ALKBH4 Antibody

What is ALKBH4 and what are its primary cellular functions?

ALKBH4 is a member of the AlkB family of non-heme Fe(II)/α-ketoglutarate-dependent dioxygenases, which function primarily in the repair of methylation damage in DNA and RNA . ALKBH4 has multiple cellular functions that researchers should be aware of when designing experiments. Specifically, ALKBH4 mediates demethylation of actin monomethylated at 'Lys-84' (K84me1), thereby regulating actomyosin processes critical for cytokinesis and cell migration . Beyond protein demethylation, ALKBH4 also demethylates DNA - specifically N(6)-methyladenosine DNA - which has implications for Polycomb silencing . Most recently, research has revealed ALKBH4's role in cancer metastasis, particularly as a suppressor in colorectal cancer through epigenetic mechanisms involving histone modification .

What specimens can be detected using ALKBH4 antibodies?

Based on validated research applications, ALKBH4 antibodies have demonstrated efficacy in detecting the protein in both human and mouse samples . When selecting an ALKBH4 antibody for your research, it's important to verify species reactivity. Commercial rabbit polyclonal antibodies against ALKBH4 have been validated for Western blotting (WB) and immunocytochemistry/immunofluorescence (ICC/IF) applications . When working with clinical specimens, researchers have successfully employed ALKBH4 antibodies for immunohistochemistry staining (IHC) on paraffin-embedded colorectal cancer tissues to assess protein expression levels in tumor versus non-tumor samples .

How should ALKBH4 protein function be interpreted in experimental contexts?

When interpreting ALKBH4 function in your experiments, it's critical to consider its dual roles in demethylation. ALKBH4 functions not only as a DNA/RNA demethylase but also as a protein demethylase with specific activity toward actin K84me1 . For proper experimental design, researchers should account for ALKBH4's interactions with transcription-associated proteins, which suggest its involvement in gene regulation . Additionally, the protein has emerged as an epigenetic modifier that decreases histone H3K4me3 modification by competitively binding to WDR5, a key component of histone methyltransferase complexes . This competitive binding mechanism results in altered histone modification patterns at specific gene promoters, including those involved in epithelial-mesenchymal transition (EMT) and metastasis .

What is the clinical significance of ALKBH4 expression in colorectal cancer?

ALKBH4 expression demonstrates significant clinical relevance in colorectal cancer (CRC) that researchers should consider when designing translational studies. Multiple lines of evidence indicate that ALKBH4 expression is markedly decreased in CRC tissues compared to paired adjacent non-tumor tissues . This decreased expression pattern has been verified through multiple methodologies, including real-time PCR, microarray analysis (GSE21510 dataset), and immunohistochemistry staining .

The clinical significance of ALKBH4 expression becomes particularly evident when analyzing its relationship with metastasis. CRC patients with distant metastasis exhibit lower mRNA expression of ALKBH4 than those without metastasis . Statistical analyses have revealed that ALKBH4 expression negatively correlates with America Joint Committee on Cancer (AJCC) stage, and Kaplan-Meier analysis demonstrates that CRC patients with low ALKBH4 expression have significantly shorter recurrence-free survival compared to those with high expression . These findings collectively establish ALKBH4 as a potential prognostic biomarker in CRC research.

How does ALKBH4 influence metastasis in experimental models?

ALKBH4's influence on metastasis has been experimentally validated through both in vitro and in vivo approaches that provide methodological frameworks for researchers. In vitro transwell invasion assays have demonstrated that knockdown of ALKBH4 significantly increases the invasion capability of CRC cell lines (HT29 and SW480), while overexpression of ALKBH4 inhibits the invasive ability of HCT116 cells .

What molecular mechanisms explain ALKBH4's tumor suppressive function?

The tumor suppressive function of ALKBH4 is mediated through complex epigenetic mechanisms that researchers should consider when designing mechanistic studies. ALKBH4 interacts with and competitively binds to WDR5, a key component of histone methyltransferase complexes responsible for H3K4me3 modification . This interaction has been confirmed through co-immunoprecipitation (Co-IP) assays in HCT116 cells .

The competitive binding results in decreased H3K4me3 histone modification at specific gene promoters. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) for WDR5 and H3K4me3 in ALKBH4-overexpressed cells revealed that 63 genes showed both decreased binding to WDR5 and reduced H3K4me3 modification . Gene Ontology (GO) analysis demonstrated that these genes are significantly associated with EMT and wound healing processes .

