C11orf53 Antibody

What is C11orf53 and why has it been renamed?

C11orf53 (Chromosome 11 Open Reading Frame 53) has been renamed as "POU Class 2 Homeobox Associating Factor 2" (POU2AF2) based on its molecular function and expression pattern. This renaming reflects its newly discovered role as a co-activator of POU2 family transcription factors, particularly POU2F3 . The protein plays a critical role in regulating gene expression at enhancer elements and has been found to be essential for SCLC-P subtype cell viability and identity . The revised nomenclature better represents its biological function rather than simply its chromosomal location.

What is the cellular localization pattern of C11orf53/POU2AF2?

C11orf53/POU2AF2 displays a complex cellular distribution pattern despite lacking an obvious nuclear localization signal (NLS). Cell fractionation studies have revealed that the protein is present in multiple cellular compartments, including the cytosol, soluble nuclear fraction, and chromatin (insoluble nuclear) fractions . This diverse localization pattern is particularly intriguing given the protein's role in transcriptional regulation. Researchers should consider this distribution when designing experiments, as different extraction methods may be required to fully capture the protein's presence across cellular compartments.

What is the relationship between C11orf53/POU2AF2 and POU2F3?

C11orf53/POU2AF2 functions as a co-activator of the POU2F3 transcription factor. ChIP-seq analysis has revealed that C11orf53/POU2AF2 binding sites significantly overlap with POU2F3 motifs (p-value 1e-3053) . This co-localization suggests a direct functional interaction between these proteins. Furthermore, co-expression studies have demonstrated that while neither factor alone can significantly activate tuft cell-specific genes, their combination leads to robust activation of tuft cell marker genes such as Trpm5, Ascl2, and Avil . The interaction appears to be specific to POU2F3, as C11orf53/POU2AF2 is selectively co-expressed with POU2F3 in both normal tuft cells and SCLC-P cancer cells .

What antibody applications are most effective for C11orf53/POU2AF2 detection?

Based on available commercial antibodies, the following applications have been validated for C11orf53/POU2AF2 detection:

When selecting an antibody, researchers should consider the specific isoform of interest, as studies have specifically highlighted the importance of the long isoform of C11orf53 in tuft cell biology and SCLC-P .

How should researchers validate C11orf53/POU2AF2 antibody specificity?

Proper validation of C11orf53/POU2AF2 antibodies is essential given the protein's recent characterization. A comprehensive validation approach should include:

• Positive and negative cell line controls: Use SCLC-P subtype cell lines like NCI-H526 and NCI-H211 as positive controls, which express high levels of C11orf53/POU2AF2. Use non-SCLC-P subtype cell lines like NCI-H510 and NCI-H1963 as negative controls, which show no detectable expression .

• CRISPR knockout validation: Generate CRISPR-mediated C11orf53/POU2AF2 knockout cells to confirm antibody specificity. This approach has been successfully employed in SCLC research to demonstrate antibody specificity .

• Multiple antibody comparison: When possible, use multiple antibodies targeting different epitopes of C11orf53/POU2AF2 and compare results, as was done in ChIP-seq studies where two home-made polyclonal antibodies showed highly correlated peaks .

• Protein overexpression: Ectopic expression of C11orf53/POU2AF2 in negative cell lines can provide additional validation of antibody specificity and sensitivity.

How does C11orf53/POU2AF2 depletion affect cellular phenotypes in SCLC-P?

Depletion of C11orf53/POU2AF2 in SCLC-P cells results in multiple cellular effects:

• Reduced cell viability: CRISPR-mediated knockout of C11orf53/POU2AF2 dramatically reduces SCLC-P cell viability in vitro .

• Morphological changes: Significant alterations in cell morphology occur following C11orf53/POU2AF2 depletion .

• Apoptosis induction: Loss of C11orf53/POU2AF2 induces apoptotic markers including cleaved PARP, caspase 3, and caspase 7 .

