FANK1 Human

What is FANK1 and what are its key structural features?

FANK1 (fibronectin type III and ankyrin repeat domains 1) is an ancient, evolutionarily conserved gene present in vertebrates that encodes a nuclear protein. The protein contains fibronectin type III domains and ankyrin repeat domains, as reflected in its name. These structural motifs are important for protein-protein interactions and DNA binding activities. FANK1 belongs to the ankyrin repeat domain containing (ANKRD) and fibronectin type III domain containing protein families . In humans, the FANK1 gene has also been known by the synonym HSD13 .

What is the expression pattern of FANK1 in human tissues?

FANK1 exhibits a highly tissue-specific expression pattern. Bioinformatic and experimental analyses have revealed that FANK1 is exclusively expressed in the testis in both mice and humans . Within the testis, FANK1 mRNA expression is specifically detected during the transition from the meiotic to the haploid phase of spermatogenesis . This strict temporal and spatial expression pattern suggests a specialized role in male germ cell development. The GTEx (Genotype-Tissue Expression) database has been used to assess expression quantitative trait loci (eQTL) for FANK1 , further confirming its testis-specific expression pattern.

What is the subcellular localization and molecular function of FANK1?

FANK1 functions as a nuclear protein and DNA binding factor that specifically recognizes the DNA sequence "AAAAAG" . Its nuclear localization and DNA binding capabilities strongly implicate FANK1 as a transcription factor during spermatogenesis . The protein appears to be exclusively expressed during the transition from the meiotic to the haploid phase of spermatogenesis , suggesting specialized regulatory functions during this critical developmental window. Functionally, FANK1 has been implicated in suppressing apoptosis in male germ cells during development .

What methods have been used to study FANK1 function in vivo?

Researchers have employed several approaches to investigate FANK1 function:

• Gene Knockdown Models: Short-hairpin RNA (shRNA)-based knockdown transgenic mice have been developed to reduce FANK1 expression without altering the genomic sequence .

• Gene Knockout Models: CRISPR/Cas9-mediated gene editing has been used to generate complete FANK1 knockout mice. The process involved:

  • Design of sgRNAs targeting exon 2 of Fank1 (specific target sequences: 5′-GTGGCTTCGGTTCTCCATTGAGG-3′ and 5′-GTCACCTTGCCCACAACAGGAGG-3′)

  • In vitro transcription of Cas9 mRNA and sgRNAs

  • Microinjection into mouse zygotes

  • Genotyping using PCR and Sanger sequencing to identify founders and confirm deletions

• Protein Analysis: Western blotting with anti-FANK1 antibodies has been used to confirm protein knockout in experimental models .

• Reproductive Phenotyping: Assessment of fertility, sperm counts, and testicular histology to evaluate reproductive function in model organisms .

How can researchers effectively detect and quantify FANK1 expression?

For comprehensive analysis of FANK1 expression, researchers should employ multiple complementary techniques:

• mRNA Detection:

  • RT-PCR and qRT-PCR using primers specific to FANK1 transcripts

  • RNA-seq for unbiased transcriptome analysis and expression quantification

  • In situ hybridization to visualize tissue and cell-specific expression patterns

• Protein Detection:

  • Western blotting using anti-FANK1 antibodies (e.g., sc-398026 from Santa Cruz Biotechnology at 1:1,000 dilution)

  • Immunohistochemistry or immunofluorescence for tissue localization

  • Protein mass spectrometry for detection and quantification

• Expression Dynamics:

  • Stage-specific analysis during spermatogenesis using synchronized cell populations

  • Single-cell RNA-seq for heterogeneity analysis in testicular cell populations

When analyzing FANK1 expression, researchers should pay particular attention to testis-specific samples and consider developmental timing, as expression is highly restricted to specific stages of spermatogenesis .

What are the key methodological considerations when generating FANK1 knockout models?

