ANKRD1 Human

What is ANKRD1 and what are its main functions in human tissues?

ANKRD1 (Ankyrin Repeat Domain 1) is a member of the muscle ankyrin repeat protein family that functions as a cardiac-specific stress-response protein with pleiotropic roles in transcriptional regulation, sarcomere assembly, and mechano-sensing in the heart . The protein exhibits dual subcellular localization, functioning in the nucleus as a transcriptional co-regulator and in the cytoplasm as part of the sarcomeric structure .

In cardiac tissue, ANKRD1 acts as a negative transcriptional regulator during cardiogenesis and becomes highly upregulated during pathological cardiac remodeling and heart failure . The expression of ANKRD1 is high in the early embryonic heart but is downregulated to relatively lower levels in the adult heart .

Beyond its cardiac functions, ANKRD1 has been identified as a mesenchymal-specific transcriptional coregulator in dermal fibroblasts where it plays a key role in driving cancer-associated fibroblast (CAF) conversion . In cancer contexts, ANKRD1 expression in CAFs is associated with poor survival in head and neck squamous cell carcinoma (HNSCC), lung, and cervical SCC patients .

For studying ANKRD1's functions, researchers typically employ RNA sequencing to analyze expression patterns, co-immunoprecipitation to identify protein interactions, and loss-of-function/gain-of-function experiments using siRNA knockdown or overexpression systems.

How is ANKRD1 expression regulated during development and in adult tissues?

ANKRD1 exhibits a distinct developmental expression profile, with high expression in the early embryonic heart followed by downregulation to relatively lower levels in the adult heart . This pattern suggests an important role in cardiogenesis where it has been implicated as a negative transcriptional regulator of cardiac gene expression . Similarly, ANKRD1 is expressed in fetal skeletal muscle but becomes barely detectable in adult skeletal muscle .

In adult tissues, ANKRD1 expression is normally maintained at low levels but becomes rapidly and highly upregulated in response to hypertrophic stimuli and during heart failure, suggesting its importance in pathological cardiac remodeling . ANKRD1 is also under direct negative control by the androgen receptor (AR) in human dermal fibroblasts (HDFs) .

To investigate the developmental regulation of ANKRD1, researchers should consider:

• Developmental time-course studies using embryonic and postnatal tissue samples

• Chromatin immunoprecipitation (ChIP) assays to identify transcription factors binding to the ANKRD1 promoter

• Reporter gene assays to study the activity of the ANKRD1 promoter under various conditions

• Analysis of tissue-specific expression using RT-qPCR, western blotting, and immunohistochemistry

Understanding the regulatory mechanisms controlling ANKRD1 expression is crucial for interpreting its role in both developmental processes and disease states.

What is the subcellular localization of ANKRD1 and how does it relate to its function?

ANKRD1 displays dual subcellular localization, being present in both the nucleus and the cytoplasm of cells, with each localization associated with distinct functions:

• Nuclear localization: In the nucleus, ANKRD1 functions as a transcriptional co-regulator, interacting with various transcription factors to modulate gene expression . ANKRD1 possesses two predicted nuclear localization signals (NLS) that facilitate its active transport into the nucleus .

• Cytoplasmic/Sarcomeric localization: In cardiomyocytes, ANKRD1 localizes to the sarcomere where it interacts with sarcomeric proteins to maintain structural organization . Within the sarcomere, ANKRD1 is part of the titin-N2A mechanosensory complex .

Importantly, ANKRD1 can shuttle between these compartments in response to stimuli. Mechanical stretch can trigger the translocation of sarcomeric ANKRD1 to the nucleus, suggesting it functions as part of a stretch-sensing unit capable of relaying biomechanical stress signals to regulate gene expression .

To study ANKRD1's subcellular localization, researchers should employ:

• Immunofluorescence microscopy with compartment-specific markers

• Subcellular fractionation followed by western blotting

• Live-cell imaging using fluorescently tagged ANKRD1

• Mutation of NLS sequences to determine the impact on localization and function

Understanding this dynamic localization is essential for characterizing ANKRD1's role in mechanotransduction and transcriptional regulation in both normal and pathological conditions.

What are the structural characteristics of the ANKRD1 protein?

