GFI1B Antibody

What is GFI1B and why is it important in hematopoiesis?

GFI1B (Growth Factor Independence 1B) is an essential proto-oncogenic transcriptional regulator necessary for the development and differentiation of erythroid and megakaryocytic lineages. It functions as a component of the RCOR-GFI-KDM1A-HDAC complex that suppresses genes involved in multilineage blood cell development through histone deacetylase recruitment . GFI1B can act as both a transcriptional repressor and activator depending on promoter and cell type context. For instance, it represses promoter activity of SOCS1 and SOCS3, potentially regulating cytokine signaling pathways .

GFI1B's importance in hematopoiesis stems from its role in controlling the balance between cell proliferation, differentiation, and apoptosis. It cooperates with GATA1 to repress target gene transcription, such as the apoptosis regulator BCL2L1, and GFI1B silencing in leukemic cell lines significantly increases apoptosis rate . Recent studies have demonstrated that GFI1B facilitates megakaryopoiesis by driving megakaryocyte-specific regulons while simultaneously repressing myeloid regulons .

What are the expression patterns of GFI1B across hematopoietic lineages?

GFI1B exhibits a highly specific expression pattern within the hematopoietic system:

GFI1B expression levels decrease with erythroid cell differentiation, as confirmed by both RNA analysis and protein detection via western blotting . Notably, GFI1B shows a complementary expression pattern to its paralog GFI1, with granulocytes expressing high levels of GFI1 and no GFI1B, while erythroid precursors and megakaryocytes express highest levels of GFI1B with minimal GFI1 .

How does GFI1B regulate its own expression and that of related factors?

GFI1B exhibits a fascinating auto-regulatory mechanism whereby it can repress its own expression . Chromatin-immunoprecipitation experiments have demonstrated that both the GFI1B and GFI1 promoters are occupied by GFI1B in vivo . This autoregulation involves direct binding of GFI1B to potential binding sites in its own promoter, as demonstrated by gel-shift analyses in vitro .

Additionally, GFI1B can cross-repress the expression of the related transcription factor GFI1, likely in a cell type-specific manner . This creates a complex regulatory network where both GFI1 and GFI1B are not only under auto-regulatory control but can also cross-regulate each other by mutually silencing their promoters, particularly in lymphoid cells .

The auto-regulation of GFI1B helps maintain appropriate expression levels during hematopoietic development and differentiation. This regulatory mechanism likely contributes to the complementary expression patterns observed between GFI1B and GFI1 across different hematopoietic lineages, ensuring proper cell fate decisions during hematopoiesis .

What are the optimal antibody-based methods for detecting GFI1B protein?

For detecting GFI1B protein, researchers can employ several antibody-based techniques with specific considerations for each:

• Western Blotting (WB): GFI1B antibodies such as mouse monoclonal IgG2b (e.g., B-7 clone) are suitable for detecting GFI1B protein from mouse, rat, and human origin . For optimal results, include positive controls (erythroid or megakaryocytic cell lines) and negative controls (granulocytes which express minimal GFI1B) .

• Immunoprecipitation (IP): GFI1B antibodies effectively immunoprecipitate native GFI1B and its associated protein complexes, including components of the RCOR-GFI-KDM1A-HDAC complex .

• Immunofluorescence (IF): For subcellular localization studies, use unconjugated or fluorescently-conjugated GFI1B antibodies (FITC, PE, or Alexa Fluor conjugates) .

• ELISA: GFI1B antibodies can be employed in enzyme-linked immunosorbent assays for quantitative detection of GFI1B in biological samples .

These methods can be optimized by adjusting antibody concentrations, incubation times, and buffer conditions based on the specific experimental requirements and sample types. When selecting a GFI1B antibody, consider target species compatibility, application-specific validation, and epitope location to ensure optimal performance for your experimental system.

How can chromatin immunoprecipitation be optimized for studying GFI1B genomic binding?

Optimizing chromatin immunoprecipitation (ChIP) for GFI1B requires specific considerations:

• Crosslinking conditions: Formaldehyde-assisted isolation of regulatory elements (FAIRE) assay has been effectively used to assess chromatin conformation at GFI1B binding sites . Standard crosslinking with 1% formaldehyde for 10 minutes at room temperature is generally effective, but optimization may be required for specific cell types.

