remarkably conserved, with Xenopus Six1 protein being 100% identical to human SIX1 in both functional domains .

Mutations in either domain can significantly impair SIX1 function. Eight BOR mutations in SIX1 have been identified, including seven missense mutations and one in-frame deletion, residing in either the SD or HD domains . The structural integrity of these domains is crucial for proper transcriptional regulation, with specific mutations altering DNA binding affinity or protein-protein interactions in distinct ways.

What developmental disorders are associated with SIX1 mutations?

SIX1 mutations primarily cause branchio-oto-renal (BOR) syndrome and the related branchio-otic (BOS) syndrome. These autosomal dominant developmental disorders are characterized by:

• Hearing loss

• Branchial arch defects

• Renal anomalies (in BOR)

Single-nucleotide mutations in human SIX1 result in amino acid substitutions in either the protein-protein interaction domain or the homeodomain, accounting for approximately 4% of BOS and BOR cases . What makes these disorders particularly challenging is the significant phenotypic variation observed between patients with identical mutations, even within the same family.

Research using Xenopus embryos expressing Six1 proteins with human BOS/BOR mutations (V17E, R110W, W122R, Y129C) has demonstrated that these mutations cause specific disruptions in gene expression patterns related to neural crest and pre-placodal ectoderm development . While auditory and vestibular structures do form in these models, significant reductions occur in:

• Otic cartilaginous capsule volume

• Otolith size

• Lumen dimensions

• Hair cell-containing sensory patches

These findings indicate that BOS/BOR-associated mutations lead to subtle but significant changes in embryonic gene expression that ultimately manifest as inner ear abnormalities.

How do SIX1 mutations contribute to cancer development and progression?

SIX1 is frequently overexpressed in human tumors and functions as a repressor of cell senescence—an antiproliferative response crucial for tumor suppression . In cancer development, SIX1's role appears multifaceted:

In Wilms tumors (pediatric kidney cancer), the SIX1-Q177R mutation has been specifically associated with relapse. This mutation occurs in the DNA-binding homeodomain and results in enhanced DNA-binding affinity, generating aberrant gene expression programs . Interestingly, the same Q177R mutation occurs in the related protein SIX2 in Wilms tumors, though at approximately half the frequency of SIX1-Q177R.

Differential gene expression analyses of SIX1/2-Q177R tumors reveal distinct transcriptional signatures compared to tumors without these mutations . This suggests that altered transcriptional regulation by mutant SIX1/2 contributes to tumor development and potentially to relapse.

SIX1 also promotes cell proliferation by upregulating cyclin and glycolytic genes, while enhancing cell migration through increased expression of matrix metalloproteinase 9 (MMP9) . These functions collectively support cancer progression through multiple mechanisms.

What is the relationship between SIX1 expression and cell senescence?

SIX1 functions as a repressor of cellular senescence, an antiproliferative mechanism that serves as a critical tumor suppression pathway . This relationship is particularly significant in understanding SIX1's role in cancer development.

While the complete molecular mechanism remains under investigation, research indicates that SIX1 promotes cellular proliferation through:

• Upregulation of cyclin genes that drive cell cycle progression

• Enhancement of glycolytic metabolism supporting rapid cell division

• Promotion of SOX2-mediated cellular functions that maintain proliferative capacity

In addition to preventing senescence, SIX1 increases cell migration capacity through upregulation of matrix metalloproteinase 9 (MMP9), either directly or indirectly . This migratory capacity further contributes to the cells' ability to evade growth arrest signals.

The anti-senescence activity of SIX1 provides one explanation for why SIX1 overexpression is frequently observed in human tumors—it allows cells to bypass a fundamental tumor-suppressive mechanism, contributing to uncontrolled proliferation and cancer progression.

What model systems are most effective for studying SIX1 function?

Based on current research, several model systems have proven particularly valuable for studying SIX1 function:

• Xenopus (frog) embryos: This model offers significant advantages for developmental studies. The Xenopus Six1 protein shares 100% identity with human SIX1 in both the protein-protein interaction domain and homeodomain . Additionally, Xenopus has evolved terrestrial hearing mechanisms that share extensive otic development, morphology, and gene expression patterns with mammals. This system allows researchers to express Six1 proteins with human mutations and assess effects on gene expression and otic morphology.