The ANKRD1 gene in humans is located on chromosome 10 and encodes a protein consisting of 319 amino acids with a theoretical molecular weight of approximately 36,252 Da . The protein contains multiple structural domains that contribute to its various functions:

• Ankyrin repeat domains: As the name suggests, ANKRD1 contains ankyrin repeat motifs, which are common protein-protein interaction domains . These domains are critical for ANKRD1's interaction with multiple binding partners.

• Nuclear localization signals (NLS): ANKRD1 possesses two predicted NLS sequences, which facilitate its active transport into the nucleus, enabling its function as a transcriptional co-regulator .

• Nuclear export signal: A predicted nuclear export signal is located within the second ankyrin repeat domain, suggesting regulated nuclear-cytoplasmic shuttling .

For structural studies of ANKRD1, researchers typically employ:

• X-ray crystallography or NMR spectroscopy to determine three-dimensional structure

• Domain deletion and mutation studies to assess the functional importance of specific regions

• Protein-protein interaction assays to map binding interfaces

• In silico structural prediction tools to model protein domains

Understanding the structural basis of ANKRD1's interactions is crucial for developing targeted approaches to modulate its activity in disease contexts.

What are the known interacting partners of ANKRD1?

ANKRD1 interacts with a diverse array of proteins, reflecting its pleiotropic functions in different cellular compartments. Key interacting partners include:

• Transcription factors:

  • Y-Box Binding Protein 1 (YB-1): ANKRD1 acts as a negative transcriptional co-factor of YB-1, potentially by sequestering it to regulate ventricular-specific myosin light chain-2 (MLC2v) gene expression .

  • AP-1 transcription factors: ANKRD1 binds to AP-1 family members, including c-Jun, and promotes their association at regulatory regions of myofibroblast CAF effector genes .

  • Tumor suppressor p53: Unlike its repressive role with other transcription factors, ANKRD1 acts as a positive transcriptional co-activator of p53, moderately up-regulating its activity and downstream expression of p21, Mdm2, and ANKRD2 .

  • Other transcription factors: ANKRD1 has been reported to interact with at least 16 other transcription factors, including HDAC1 and Smad3 .

• Sarcomeric proteins:

  • Myopalladin: The interaction between ANKRD1 and myopalladin is essential for maintaining sarcomeric structure .

  • Titin: ANKRD1 interacts with the N2A domain of titin as part of the mechanosensory complex .

• Other binding partners:

  • Talin 1 and FHL2: Mutations in ANKRD1 (P105S and M184I) result in loss of binding to these proteins, which may contribute to dilated cardiomyopathy development .

To study protein-protein interactions involving ANKRD1, researchers commonly use:

• Co-immunoprecipitation assays

• Yeast two-hybrid screening

• GST pulldown assays

• Proximity ligation assays

• Bimolecular fluorescence complementation (BiFC)

Understanding these interactions provides insight into ANKRD1's role in transcriptional regulation, sarcomere organization, and mechanosensing, and may reveal potential therapeutic targets for associated diseases.

What methodologies are most effective for investigating ANKRD1's role in cancer-associated fibroblast transformation?

Investigating ANKRD1's role in cancer-associated fibroblast (CAF) transformation requires a multifaceted approach combining molecular, cellular, and in vivo techniques. Based on recent research, the following methodologies are recommended:

• Primary fibroblast isolation and CAF conversion models:

  • Establish primary cultures of normal human dermal fibroblasts (HDFs)

  • Develop co-culture systems with cancer cells or condition fibroblasts with tumor-derived factors to induce CAF phenotypes

  • Monitor changes in ANKRD1 expression during CAF transformation using RT-qPCR and western blotting

  • Compare ANKRD1 levels between normal fibroblasts and patient-derived CAFs

• Genetic manipulation approaches:

  • Use siRNA or shRNA for ANKRD1 knockdown in established CAFs to assess reversal of phenotype

  • Employ CRISPR/Cas9 genome editing to generate ANKRD1-knockout fibroblast lines

  • Conduct ANKRD1 overexpression studies in normal fibroblasts to determine if this drives CAF-like features

  • Create inducible expression systems to study temporal aspects of ANKRD1 function

• Mechanistic studies:

  • Perform ChIP-seq analysis to identify genomic binding sites of ANKRD1 in CAFs

  • Use Co-IP and proximity ligation assays to confirm ANKRD1's interaction with AP-1 transcription factors

  • Conduct reporter gene assays to assess ANKRD1's impact on the expression of CAF marker genes

  • Analyze downstream signaling pathways affected by ANKRD1 modulation

• Functional assays:

  • Employ 3D organotypic culture systems to assess how ANKRD1 manipulation affects interactions between fibroblasts and cancer cells

  • Perform extracellular matrix (ECM) remodeling assays to evaluate how ANKRD1 influences ECM production and organization

  • Conduct migration and invasion assays to determine the impact of ANKRD1 on CAF motility

• In vivo models:

  • Develop orthotopic xenograft models using cancer cells co-injected with ANKRD1-manipulated fibroblasts

  • Create fibroblast-specific ANKRD1 knockout or overexpression mouse models

  • Analyze tumor growth, metastasis, and immune infiltration in these models

  • Perform ex vivo analyses of tumor stroma to assess CAF phenotypes

These methodologies collectively provide a comprehensive framework for investigating ANKRD1's role in CAF transformation and its potential as a therapeutic target in cancer.

How can researchers differentiate between the nuclear and cytoplasmic functions of ANKRD1?

Distinguishing between the nuclear and cytoplasmic functions of ANKRD1 requires specialized techniques that can separate and analyze these distinct roles. Here are recommended methodological approaches:

• Compartment-specific mutant construction:

  • Generate ANKRD1 mutants with modified nuclear localization signals (NLS) to restrict the protein to the cytoplasm

  • Create nuclear export signal (NES) mutants to concentrate ANKRD1 in the nucleus

  • Develop fusion proteins with compartment-specific targeting sequences

  • Validate localization patterns using immunofluorescence microscopy

• Protein-protein interaction analysis by compartment:

  • Perform subcellular fractionation followed by co-immunoprecipitation to identify location-specific interaction partners

  • Use BioID or APEX2 proximity labeling techniques fused to compartment-restricted ANKRD1 variants

  • Conduct yeast two-hybrid screens with domains involved in specific compartmental functions

  • Apply fluorescence resonance energy transfer (FRET) microscopy to visualize interactions in living cells

• Transcriptional regulation studies:

  • Perform ChIP-seq to identify genomic binding sites of nuclear ANKRD1

  • Conduct RNA-seq after expression of nuclear-restricted versus cytoplasmic-restricted ANKRD1

  • Use reporter gene assays to assess the impact of compartment-specific mutants on target gene expression

  • Analyze the effect of ANKRD1 subcellular localization on chromatin accessibility using ATAC-seq

• Sarcomeric function analysis:

  • Assess sarcomere organization using super-resolution microscopy in cells expressing cytoplasmic-restricted ANKRD1

  • Perform live-cell imaging to track dynamics of sarcomeric proteins in the presence of different ANKRD1 variants

  • Use atomic force microscopy to measure mechanical properties of sarcomeres with modified ANKRD1 localization

  • Conduct protein turnover studies to determine how cytoplasmic ANKRD1 affects sarcomere protein stability

• Mechanosensing studies:

  • Apply controlled mechanical stretch to cardiomyocytes expressing localization-specific ANKRD1 mutants

  • Monitor translocation of wild-type ANKRD1 versus localization mutants in response to mechanical stimuli

  • Assess downstream gene expression changes after mechanical stimulation with different ANKRD1 variants

  • Measure calcium handling and contractile responses in cells with altered ANKRD1 compartmentalization

What are the current approaches for studying ANKRD1 mutations in dilated cardiomyopathy?