• Sonication parameters: Carefully optimize sonication conditions to generate DNA fragments of appropriate size (typically 200-500 bp). Test different sonication cycles and intensities to achieve consistent fragmentation.

• Antibody selection: Use ChIP-validated GFI1B antibodies, with consideration of epitope accessibility in the crosslinked chromatin context. Antibodies targeting the N-terminal region of GFI1B may be preferable as the C-terminus interacts with other proteins.

• Quantification method: Analyze immunoprecipitated DNA by quantitative real-time PCR using primers and probes designed with Primer Express software . Results should be normalized to input DNA and control sequences such as 18S ribosomal RNA gene or GAPDH .

• Controls: Include positive controls (known GFI1B binding sites such as its own promoter) and negative controls (regions not bound by GFI1B) .

For multi-species studies, design species-specific primers and probes, as demonstrated in studies examining both human GFI1B and mouse Gfi1b loci . Sequential ChIP (ChIP-reChIP) can be used to determine if GFI1B and its interacting partners (such as LSD1 or β-catenin) co-occupy the same genomic regions .

What approaches can be used to study GFI1B-dependent gene regulation?

Studying GFI1B-dependent gene regulation requires integrated approaches:

• RNA-sequencing after GFI1B manipulation: Compare transcriptomes after GFI1B overexpression, knockdown, or mutation. For example, researchers have expressed GFI1B variants (T174N, H181Y, R184P, Q287*) in MEG01 megakaryoblasts to identify differentially affected gene programs .

• ChIP-sequencing: Map genome-wide GFI1B binding sites to identify direct regulatory targets. Integration with histone modification ChIP-seq data can reveal the chromatin context of GFI1B binding.

• CRISPR-based approaches:

  • CRISPR-mediated knockout of GFI1B for loss-of-function studies

  • CRISPR interference (CRISPRi) for precise repression of GFI1B expression

  • CRISPR activation (CRISPRa) for targeted upregulation of GFI1B

  • CRISPR-mediated insertion of mutations to study variant effects

• Reporter assays: Measure the impact of GFI1B on putative target gene promoters using luciferase reporter constructs. GFI1B can activate TCF-dependent transcription, which can be enhanced by Wnt3a treatment .

• Modulatory compounds: Use small molecules that affect GFI1B function, such as GSK-LSD1 that inhibits the GFI1B-LSD1 interaction, to examine consequent changes in gene expression .

• Single-cell approaches: Apply single-cell RNA-sequencing to understand cell-type-specific GFI1B functions. This approach revealed that GFI1B Q287* mutation causes a 4.5-fold decrease in the megakaryocyte/myeloid cell ratio in induced pluripotent stem cell-derived hematopoietic cultures .

These approaches have demonstrated that GFI1B and LSD1 facilitate gene programs relevant for megakaryopoiesis while simultaneously repressing programs that induce myeloid differentiation .

How can GFI1B antibodies be used to investigate protein-protein interactions within transcriptional complexes?

GFI1B antibodies can reveal the complex network of protein-protein interactions within transcriptional regulatory complexes:

• Co-immunoprecipitation (Co-IP): Use GFI1B antibodies to pull down GFI1B-containing complexes, followed by western blotting for potential interacting partners. This approach has revealed GFI1B's interaction with components of the RCOR-GFI-KDM1A-HDAC complex and with β-catenin and its co-factors Pontin52, CHD8, TLE3, and CtBP1 .

• Proximity ligation assay (PLA): Visualize and quantify GFI1B interactions with partner proteins in situ, providing spatial information about complex formation within cellular compartments.

• BioID or APEX proximity labeling: Fuse GFI1B to a biotin ligase (BioID) or an engineered peroxidase (APEX) to biotinylate proteins in close proximity, which can then be purified and identified by mass spectrometry.