• Cell culture systems:

  • MCF7 cells: Effective for studying protein-protein interactions and subcellular localization

  • NIH3T3 cells: Useful for luciferase reporter assays assessing transcriptional activity

  • Renal cell models: Valuable for studying SIX1's role in kidney injury and repair

• E. coli expression systems: For biochemical and structural analyses, bacterial expression systems enable production of purified SIX1 proteins for in vitro DNA binding and protein-protein interaction studies .

Each system offers distinct advantages depending on the research question. Xenopus embryos provide an in vivo developmental context, cell culture systems enable controlled mechanistic studies, and bacterial systems facilitate biochemical characterization.

What techniques are recommended for assessing SIX1 DNA binding affinity?

Electrophoretic mobility shift assay (EMSA) represents the gold standard for assessing SIX1 DNA binding affinity. The optimal protocol includes:

• Preparation of labeled DNA probes:

  • Oligonucleotides containing SIX1 binding sites are radiolabeled (typically with 32P)

  • Annealed to form double-stranded probes

• Binding reaction setup:

  • Purified SIX1 protein (with or without cofactors like EYA) is incubated with labeled DNA

  • For quantitative affinity determination, protein concentrations should range from 15 to 7500 fmol while maintaining constant DNA (4 fmol/reaction)

• Competition assays:

  • Include unlabeled wild-type oligonucleotide or scrambled versions at 200× concentration (800 fmol/reaction) to assess binding specificity

• Electrophoresis conditions:

  • Run for 90 minutes at 60 volts at 4°C

  • Use 6% nondenaturing polyacrylamide gel

• Detection methodology:

  • Dry gels and expose to phosphorimaging plates

  • Visualize using a phosphorimager

This approach enables researchers to assess both qualitative binding capacity and quantitative binding affinity under various conditions. The technique has successfully demonstrated that the Q177R mutation in SIX1, associated with Wilms tumors, enhances DNA binding affinity .

How can researchers effectively measure SIX1-mediated transcriptional activation?

Luciferase reporter assays provide the most reliable approach for measuring SIX1-mediated transcriptional activation. The optimal protocol involves:

• Reporter construct preparation:

  • Develop luciferase reporter constructs containing SIX1-responsive elements in the promoter region

• Transfection protocol:

  • Co-transfect cells (e.g., NIH3T3) with:

    • Luciferase reporter construct

    • Renilla luciferase construct (for normalization)

    • SIX1 expression construct (wild-type or mutant): 100 ng/well

    • Cofactor expression construct (e.g., FLAG-Eya2) or empty vector: 150 ng/well

• Analytical procedure:

  • Lyse cells 48 hours post-transfection

  • Analyze extracts for luciferase and Renilla activities

  • Calculate normalized luciferase activity

• Validation of protein expression:

  • Confirm SIX1 expression via western blotting with anti-SIX1 antibodies

  • Verify equal loading with anti-α-actin antibodies

This methodology has successfully demonstrated that certain BOR-associated mutations (V17E, R110W, Y129C) render SIX1 unable to activate transcription even in the presence of EYA cofactors . For assessing endogenous target gene expression, real-time PCR using TaqMan gene expression assays provides complementary data on transcriptional outcomes.

How do different SIX1 mutations affect its transcriptional activity?

SIX1 mutations exhibit distinct effects on transcriptional activity, depending on how they alter protein structure, protein-protein interactions, or DNA binding. Research has revealed:

• Transcriptional deficiency: V17E, R110W, and Y129C mutations render SIX1 unable to activate transcription in the presence of Eya1 or EYA2 cofactors, despite retaining nuclear localization . This indicates that these mutations disrupt either cofactor interaction or DNA binding without affecting nuclear import.

• Differential effects on target genes: When expressed in Xenopus embryos, four BOS/BOR substitutions (V17E, R110W, W122R, Y129C) cause specific and often different disruptions in gene expression :

  • V17E showed similar activity to wild-type Six1 on the neural border gene msx1

  • R110W and W122R demonstrated distinctly different patterns of activity

• Protein stability effects: Expression tests in E. coli revealed that H73P and R110Q mutants failed to express, suggesting these mutations might fundamentally affect protein folding or stability .

• Enhanced DNA binding: The Q177R mutation (associated with Wilms tumors) increases DNA-binding affinity, leading to aberrant gene activation patterns .

These varied effects demonstrate that SIX1 mutations can disrupt transcriptional function through multiple mechanisms, explaining the diverse phenotypic outcomes observed in patients.