When developing FANK1 knockout models, researchers should consider:

• Targeting Strategy:

  • Exon selection is critical; targeting exon 2 has proven effective

  • Design of multiple sgRNAs to increase editing efficiency

  • Verification of target site specificity to minimize off-target effects

• Validation Approaches:

  • Genotyping PCR with primers flanking the expected deletion (e.g., F: 5′-GGTCCACAGTTGTTGTTGCT-3′ and R: 5′-ATTCCAAGAGTCCATCGGTTCA-3′)

  • Sanger sequencing to confirm precise molecular alterations

  • Western blotting to verify complete protein loss

  • RT-PCR to check for potential cryptic splicing or compensatory transcripts

• Strain Considerations:

  • Backcrossing to eliminate potential off-target mutations

  • Generation of pure heterozygous mice before homozygous breeding

  • Consideration of genetic background effects on phenotype

• Control Selection:

  • Use of wild-type littermates as optimal controls

  • Inclusion of heterozygous animals to assess dosage effects

Researchers should be aware that different methodological approaches (knockdown vs. knockout) have yielded contradictory results for FANK1 function, necessitating careful experimental design and interpretation .

What evidence suggests FANK1 has been under balancing selection in humans?

Genomic analyses have revealed compelling evidence that FANK1 has been subject to balancing selection in humans:

• Composite Likelihood Ratio Tests: When applying sophisticated methods specifically designed to detect balancing selection to whole-genome human data, FANK1 emerged as one of the strongest candidates outside the HLA region .

• Polymorphism Patterns: The spatial distribution of polymorphisms in and around FANK1 shows signatures consistent with long-term balancing selection .

• Functional Implications: FANK1's role in suppressing apoptosis during meiosis in males, combined with its potential involvement in segregation distortion, provides a mechanistic basis for balancing selection. Researchers have hypothesized that balancing selection acts on this locus to stabilize segregation distortion and mitigate negative fitness effects of distorter alleles .

This evolutionary signature makes FANK1 particularly interesting from both functional and population genetics perspectives, suggesting that genetic variation at this locus has been actively maintained by selection.

How many SNPs have been associated with FANK1 expression variation in humans?

Research has identified 54 SNPs (Single Nucleotide Polymorphisms) associated with FANK1 expression in humans . These expression quantitative trait loci (eQTLs) were identified using data from the Genotype-Tissue Expression (GTEx) project, which provides RNA-seq and genotyping data from human samples .

The SNPs showing association with FANK1 expression represent potentially functionally relevant genetic variation that may contribute to differences in FANK1 activity or regulation across individuals. Researchers investigating FANK1 should consider these genetic variants when designing studies involving human populations, as they may influence experimental outcomes or explain inter-individual differences in FANK1-related phenotypes.

What is known about FANK1 conservation across species?

FANK1 represents an ancient, evolutionarily conserved gene that is present across vertebrate species . Its structural and functional conservation suggests fundamental biological importance. Comparative genomic analyses have revealed:

• Sequence Conservation: The DNA binding domain and key functional motifs show high sequence conservation, particularly the domains that recognize the specific DNA sequence "AAAAAG" .

• Expression Pattern Conservation: Both mouse and human FANK1 show testis-specific expression, particularly during the transition from meiotic to haploid phases of spermatogenesis .

• Functional Conservation: The role in male germ cell development appears to be conserved, though the specific reproductive phenotypes associated with gene disruption may vary between species .

This evolutionary conservation makes FANK1 a valuable target for comparative functional studies and suggests that insights gained from model organisms may have translational relevance to human reproductive biology.

How do researchers explain the contradictory phenotypes between FANK1 knockdown and knockout models?

The discrepancy between FANK1 knockdown and knockout models represents a significant research puzzle:

• Observed Phenotypic Differences:

  • Knockdown model: shRNA-based FANK1 reduction resulted in oligospermia caused by increased apoptotic germ cells .