• Sequential ChIP (ChIP-reChIP): Determine if GFI1B and its interacting partners co-occupy the same genomic regions using sequential ChIP with GFI1B antibodies followed by antibodies against potential partners such as LSD1 or β-catenin .

• Mass spectrometry analysis: Perform immunoprecipitation with GFI1B antibodies followed by mass spectrometry to identify novel interaction partners and post-translational modifications that regulate GFI1B function.

These approaches have revealed that GFI1B forms functionally significant complexes, including a tripartite β-catenin/GFI1B/LSD1 complex that regulates Wnt/β-catenin target genes . The interaction between GFI1B and LSD1 is particularly crucial, as it enables GFI1B to activate TCF-dependent transcription, which can be enhanced by Wnt3a treatment .

What methodological approaches can be used to study the role of GFI1B in megakaryocyte differentiation and function?

Investigating GFI1B's role in megakaryocyte differentiation and function requires specialized methodological approaches:

• In vitro differentiation systems: Induce megakaryocyte differentiation from hematopoietic progenitors using thrombopoietin (TPO), assessing GFI1B expression and function at different timepoints.

• GFI1B variant analysis: Compare normal GFI1B with disease-associated variants (e.g., T174N, H181Y, R184P, Q287*) in megakaryoblast cell lines such as MEG01 to reveal differential effects on gene expression programs .

• Small molecule modulators: Inhibit the GFI1B-LSD1 interaction with small molecules like GSK-LSD1 to elucidate the importance of this interaction for megakaryocyte development .

• Single-cell RNA-sequencing: Reveal the impact of GFI1B mutations on cell fate decisions, as demonstrated by the observation that GFI1B Q287* mutation caused a 4.5-fold decrease in the megakaryocyte/myeloid cell ratio in iPSC-derived megakaryocytes .

• Functional assays: Assess megakaryocyte function through integrin-dependent spreading assays. GFI1B-deficient megakaryocytes show defects that can be rescued by Wnt3a treatment .

• Regulon activity analysis: Evaluate transcription factor regulon activity to understand how GFI1B facilitates megakaryopoiesis by driving megakaryocyte-specific regulons while repressing myeloid regulons .

• Morphological and ploidy analysis: Examine megakaryocyte morphology and ploidy using flow cytometry and microscopy to assess maturation under different GFI1B expression conditions.

These approaches have demonstrated that GFI1B Q287* uniquely fails to repress myeloid traits, and that inhibiting the GFI1B-LSD1 interaction results in activation of myeloid genes in normal iPSC-derived megakaryocytes similar to what was observed for GFI1B Q287* .

How can researchers investigate the metabolic regulatory functions of GFI1B in hematopoiesis and leukemia?

Studying GFI1B's role as a metabolic regulator requires specialized approaches combining molecular and metabolic analyses:

• Metabolic flux analysis: Use Seahorse XF analyzer to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in GFI1B-manipulated cells to assess changes in oxidative phosphorylation and glycolysis .

• Substrate dependency tests: Apply specific inhibitors such as etomoxir (fatty acid oxidation inhibitor) and UK5099 (mitochondrial pyruvate carrier inhibitor) to determine metabolic substrate dependency changes upon GFI1B manipulation .

• Mitochondrial analyses: Assess mitochondrial mass, membrane potential, and morphology using flow cytometry and microscopy to understand GFI1B's impact on mitochondrial function.

• Genetic rescue experiments: Overexpress GFI1B in human AML cell lines (e.g., OCI/AML3 and KG1) to determine if GFI1B restoration reverses metabolic phenotypes, such as reduced fatty acid dependency .

• Metabolite profiling: Perform mass spectrometry-based metabolomics to identify specific metabolite changes associated with GFI1B expression levels.

• In vivo metabolic studies: Use mouse models with conditional Gfi1b deletion in hematopoietic tissues to assess metabolic alterations in hematopoietic stem cells and their progeny .

These approaches have revealed that loss of Gfi1b leads to increased metabolic activation in HSCs and that GFI1B deficiency increases mitochondrial substrate dependency on fatty acids in hematopoietic progenitor cells . GFI1B overexpression significantly reduces fatty acid dependency in AML cell lines, suggesting a key role in regulating fatty acid metabolism .