What methodologies are most effective for studying SIX1 protein-protein interactions?

Several complementary methodologies have proven effective for studying SIX1 protein-protein interactions:

• Co-immunoprecipitation assays:

  • Transfect cells with SIX1 mutant constructs and FLAG-tagged EYA proteins

  • Extract proteins under conditions that preserve native interactions

  • Immunoprecipitate with appropriate antibodies

  • Detect interactions via western blotting

• Nuclear/cytoplasmic fractionation:

  • Co-transfect SIX1 and FLAG-EYA1

  • Generate separate nuclear and cytoplasmic extracts

  • Analyze by western blotting to determine how SIX1 affects EYA1 localization

  • Probe with compartment-specific markers (HDAC1 for nucleus, α-actin for cytoplasm)

• Recombinant protein purification and binding assays:

  • Express SIX1 as GST fusion proteins in E. coli

  • Purify using glutathione-Sepharose resin

  • Cleave with PreScission protease

  • Further purify by ion exchange chromatography using RESOURCE S columns

  • Conduct in vitro binding assays with purified proteins

• Transcriptional reporter assays:

  • Measure functional interactions by assessing how cofactors influence SIX1-mediated transcription

  • Co-transfect SIX1, cofactors, and luciferase reporters

  • Quantify transcriptional activity as a readout of productive interaction

These methods provide complementary approaches to understanding SIX1's interaction network and how mutations disrupt specific protein-protein interactions important for developmental processes.

How does SIX1 contribute to kidney injury repair mechanisms?

Research has revealed that SIX1 is reactivated in tubular epithelial cells following ischemia/reperfusion (I/R) injury, where it plays multiple beneficial roles:

• Cell proliferation promotion:

  • SIX1 upregulates cyclin genes essential for cell cycle progression

  • Increases expression of glycolytic genes that support energy production for proliferation

• Enhanced cell migration:

  • Upregulates matrix metalloproteinase 9 (MMP9) expression either directly or indirectly

  • MMP9 facilitates cell movement necessary for repair processes

• Anti-inflammatory functions:

  • SIX1 targets promoters of amino-terminal enhancer of split (AES) and fused in sarcoma (FUS)

  • These proteins function as cofactors of nuclear factor-κB (NF-κB) subunit RELA

  • SIX1 inhibits the transactivation function of RELA

  • This reduces expression of monocyte chemotactic protein-1 (MCP-1)

  • Ultimately suppresses infiltration of monocytes/macrophages

Experimental overexpression of SIX1 in mouse kidneys resulted in:

• Significantly increased expression of cyclin and glycolytic genes

• Suppressed infiltration of monocytes/macrophages at 1, 2, and 3 days after reperfusion

• Reduced kidney damage from I/R injury

These findings suggest SIX1 may represent a potential therapeutic target for ameliorating kidney damage in acute kidney injury by simultaneously promoting repair and limiting inflammatory damage.

How do SIX1-Q177R mutations affect gene expression in Wilms tumors?

The SIX1-Q177R mutation, associated with relapsed Wilms tumors, fundamentally alters gene expression through enhanced DNA-binding affinity. Research characterizing this mutation has shown:

• Differential gene expression patterns:

  • SIX1/2-Q177R tumors display distinct transcriptional signatures

  • This has been confirmed through analyses of both chemotherapy-treated tumors and chemotherapy-naïve high-risk favorable histology Wilms tumors

• Shared binding targets with SIX2:

  • SIX1 and SIX2 share ~100% amino acid conservation in their DNA-binding homeodomain

  • Both proteins bind largely overlapping genomic sites

  • SIX2 targets ~90% of genes also targeted by SIX1 in human fetal kidney

• Potential interaction with miRNA processing:

  • Approximately 30% of SIX1/2-Q177R tumors harbor inactivating mutations in microRNA-processing genes

  • This suggests a complex interaction between altered SIX1/2 activity and dysregulated miRNA processing

• Developmental origin implications:

  • Aberrant gene expression programs linked to the Q177R mutation appear related to nephrogenic development

  • This aligns with evidence supporting the nephrogenic origin of Wilms tumors

The specific molecular consequences of enhanced SIX1 DNA binding include altered regulation of genes involved in nephrogenesis, potentially contributing to both tumor initiation and relapse mechanisms.

Data Table: Differential Effects of SIX1 Mutations on Functional Properties