• Cell cycle arrest: Flow cytometry analysis reveals cell cycle disruption in C11orf53/POU2AF2-depleted cells .

• Reduced tumor growth: In xenograft models, C11orf53/POU2AF2 depletion significantly represses tumor growth (P=0.0033) and delays disease progression (P=0.0023) .

These findings collectively establish C11orf53/POU2AF2 as an essential factor for SCLC-P cell survival and proliferation, highlighting its potential as a therapeutic target.

What is the role of C11orf53/POU2AF2 in chromatin regulation?

C11orf53/POU2AF2 functions as a key regulator of chromatin structure and accessibility:

• Enhancer binding: ChIP-seq analysis identified 8,820 specific C11orf53/POU2AF2 binding sites in NCI-H526 cells, predominantly at intergenic or intronic regions, suggesting enhancer-based regulation .

• H3K27ac regulation: Depletion of C11orf53/POU2AF2 reduces enhancer H3K27ac levels, a mark of active enhancers, indicating its role in maintaining enhancer activity .

• Chromatin accessibility: C11orf53/POU2AF2 is required for maintaining chromatin accessibility at its target sites .

• Super enhancer regulation: C11orf53/POU2AF2 appears to regulate lineage-specific gene expression through super enhancers, regions of clustered enhancers that drive cell identity genes .

• Transcription factor recruitment: Evidence suggests C11orf53/POU2AF2 may facilitate the recruitment or function of POU2F3 at chromatin, enabling the activation of tuft cell-specific genes .

What are the optimized protocols for C11orf53/POU2AF2 ChIP-seq experiments?

Based on published research, an optimized ChIP-seq protocol for C11orf53/POU2AF2 should include:

• Antibody selection: Use validated antibodies specifically targeting C11orf53/POU2AF2. In published studies, home-made polyclonal antibodies have shown consistent performance with highly correlated peaks .

• Cross-validation approach: Employ multiple antibodies targeting different regions of C11orf53/POU2AF2 and compare peak overlaps to ensure specificity. This approach has been successfully used to identify 8,820 specific C11orf53/POU2AF2 peaks in NCI-H526 cells .

• Cell type considerations: SCLC-P subtype cell lines like NCI-H526 are optimal for C11orf53/POU2AF2 ChIP-seq due to their high expression levels .

• Motif analysis integration: Include motif analysis to identify co-occurring transcription factor binding sites, particularly POU2F3 motifs which have been identified as significantly enriched at C11orf53/POU2AF2 binding sites .

• Integration with other epigenomic data: Combine ChIP-seq with ATAC-seq or H3K27ac ChIP-seq to correlate C11orf53/POU2AF2 binding with changes in chromatin accessibility and enhancer activity .

How might the C11orf53(POU2AF2)/POU2F3 heterodimer serve as a therapeutic target in SCLC?

The C11orf53(POU2AF2)/POU2F3 heterodimer represents a promising therapeutic target for SCLC-P subtype tumors based on several lines of evidence:

• Selective expression: The heterodimer is selectively expressed in SCLC-P subtype tumors, potentially allowing for targeted therapy with minimal effects on normal tissues .

• Essential function: CRISPR studies have demonstrated that both C11orf53/POU2AF2 and POU2F3 are essential for SCLC-P cell viability, with depletion significantly reducing tumor growth in xenograft models .

• Transcriptional dependency: SCLC-P tumors show strong dependency on C11orf53/POU2AF2 and POU2F3 co-expression for maintaining their cellular identity and transcriptional program .

• Protein-protein interaction target: The interaction between C11orf53/POU2AF2 and POU2F3 could potentially be disrupted by small molecules or peptides, offering a specific therapeutic approach for SCLC-P tumors .

• Synthetic lethality: Understanding the transcriptional networks regulated by the C11orf53/POU2AF2-POU2F3 heterodimer may reveal synthetic lethal interactions that could be therapeutically exploited .