What are the critical validation steps for confirming GFI1B antibody specificity?

Ensuring GFI1B antibody specificity is crucial for reliable experimental results. Key validation steps include:

• Positive and negative control cells: Test antibodies on cells known to express high levels of GFI1B (megakaryocytes, erythroid progenitors) versus those with minimal expression (granulocytes, most T-cells) .

• Genetic validation: Use GFI1B knockdown/knockout cells to confirm antibody specificity. The absence of signal in these samples provides strong evidence for specificity.

• Overexpression validation: Complement knockout validation with overexpression studies where GFI1B is ectopically expressed, which should result in increased signal.

• Peptide competition: Pre-incubate the antibody with the immunizing peptide to block specific binding and eliminate true positive signals.

• Cross-reactivity assessment: Test the antibody against related proteins, particularly GFI1, to ensure it doesn't cross-react with this close paralog.

• Multiple antibody comparison: Use antibodies targeting different epitopes of GFI1B and compare detection patterns to confirm consistent results.

• Western blot molecular weight verification: Confirm that the detected protein band corresponds to the expected molecular weight of GFI1B (approximately 37 kDa) or its known isoforms.

• Immunoprecipitation-mass spectrometry: For the most rigorous validation, analyze immunoprecipitated protein by mass spectrometry to confirm GFI1B identity.

These validation steps ensure that experimental observations genuinely reflect GFI1B biology rather than non-specific or off-target effects. This is particularly important given GFI1B's role in multiple cellular processes and its structural similarity to GFI1 .

What are common technical challenges when studying GFI1B in primary hematopoietic cells and how can they be addressed?

Studying GFI1B in primary hematopoietic cells presents several technical challenges with specific solutions:

When working with primary hematopoietic stem cells, it's essential to validate findings from cell lines in primary cells whenever possible, as GFI1B expression patterns and function may differ between immortalized cell lines and their primary counterparts .

How can researchers differentiate between GFI1B and GFI1 detection in experimental systems?

Distinguishing between GFI1B and GFI1 detection is crucial due to their structural similarity and potentially overlapping functions:

• Antibody selection: Choose antibodies specifically validated against both proteins to ensure no cross-reactivity. Antibodies targeting the non-conserved regions between these paralogs provide the highest specificity.

• Epitope mapping: Understand the exact epitope recognized by the antibody. Most commercial GFI1B antibodies are raised against epitopes that differ from GFI1, such as the mouse monoclonal B-7 antibody that specifically detects GFI1B .

• Expression pattern analysis: Leverage the distinct expression patterns of these proteins - GFI1B is highly expressed in erythroid precursors and megakaryocytes but absent in granulocytes, while GFI1 shows the opposite pattern .

• Molecular weight discrimination: GFI1B (37 kDa) and GFI1 (45 kDa) have different molecular weights that can be distinguished on western blots with adequate resolution.

• Isoform-specific PCR: For mRNA detection, design primers targeting unique regions of each transcript to ensure specificity in qPCR analysis.

• Knockout/knockdown controls: Include samples with specific knockdown or knockout of either GFI1B or GFI1 to validate signal specificity.

These approaches ensure accurate attribution of experimental observations to the correct protein, which is essential given that GFI1B and GFI1 have both distinct and overlapping functions in hematopoiesis .

How can GFI1B antibodies be used to investigate inherited platelet disorders?

GFI1B antibodies offer valuable tools for investigating inherited platelet disorders through multiple approaches:

• Diagnostic immunophenotyping: Use GFI1B antibodies to identify abnormal megakaryocyte development and platelet production in patient samples through flow cytometry or immunofluorescence microscopy.

• Mutation-specific analysis: Develop antibodies that specifically recognize normal versus mutant GFI1B to distinguish heterozygous carriers from homozygous patients. This is particularly relevant for GFI1B variants (T174N, H181Y, R184P, Q287*) associated with platelet disorders .

• Functional studies: Employ immunofluorescence microscopy using GFI1B antibodies to reveal abnormal localization or expression in patient-derived megakaryocytes, correlating with platelet dysfunction.