  • Knockout model: Complete FANK1 deletion using CRISPR/Cas9 produced mice with normal fertility, sperm counts, and no significant changes in germ cell apoptosis .

• Potential Mechanistic Explanations:

  • Genetic Compensation: Knockout models may trigger compensatory upregulation of functionally related genes that doesn't occur in knockdown models. This genetic robustness can mask phenotypes in complete knockouts .

  • Off-Target Effects: shRNA-based knockdown approaches may cause unintended off-target effects that contribute to the observed phenotype .

  • Developmental Adaptation: Complete absence of FANK1 from early development may allow for more effective compensatory adaptations compared to post-developmental reduction of expression.

  • Partial vs. Complete Loss: Knockdown models retain some residual FANK1 expression which may disrupt normal function in ways different from complete absence.

• Molecular Evidence:

  • Different patterns of Dusp1, Klk1b21, and Klk1b27 mRNA expression were detected between knockdown and knockout models, suggesting distinct molecular responses to these different gene targeting approaches .

Researchers investigating FANK1 should consider both models and, ideally, employ complementary approaches to fully understand the gene's function.

What is the current understanding of FANK1's role in apoptosis regulation during spermatogenesis?

FANK1 has been identified as a gene that suppresses apoptosis during spermatogenesis, particularly during meiosis in males . The current understanding of this function includes:

• Observational Evidence:

  • shRNA-based knockdown of FANK1 results in increased apoptotic germ cells , suggesting an anti-apoptotic role.

  • The protein's expression pattern coincides with critical periods of apoptotic regulation during male germ cell development .

• Molecular Mechanisms:

  • As a DNA binding protein recognizing the "AAAAAG" sequence , FANK1 likely influences transcription of genes involved in apoptotic pathways.

  • Its nuclear localization further supports a direct role in transcriptional regulation of apoptosis-related genes .

• Contradictory Evidence:

  • Complete knockout of FANK1 does not significantly increase apoptotic cell numbers in the testis , challenging the simplistic view of FANK1 as an essential apoptosis suppressor.

• Research Implications:

  • The contradictory evidence suggests context-dependent functions or compensatory mechanisms that require further investigation.

  • Studies of FANK1's interaction partners and transcriptional targets are needed to clarify its precise role in apoptotic regulation.

Understanding these nuances is crucial for researchers designing experiments to investigate FANK1's role in germ cell survival and male fertility.

What evidence supports FANK1's potential role in segregation distortion?

The hypothesis that FANK1 may be involved in segregation distortion is supported by several lines of evidence:

• Evolutionary Signatures: Genomic analyses have identified FANK1 as a locus under balancing selection, which is consistent with genes involved in segregation distortion. Researchers have hypothesized that balancing selection acts on this locus to stabilize segregation distortion and negative fitness effects of distorter alleles .

• Expression Pattern: FANK1's specific expression during meiosis in males coincides with the cellular stage where segregation distortion would manifest, as this is when genetic elements compete for transmission to the next generation.

• Functional Characteristics: As a DNA binding protein that recognizes specific sequences , FANK1 could potentially influence chromosome dynamics or gene expression patterns relevant to meiotic drive systems.

• Marginal Signs: Researchers have noted "marginal signs of segregation distortion" associated with FANK1 , though detailed molecular and genetic evidence is still emerging.

This hypothesis represents an intriguing area for future research, potentially connecting FANK1's molecular function to broader evolutionary dynamics in mammalian genomes.

What research approaches might resolve the contradictions in FANK1 functional studies?