Specifically, ALKBH4 has been shown to inhibit miR-21 by decreasing H3K4me3 modification in its promoter region, with real-time PCR confirming reduced miR-21 expression following ALKBH4 overexpression . Clinical validation has shown a negative correlation between ALKBH4 and miR-21 expression in both the GSE21510 dataset and clinical cohorts . This epigenetic regulatory mechanism provides a molecular basis for ALKBH4's tumor suppressive function through inhibition of EMT and metastasis.

What protein-protein interactions have been identified for ALKBH4?

Yeast two-hybrid (Y2H) screens have identified multiple high-confidence protein interaction partners for ALKBH4, particularly those involved in transcription and chromatin modification . These interactions provide important mechanistic insights for researchers studying ALKBH4's cellular functions.

Notably, Y2H screens performed with an enzymatically inactive ALKBH4 mutant (ALKBH4 H169A/D171A) retrieved a similar set of proteins, indicating that these interactions occur independently of ALKBH4's oxygenase activity . The interaction domains identified in these screens consistently mapped to chromatin-associated domains in the partner proteins, reinforcing ALKBH4's role in transcriptional regulation and chromatin modification .

How does ALKBH4 regulate histone modifications?

ALKBH4 regulates histone modifications through a specific mechanism involving its interaction with WDR5, providing researchers with a model for investigating epigenetic regulation. Western blot assays have demonstrated that overexpression of ALKBH4 results in significant reduction of H3K4me3, but not H3K79me2 . Conversely, downregulation of ALKBH4 significantly increases H3K4me3 expression without affecting H3K79me2 .

The mechanistic basis for this selective effect on H3K4me3 involves ALKBH4's direct interaction with WDR5, a key component of histone methyltransferase complexes responsible for H3K4 trimethylation . This interaction has been experimentally verified through Co-IP assays, which demonstrated that WDR5 and ALKBH4 interact with each other in HCT116 cells . Genome-wide ChIP-seq analysis for WDR5 and H3K4me3 in ALKBH4-overexpressed cells revealed that ALKBH4 modulates WDR5 genomic binding and H3K4me3 modification at specific gene promoters .

The model emerging from these studies suggests that ALKBH4 competitively binds to WDR5, thereby decreasing H3K4me3 histone modification on target genes, particularly those involved in EMT and metastasis. This competitive binding mechanism represents a novel pathway through which ALKBH4 exerts its tumor suppressive function .

What is the relationship between ALKBH4 and miR-21 in gene regulation?

ALKBH4 specifically regulates miR-21 expression through epigenetic mechanisms, providing researchers with a model system for studying how demethylases influence gene expression. Real-time PCR analysis has demonstrated that overexpression of ALKBH4 decreases miR-21, a microRNA known to promote EMT and metastasis .

The molecular mechanism underlying this regulation involves ALKBH4-mediated changes in histone modifications at the miR-21 promoter. ChIP assays have shown that miR-21 can be detected in chromatin samples immunoprecipitated using antibodies against WDR5 or H3K4me3 . Importantly, overexpression of ALKBH4 reduces the binding efficiency of both WDR5 and H3K4me3 to the miR-21 promoter .

Clinical validation of this regulatory relationship has been obtained through correlation analyses in both the GSE21510 dataset and clinical cohorts, which revealed a negative correlation between ALKBH4 and miR-21 expression . Furthermore, miR-21 expression was found to be elevated in CRC tissues compared to adjacent non-tumor tissues, with higher expression in metastatic versus non-metastatic cases . This ALKBH4-miR-21 regulatory axis represents a specific example of how ALKBH4's epigenetic functions influence gene expression programs relevant to cancer progression.

What are the optimal techniques for detecting ALKBH4 expression in clinical samples?

Multiple complementary techniques have been validated for detecting ALKBH4 expression in clinical samples, providing researchers with methodological options based on their specific needs:

• Real-time PCR: For mRNA expression analysis, real-time PCR has been successfully applied to detect ALKBH4 expression differences between CRC tissues and paired adjacent non-tumor tissues . This method is particularly useful for quantitative assessment of expression levels.

• Immunohistochemistry (IHC): For protein-level detection, IHC staining on paraffin-embedded tissues has effectively demonstrated differences in ALKBH4 expression between tumor and non-tumor tissues . This method allows for spatial visualization of expression patterns within tissue architecture.

• Microarray analysis: Public datasets such as GSE21510 from the GEO database have been used to validate ALKBH4 expression patterns observed in primary samples . This approach allows for comparison with larger cohorts and integration with other molecular data.