• Chromatin occupancy analysis: Perform ChIP with GFI1B antibodies followed by sequencing (ChIP-seq) to map genomic binding sites in normal versus patient-derived cells, revealing how mutations affect transcriptional regulation.

• Protein-protein interaction studies: Use co-immunoprecipitation with GFI1B antibodies to identify altered interaction partners in mutant GFI1B, providing insights into disease mechanisms.

Research has shown that GFI1B mutations affecting its interaction with LSD1 result in abnormal megakaryopoiesis, with the Q287* variant uniquely failing to repress myeloid traits . Small molecule inhibition of the GFI1B-LSD1 interaction with GSK-LSD1 leads to activation of myeloid genes in normal megakaryocytes, mimicking the effect of the Q287* mutation .

What approaches can be used to study GFI1B's role in leukemogenesis and its potential as a therapeutic target?

Investigating GFI1B's role in leukemogenesis requires specialized approaches:

• Expression correlation studies: Quantify GFI1B protein levels in leukemia samples using antibody-based techniques and correlate with clinical outcomes. Low-level or loss of GFI1B promotes AML development and negatively influences the prognosis of myelodysplastic syndrome (MDS)/AML patients .

• Genetic manipulation in leukemia models: Employ CRISPR/Cas9 or shRNA approaches to modulate GFI1B expression in leukemia cell lines and patient-derived xenografts, followed by assessment of cellular phenotypes.

• Metabolic profiling: Use GFI1B antibodies in combination with metabolic assays to understand how GFI1B regulates metabolism in leukemic cells. Loss of Gfi1b leads to increased metabolic activation and elevated mitochondrial substrate dependency on fatty acids .

• Therapeutic targeting assessment: Evaluate the effects of small molecules that modulate GFI1B function or its interaction with co-factors like LSD1, using antibodies to monitor target engagement and downstream effects.

• Combinatorial therapy studies: Investigate how GFI1B-targeted approaches might synergize with conventional chemotherapy or other targeted therapies, using antibodies to monitor pathway activation.

These approaches build on findings that GFI1B expression is negatively related to the prognosis of AML patients, and that GFI1B deficiency promotes leukemia development, possibly through metabolic rewiring of hematopoietic stem and progenitor cells .

How can GFI1B antibodies contribute to understanding the dysregulation of the Wnt/β-catenin pathway in hematological disorders?

GFI1B antibodies provide valuable tools for investigating the intersection of GFI1B and Wnt/β-catenin signaling in hematological disorders:

• Complex formation analysis: Perform co-immunoprecipitation with GFI1B antibodies to identify abnormal interactions between GFI1B and Wnt/β-catenin pathway components (β-catenin, Pontin52, CHD8, TLE3, CtBP1) in patient samples .

• Dual ChIP studies: Conduct sequential chromatin immunoprecipitation with GFI1B and β-catenin antibodies to map co-occupied genomic regions and reveal dysregulation in disease states.

• Reporter assays: Use GFI1B antibodies to correlate protein levels with TCF-dependent transcriptional activation in normal versus diseased cells. Research shows GFI1B can activate TCF-dependent transcription, especially when enhanced by Wnt3a treatment .

• Wnt response studies: Evaluate how cells with normal versus abnormal GFI1B respond to Wnt stimulation. Research has shown that Wnt3a treatment can restore normal cellularity in Gfi1b-deficient cells and enable Gfi1b-deficient megakaryocytes to regain their ability to spread on integrin substrates .

• Tripartite complex analysis: Investigate the integrity of the β-catenin/GFI1B/LSD1 complex in hematological disorders using antibodies against all three components .

These approaches are supported by evidence that Gfi1b forms complexes with β-catenin and its co-factors and regulates Wnt/β-catenin-dependent gene expression, controlling both the cellularity and functional integrity of hematopoietic stem cells and megakaryocytes .

How can GFI1B antibodies be integrated into single-cell protein analysis workflows?