Investigating ANKRD1 mutations in dilated cardiomyopathy (DCM) requires an integrated approach spanning genetic screening, functional characterization, and disease modeling. Here are the current methodological approaches:

• Genetic screening and variant identification:

  • Targeted sequencing of the ANKRD1 coding region in DCM patient cohorts

  • Whole exome or genome sequencing to identify novel ANKRD1 variants

  • Use of denaturing high-performance liquid chromatography and direct DNA sequencing as described in the literature

  • Bioinformatic prediction of variant pathogenicity using tools like SIFT, PolyPhen-2, and CADD

  • Population frequency analysis using databases such as gnomAD to identify rare variants

• Functional characterization of ANKRD1 mutations:

  • Yeast two-hybrid assays to assess the impact of mutations on protein-protein interactions

  • Co-immunoprecipitation studies to confirm altered binding to known partners like Talin 1 and FHL2

  • GST pulldown assays to quantify changes in binding affinity

  • Immunofluorescence microscopy to determine if mutations affect intracellular localization

  • Luciferase reporter assays to measure effects on transcriptional regulation

• Cellular models:

  • Expression of mutant ANKRD1 in primary cardiomyocytes to assess effects on sarcomere structure

  • siRNA knockdown of endogenous ANKRD1 followed by expression of mutant versions

  • Analysis of stretch-induced gene expression in myoblastoid cell lines expressing mutant ANKRD1

  • Assessment of mechanosensing capabilities using stretch chambers or micropost arrays

  • Calcium handling studies to determine functional consequences of mutations

• Advanced disease models:

  • Generation of patient-specific induced pluripotent stem cells (iPSCs) and differentiation into cardiomyocytes

  • CRISPR/Cas9 gene editing to introduce or correct specific ANKRD1 mutations in iPSCs

  • Development of engineered heart tissues (EHTs) to assess contractile properties

  • Creation of transgenic mouse models expressing human ANKRD1 mutations

  • Cardiac-specific expression of mutant ANKRD1 using AAV-mediated gene delivery

These approaches collectively enable researchers to understand how ANKRD1 mutations contribute to DCM pathogenesis and potentially identify novel therapeutic strategies for affected patients.

How should researchers design experiments to investigate ANKRD1's mechanosensing capabilities?

Investigating ANKRD1's mechanosensing capabilities requires specialized techniques that can apply controlled mechanical stimuli and monitor subsequent molecular responses. Here are recommended experimental designs:

• In vitro mechanical stimulation systems:

  • Uniaxial stretch chambers: Culture cardiomyocytes or fibroblasts on elastic membranes and apply controlled cyclic stretch

  • Micropost arrays: Seed cells on micropillar substrates to measure traction forces and cellular responses

  • Atomic force microscopy (AFM): Apply precise localized forces to cells and measure immediate responses

  • Magnetic tweezers: Attach magnetic beads to specific cellular structures and apply controlled forces

  • Hydrogels with tunable stiffness: Culture cells on substrates with defined mechanical properties to study how matrix rigidity affects ANKRD1 function

• Real-time monitoring of ANKRD1 translocation:

  • Live-cell imaging using fluorescently tagged ANKRD1 (e.g., GFP-ANKRD1 fusion protein)

  • FRAP (Fluorescence Recovery After Photobleaching) analysis to study dynamics of ANKRD1 movement

  • Development of FRET-based biosensors to detect conformational changes in ANKRD1 upon mechanical stimulation

  • Super-resolution microscopy to visualize ANKRD1 integration into mechanosensory complexes

• Molecular interaction studies under mechanical loading:

  • Proximity ligation assays to detect in situ interactions between ANKRD1 and binding partners during stretch

  • Co-immunoprecipitation experiments from stretched versus non-stretched cells

  • ChIP assays following mechanical stimulation to identify stretch-responsive genomic binding sites

  • Analysis of posttranslational modifications of ANKRD1 in response to mechanical stress

  • Mass spectrometry-based interactome analysis under various mechanical conditions

• Genetic manipulation approaches:

  • CRISPR/Cas9-mediated knockout of ANKRD1 followed by mechanical stimulation

  • Expression of domain-specific mutants to identify regions critical for mechanosensing

  • siRNA-mediated knockdown of potential mechanosensory complex partners

  • Rescue experiments with wild-type versus mutant ANKRD1 in knockout backgrounds

  • Generation of inducible expression systems to control ANKRD1 levels during mechanical testing

• Downstream signaling and gene expression analysis:

  • RNA-seq to identify mechano-responsive genes regulated by ANKRD1

  • Phosphoproteomics to map signaling pathways activated by mechanical stimulation in the presence/absence of ANKRD1

  • Real-time PCR arrays focused on mechanosensitive genes following ANKRD1 manipulation

  • Reporter gene assays using mechano-responsive promoters

By implementing these experimental approaches, researchers can comprehensively characterize ANKRD1's role in mechanosensing and elucidate how this function contributes to both normal physiology and disease states.