To address the conflicting results between knockdown and knockout models of FANK1, researchers should consider:

• Comparative Transcriptomic Analysis:

  • RNA-seq of testicular tissue from wild-type, knockdown, and knockout models at multiple developmental stages

  • Single-cell RNA-seq to identify cell type-specific responses and potential compensatory mechanisms

  • Analysis focused on apoptosis-related genes and potential compensatory pathways

• Conditional and Inducible Systems:

  • Generation of conditional knockout models allowing for stage-specific FANK1 deletion

  • Inducible systems to eliminate developmental compensation effects

  • Tissue-specific rescue experiments in knockout backgrounds

• Molecular Interaction Studies:

  • Identification of FANK1 protein interaction partners using techniques like IP-MS

  • ChIP-seq to map genomic binding sites and regulated genes

  • Proteomic analysis to identify post-translational modifications affecting function

• Combined Approach Models:

  • Development of models with both genomic modification and expression modulation

  • Titrated expression systems to determine threshold effects

  • Heterozygous analysis to assess dosage sensitivity

• Cross-Species Validation:

  • Comparative studies across multiple model organisms

  • Humanized mouse models expressing human FANK1 variants

These multifaceted approaches would provide deeper insights into FANK1's true biological functions and help resolve current contradictions in the literature.

How might researchers further investigate FANK1's potential role in segregation distortion?

To explore FANK1's hypothesized role in segregation distortion, researchers should consider these advanced approaches:

• Transmission Ratio Analysis:

  • Large-scale breeding studies with FANK1 variant carriers to detect non-Mendelian inheritance patterns

  • Single-sperm genotyping to directly assess meiotic drive effects

  • Quantitative analysis of allele frequencies in sperm populations

• Meiotic Investigation:

  • Cytogenetic analysis of meiotic cells in FANK1 variant carriers

  • Chromosome segregation imaging using fluorescent tags

  • Assessment of synapsis, recombination, and checkpoint control in the presence of FANK1 variants

• Molecular Mechanism Studies:

  • Identification of FANK1-interacting chromatin regions during meiosis

  • Analysis of DNA binding specificity to sequences near centromeres or other segregation-relevant regions

  • Investigation of potential interactions with known segregation distortion systems

• Population Genetic Approaches:

  • Analysis of FANK1 haplotype structure and frequency across diverse human populations

  • Assessment of linkage disequilibrium patterns around FANK1

  • Identification of signatures of selective sweeps or balancing selection

• Experimental Evolution:

  • Long-term selection experiments in model organisms with FANK1 variants

  • Tracking of allele frequency changes across generations

  • Assessment of fitness consequences in different genetic backgrounds

These investigations would provide crucial insights into whether and how FANK1 influences genetic transmission and contributes to evolutionary dynamics in mammalian genomes.

What technologies and approaches are most promising for future FANK1 research?

Emerging technologies and methodological approaches that hold particular promise for advancing FANK1 research include:

• Advanced Genomic Engineering:

  • Base editing and prime editing for precise introduction of specific FANK1 variants

  • Epigenome editing to modulate FANK1 expression without altering sequence

  • Large-scale CRISPR screens of potential FANK1 interaction partners

• Single-Cell Multi-Omics:

  • Integrated single-cell RNA-seq, ATAC-seq, and proteomics to map FANK1's role in cellular regulatory networks

  • Spatial transcriptomics to understand FANK1 expression in the architectural context of the testis

  • Single-cell lineage tracing to track developmental consequences of FANK1 variation

• Advanced Imaging:

  • Live-cell imaging of FANK1-tagged proteins during spermatogenesis

  • Super-resolution microscopy to visualize nuclear localization and chromatin interactions

  • Intravital imaging to observe cellular dynamics in intact tissues

• Computational Approaches:

  • Machine learning analysis of large-scale genomic datasets to identify FANK1-associated patterns

  • Molecular dynamics simulations of FANK1 protein interactions

  • Network analysis to position FANK1 within broader regulatory systems

• Translational Methods:

  • Development of human testicular organoids to study FANK1 in a physiologically relevant context

  • Patient-derived cellular models from individuals with FANK1 variants

  • Population-scale studies connecting FANK1 variants to reproductive outcomes

By leveraging these advanced technologies and approaches, researchers can develop a more comprehensive understanding of FANK1's fundamental biology and potential clinical relevance to human reproductive health.