Integrating GFI1B antibodies into single-cell protein analysis creates powerful new research opportunities:

• Mass cytometry (CyTOF): Use metal-conjugated GFI1B antibodies in CyTOF panels to simultaneously measure GFI1B expression alongside dozens of other proteins at single-cell resolution, revealing heterogeneity within seemingly homogeneous populations.

• Single-cell western blotting: Employ microfluidic platforms that perform western blotting on individual cells to quantify GFI1B expression levels and correlate with cellular phenotypes.

• Proximity extension assays (PEA): Utilize pairs of GFI1B antibodies coupled with DNA oligonucleotides to enable highly sensitive detection of GFI1B protein in single cells through PCR amplification.

• Spatial proteomics: Integrate GFI1B antibodies into multiplexed immunofluorescence or imaging mass cytometry workflows to visualize protein expression in tissue context, preserving spatial relationships between cells.

• CITE-seq approach: Conjugate GFI1B antibodies to unique DNA barcodes to simultaneously measure protein expression and transcriptomes in the same cells.

These advanced approaches can reveal previously unappreciated heterogeneity in GFI1B expression and function across hematopoietic populations, building on findings from single-cell RNA-sequencing of iPSC-derived megakaryocytes . Such techniques are particularly valuable for understanding how GFI1B mutations affect cell fate decisions in hematological disorders.

What are the implications of GFI1B's role in metabolic regulation for therapeutic development?

GFI1B's emerging role as a metabolic regulator has significant implications for therapeutic development:

• Targeting GFI1B-mediated metabolic pathways: GFI1B deficiency increases mitochondrial substrate dependency on fatty acids in hematopoietic progenitor cells . Therapies targeting fatty acid metabolism may therefore be effective in GFI1B-low leukemias.

• Combination therapy approaches: Combining metabolic inhibitors with conventional therapies may enhance efficacy in GFI1B-dysregulated disorders. The metabolic vulnerabilities created by GFI1B deficiency could be exploited therapeutically.

• GFI1B restoration strategies: For conditions characterized by GFI1B deficiency, therapies aimed at restoring GFI1B expression or function might normalize metabolic phenotypes. Research shows that GFI1B overexpression significantly reduces fatty acid dependency in AML cell lines .

• Biomarker development: GFI1B expression levels or metabolic signatures associated with GFI1B status could serve as biomarkers for patient stratification and treatment selection.

• Small molecule modulators: Compounds that affect the interaction between GFI1B and its cofactors may offer therapeutic opportunities. For instance, modulators of the GFI1B-LSD1 interaction could influence both transcriptional and metabolic outcomes.

The connection between GFI1B and metabolism offers a novel therapeutic angle for hematological disorders, particularly AML where GFI1B expression is negatively related to patient prognosis . Metabolic profiling of patient samples using GFI1B antibodies in conjunction with metabolic assays could help identify patients who might benefit from metabolism-targeted therapies.

How might computational approaches enhance the interpretation of GFI1B antibody-based experimental data?

Computational methods can significantly enhance the value of GFI1B antibody-based data:

• Network analysis: Integrate GFI1B protein interaction data from co-immunoprecipitation studies with transcriptomic data to build comprehensive regulatory networks, revealing indirect effects of GFI1B perturbation.

• Machine learning classification: Develop algorithms to identify patterns in GFI1B expression, localization, or modification data that correlate with cellular phenotypes or disease outcomes.

• Multi-omics data integration: Combine antibody-based proteomic data with transcriptomic, epigenomic, and metabolomic datasets to create comprehensive models of GFI1B function.

• Structural biology predictions: Use antibody epitope mapping data to refine structural predictions of GFI1B and its complexes, informing rational design of function-modulating compounds.

• Sequence-structure-function relationships: Analyze how sequence variations in GFI1B (such as the disease-associated variants T174N, H181Y, R184P, Q287*) affect protein structure and function .

• Systems biology modeling: Develop mathematical models of GFI1B regulatory circuits, incorporating antibody-derived quantitative data on protein levels and interactions.

These computational approaches can help decode the complex regulatory networks involving GFI1B, such as its role in controlling both the cellularity and functional integrity of hematopoietic stem cells and megakaryocytes by regulating Wnt/β-catenin signaling .