What techniques are recommended for analyzing ANKRD1's transcriptional regulatory functions?

Investigating ANKRD1's role as a transcriptional co-regulator requires specialized techniques to identify target genes, characterize protein-DNA interactions, and assess functional outcomes. Here are recommended methodological approaches:

• Genome-wide binding site identification:

  • Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) to map ANKRD1 binding sites across the genome

  • CUT&RUN or CUT&Tag as alternatives to traditional ChIP with potentially improved signal-to-noise ratio

  • ChIP-exo or ChIP-nexus for higher resolution mapping of binding sites

  • Re-ChIP (sequential ChIP) to identify genomic loci where ANKRD1 co-localizes with interacting transcription factors

  • Assay for Transposase-Accessible Chromatin (ATAC-seq) to correlate ANKRD1 binding with chromatin accessibility

• Transcriptional impact assessment:

  • RNA-seq following ANKRD1 overexpression, knockdown, or knockout to identify regulated genes

  • Nascent RNA sequencing (e.g., PRO-seq, GRO-seq) to capture immediate transcriptional responses

  • Single-cell RNA-seq to understand cell-to-cell variability in ANKRD1-mediated gene regulation

  • Temporal RNA-seq to track dynamic gene expression changes after modulating ANKRD1 levels

• Protein-protein interaction characterization:

  • Co-immunoprecipitation followed by mass spectrometry to identify novel transcriptional partners

  • Proximity labeling methods (BioID, APEX) to capture transient interactions in the nuclear compartment

  • Microscopy-based approaches (FRET, FLIM) to visualize interactions with transcription factors in living cells

  • Sequential ChIP to determine co-occupancy of ANKRD1 with other factors at specific genomic loci

  • Protein fragment complementation assays to validate direct interactions

• Functional reporter systems:

  • Luciferase reporter assays with promoters/enhancers of identified target genes

  • CRISPR activation (CRISPRa) or interference (CRISPRi) targeting ANKRD1-bound regulatory elements

  • Enhancer-promoter interaction analysis using chromosome conformation capture techniques (3C, 4C, Hi-C)

  • Analysis of how ANKRD1 affects the expression of its target genes in disease versus normal conditions

• Context-specific regulation studies:

  • Analysis of ANKRD1's transcriptional function under various stimuli (mechanical stretch, cytokines, growth factors)

  • Comparison of ANKRD1-regulated genes across different cell types (cardiomyocytes vs. fibroblasts)

  • Examination of target gene regulation in disease models vs. normal conditions

  • Investigation of how ANKRD1 mutations affect transcriptional regulatory capabilities

By systematically applying these techniques, researchers can develop a comprehensive understanding of ANKRD1's role as a transcriptional regulator in both normal physiology and disease states.

How does ANKRD1's interaction with AP-1 transcription factors influence gene expression in cancer models?

Investigating the functional consequences of ANKRD1's interaction with AP-1 transcription factors in cancer contexts requires a multifaceted approach. Here's a systematic research strategy:

• Molecular characterization of the ANKRD1-AP-1 interaction:

  • Co-immunoprecipitation assays to confirm binding between ANKRD1 and specific AP-1 family members (c-Jun, c-Fos) in cancer-associated fibroblasts (CAFs)

  • Domain mapping studies to identify the specific regions of ANKRD1 that interact with AP-1 factors

  • In vitro binding assays with purified recombinant proteins to determine binding affinities

  • FRET or BiFC assays to visualize the interaction in living cells

• Genome-wide binding pattern analysis:

  • ChIP-seq for both ANKRD1 and AP-1 factors to identify co-occupied genomic regions

  • Sequential ChIP to confirm simultaneous binding of both factors to the same DNA regions

  • ATAC-seq to correlate binding with changes in chromatin accessibility

  • Motif analysis to characterize the DNA sequence preferences of ANKRD1-AP-1 complexes

• Transcriptional outcome assessment:

  • RNA-seq following manipulation of ANKRD1 levels to identify genes regulated by the ANKRD1-AP-1 complex

  • Comparison of gene expression changes after knockdown of ANKRD1 versus AP-1 factors

  • Luciferase reporter assays with promoters containing AP-1 binding sites, with and without ANKRD1

  • Assessment of RNA polymerase II recruitment and phosphorylation status at target genes

• Mechanistic studies in cancer models:

  • Generation of ANKRD1 mutants unable to interact with AP-1 factors but retaining other functions

  • Expression of these mutants in CAFs to determine the specific contribution of the AP-1 interaction

  • Analysis of how the ANKRD1-AP-1 interaction affects the pro-tumorigenic properties of CAFs

  • Investigation of whether ANKRD1 alters AP-1's interaction with other co-factors or the basal transcriptional machinery

• In vivo validation studies:

  • Orthotopic tumor models using cancer cells co-injected with CAFs expressing wild-type or mutant ANKRD1

  • Analysis of tumor growth, invasion, angiogenesis, and metastasis

  • Immunohistochemical assessment of AP-1 target gene expression in tumor stroma

  • Correlation of ANKRD1 and AP-1 factor expression in human cancer specimens

  • Evaluation of patient outcomes based on ANKRD1-AP-1 signature in tumor stroma

This research framework provides a comprehensive approach to understanding how ANKRD1's interaction with AP-1 transcription factors drives gene expression programs that contribute to cancer progression.

What are the recommended protocols for studying ANKRD1's role in sarcomere organization?

Investigating ANKRD1's function in sarcomere organization requires specialized techniques spanning from molecular interactions to structural integrity assessment. Here are recommended protocols and methodological approaches:

• Visualization and quantification of sarcomere structure:

  • Immunofluorescence microscopy using antibodies against ANKRD1 and sarcomeric proteins (α-actinin, titin, myosin, etc.)

  • Super-resolution microscopy techniques (STED, STORM, SIM) for nanoscale visualization of ANKRD1 localization

  • Transmission electron microscopy to assess ultrastructural changes in sarcomere organization

  • Live-cell imaging using fluorescently tagged sarcomeric proteins to monitor dynamics

  • Quantitative image analysis to measure sarcomere length, width, and organization patterns

• Genetic manipulation approaches:

  • siRNA or shRNA knockdown of ANKRD1 in primary cardiomyocytes to assess impact on sarcomere integrity

  • CRISPR/Cas9-mediated knockout of ANKRD1 in cell lines or primary cells

  • Rescue experiments expressing wild-type or mutant ANKRD1 in knockdown/knockout backgrounds

  • Overexpression of ANKRD1 to determine dose-dependent effects on sarcomere structure

  • Domain-specific mutants to identify regions critical for sarcomere organization

• Protein-protein interaction studies:

  • Co-immunoprecipitation of ANKRD1 with sarcomeric binding partners (myopalladin, titin)

  • Proximity ligation assays to visualize and quantify interactions in situ

  • FRET-based approaches to monitor dynamic interactions in living cells

  • In vitro binding assays with purified proteins to determine direct interactions and binding affinities

  • Cross-linking mass spectrometry to map interaction interfaces at amino acid resolution

• Functional assessment of sarcomere integrity:

  • Atomic force microscopy to measure mechanical properties of sarcomeres with modified ANKRD1 levels

  • Traction force microscopy to evaluate force generation and transmission

  • Calcium transient measurements to assess excitation-contraction coupling

  • Video-based edge detection to quantify contractile function in isolated cardiomyocytes

  • Force measurements in engineered heart tissues with controlled ANKRD1 expression

• Disease model applications:

  • Introduction of DCM-associated ANKRD1 mutations to assess effects on sarcomere structure

  • Mechanical stretch experiments to model pathological loading conditions

  • Engineered heart tissues with ANKRD1 modifications subjected to various stressors

  • Patient-derived iPSC-cardiomyocytes harboring ANKRD1 mutations

  • Analysis of sarcomere organization in myocardial samples from disease models

By systematically applying these techniques, researchers can comprehensively characterize ANKRD1's role in maintaining sarcomere structural integrity and understand how perturbations in this function contribute to cardiomyopathies and heart failure.