Future drug development strategies could focus on disrupting the formation or function of the C11orf53(POU2AF2)/POU2F3 heterodimer, potentially offering a novel therapeutic avenue for this aggressive SCLC subtype.

What are the key challenges in developing functional assays for C11orf53/POU2AF2 studies?

Researchers face several challenges when developing functional assays for C11orf53/POU2AF2:

• Protein size and structure: C11orf53/POU2AF2 is a relatively small protein with no obvious nuclear localization signal, making structural studies challenging .

• Multiple cellular localizations: The protein's presence in multiple cellular compartments (cytosol, soluble nuclear, and chromatin fractions) complicates the development of localization-specific assays .

• Co-factor dependency: C11orf53/POU2AF2 functions primarily in cooperation with POU2F3, requiring assays that can capture this interaction and co-dependency .

• Cell type specificity: Functional assays need to account for the highly restricted expression pattern of C11orf53/POU2AF2, which is limited to tuft cells and SCLC-P tumor cells .

• Context-dependent function: The protein appears to have different effects depending on cellular context, requiring careful selection of model systems for functional studies .

To overcome these challenges, researchers should consider developing assays that simultaneously monitor C11orf53/POU2AF2 and POU2F3 activities, potentially focusing on transcriptional reporter systems that reflect their co-dependent function in regulating tuft cell-specific genes.

How does the C11orf53/POU2AF2 and POU2F3 relationship compare to other transcriptional co-activator systems?

The C11orf53/POU2AF2 and POU2F3 relationship represents an emerging model of transcriptional co-activation with several distinctive features:

• Lineage restriction: Unlike many broadly expressed co-activators, C11orf53/POU2AF2 shows a highly restricted expression pattern tightly correlated with POU2F3, suggesting a specialized co-evolutionary relationship .

• Mutual dependency: Evidence suggests a co-dependent relationship where neither factor can effectively function without the other, as demonstrated by co-expression studies showing that expression of either factor alone fails to activate target genes .

• Enhancer-focused activity: C11orf53/POU2AF2 appears to function primarily at enhancer elements rather than promoters, with ChIP-seq analysis showing predominant binding at intergenic and intronic regions .

• Cell identity determination: The heterodimer appears fundamental to establishing and maintaining tuft cell identity, controlling a cell-type-specific transcriptional program essential for both normal tuft cells and SCLC-P tumor cells .

Further comparative studies between this system and other co-activator/transcription factor pairs will likely yield important insights into transcriptional control mechanisms and lineage specification.

What role might C11orf53/POU2AF2 play in normal development and differentiation of tuft cells?

Based on expression data and functional studies, C11orf53/POU2AF2 likely plays a critical role in normal tuft cell development and differentiation:

• Tissue-specific expression: C11orf53/POU2AF2 is selectively expressed in tuft cells of multiple tissues including the small intestine, trachea, thymus, and colon, suggesting a conserved role in tuft cell biology across different organ systems .

• Co-expression with POU2F3: RNA-FISH studies have confirmed co-expression of C11orf53/POU2AF2 and POU2F3 in mouse tissues, indicating a conserved transcriptional partnership .

• Gene activation function: Co-expression of C11orf53/POU2AF2 and POU2F3 activates tuft-cell-specific genes such as Trpm5, Ascl2, and Avil, which are critical for tuft cell function .

• Chromatin modification: C11orf53/POU2AF2's role in regulating H3K27ac levels and chromatin accessibility suggests it may function in establishing the epigenetic landscape necessary for tuft cell differentiation .

• Evolutionary conservation: The consistent co-expression pattern across species suggests an evolutionarily conserved function in specifying this rare cell lineage .

Future developmental studies focusing on the timing and regulation of C11orf53/POU2AF2 expression during tuft cell differentiation will be valuable for understanding both normal development and the origin of SCLC-P tumors.