When analyzing clinical samples, researchers should consider the relationship between ALKBH4 expression and clinicopathological parameters. Studies have demonstrated correlations between ALKBH4 expression levels and disease features including distant metastasis and AJCC stage . Additionally, survival analyses can reveal associations between ALKBH4 expression and clinical outcomes such as recurrence-free survival .

What experimental models are recommended for studying ALKBH4 function?

Both in vitro and in vivo experimental models have been validated for studying ALKBH4 function, providing researchers with established methodological frameworks:

In vitro models:

• Cell line manipulation: CRC cell lines including HT29, SW480, and HCT116 have been successfully used for ALKBH4 gain-of-function and loss-of-function studies .

• Functional assays: Transwell invasion assays have effectively demonstrated the impact of ALKBH4 expression on invasive capacity .

• Protein interaction studies: Yeast two-hybrid screens and co-immunoprecipitation assays have successfully identified and validated ALKBH4 binding partners .

In vivo models:

The complementary use of these models allows for comprehensive investigation of ALKBH4's functions from molecular interactions to physiological outcomes.

What techniques are effective for studying ALKBH4's epigenetic functions?

Several advanced molecular techniques have been validated for investigating ALKBH4's epigenetic functions, providing methodological guidance for researchers:

• Western blot analysis: This technique has effectively demonstrated changes in histone modification patterns (specifically H3K4me3 and H3K79me2) in response to ALKBH4 manipulation .

• Co-immunoprecipitation (Co-IP): Co-IP assays have successfully detected interactions between ALKBH4 and WDR5, providing evidence for direct protein-protein binding .

• Chromatin immunoprecipitation (ChIP): ChIP assays have been used to analyze the binding of WDR5 and H3K4me3 to specific gene promoters like miR-21 .

• ChIP-seq analysis: This genome-wide approach has identified genes exhibiting decreased WDR5 binding and reduced H3K4me3 modification following ALKBH4 overexpression . The analysis revealed 63 genes with both decreased WDR5 binding and reduced H3K4me3 modification.

• Gene Ontology (GO) analysis: GO analysis of ChIP-seq data has identified biological processes associated with genes regulated by ALKBH4, including EMT and wound healing .

These techniques collectively provide a comprehensive toolkit for investigating ALKBH4's epigenetic functions, from specific protein interactions to genome-wide effects on histone modifications and downstream gene regulation.

What factors should be considered when interpreting ALKBH4 expression data?

When interpreting ALKBH4 expression data, researchers should consider several factors that might influence results:

• Tissue heterogeneity: ALKBH4 expression varies between tumor and normal tissues, but also potentially within different regions of the same tumor . Microdissection techniques may be necessary for precise expression analysis.

• Stage-dependent expression: ALKBH4 expression negatively correlates with AJCC stage in CRC, suggesting that expression patterns may change during disease progression . Therefore, proper staging information is crucial for data interpretation.

• Relationship with metastasis: CRC patients with distant metastasis exhibit lower ALKBH4 expression than those without metastasis . This relationship should be considered when analyzing expression data in the context of disease progression.

• Multiple functions: ALKBH4 has diverse cellular functions, including DNA/RNA demethylation, protein demethylation, and histone modification regulation . Expression data should be interpreted with awareness of these multiple functions.

• Methodological considerations: Different detection methods (RNA versus protein) may yield different results due to post-transcriptional regulation. Both mRNA (RT-PCR) and protein (IHC) analyses are recommended for comprehensive assessment .

How can researchers effectively validate ALKBH4 antibody specificity?

Ensuring ALKBH4 antibody specificity is critical for obtaining reliable experimental results. Researchers should implement a multi-faceted validation approach:

• Genetic manipulation controls: Compare antibody staining in wildtype cells versus those with ALKBH4 knockdown or knockout. Reduction or absence of signal in ALKBH4-depleted cells provides strong evidence for specificity .

• Overexpression validation: Complementary to knockdown approaches, overexpression of ALKBH4 should result in increased signal detection. This approach is particularly valuable when using tagged versions of ALKBH4 that can be detected with tag-specific antibodies for cross-validation .

• Multiple antibody validation: When possible, use multiple antibodies targeting different epitopes of ALKBH4 and compare their staining patterns. Concordant results strengthen confidence in specificity.

• Immunoprecipitation followed by mass spectrometry: This approach can confirm that the antibody is indeed pulling down ALKBH4 rather than cross-reacting with other proteins.

• Peptide competition assays: Pre-incubation of the antibody with the peptide used for immunization should abolish specific staining if the antibody is truly specific.

These complementary approaches provide robust validation of antibody specificity, which is essential for reliable detection of ALKBH4 in various experimental contexts.