BTF3 Human

What is BTF3 and what are its main functions in human cells?

BTF3 (Basic Transcription Factor 3) is a 27 kDa protein encoded by the BTF3 gene in humans. It serves dual functions in cellular processes:

• Transcriptional role: Forms a stable complex with RNA polymerase II and functions as a transcriptional initiation factor by binding to promoter elements like TATA and CAAT box sequences .

• Translational role: Acts as the β-subunit of the Nascent-polypeptide Associated Complex (βNAC), involved in protein regulation during translation to aid correct folding and prevent misfolding of polypeptide chains .

BTF3 is evolutionarily conserved across multiple organisms, highlighting its fundamental importance in cellular processes . It interacts with CSNK2B (Casein Kinase 2 Beta) and is associated with multiple biological processes including early development, cell proliferation, and apoptosis .

What isoforms of BTF3 exist and how do they differ functionally?

BTF3 exists in two major isoforms resulting from alternative splicing:

• BTF3a: The transcriptionally active form that initiates transcription by binding to promoter elements.

• BTF3b: Lacks the 44 N-terminal amino acids of BTF3a and is transcriptionally inactive, although it can still bind to RNA polymerase II .

While BTF3a is involved in transcriptional regulation, both isoforms play roles in tumorigenesis. Research in prostate cancer has shown that while both BTF3a and BTF3b promote cell growth, BTF3b specifically regulates the transcriptional expression of Replication Factor C (RFC) family genes involved in DNA replication and damage repair processes .

Why is BTF3 essential for embryonic development?

BTF3 plays a critical role in embryonic development across multiple species:

• Embryonic lethality: Mutations or deletions in the BTF3 gene lead to embryonic death at early developmental stages in mice, Drosophila, and C. elegans .

• Stem cell maintenance: BTF3 is highly expressed in embryonic stem cells and is part of an ESC-like transcriptional program active in both human and mouse ESCs .

• Development regulation: BTF3 is involved in various biotic and abiotic stress processes, as well as different physiological and developmental mechanisms .

The non-redundant nature of BTF3's functions suggests it plays essential roles in transcription and protein synthesis regulation that cannot be compensated by other factors during early embryonic development.

How does BTF3 expression correlate with cancer progression and prognosis?

BTF3 overexpression has been documented in multiple cancer types and correlates with disease progression:

Table 1: Clinical significance of BTF3 expression in prostate cancer

• Colorectal cancer: BTF3 expression is significantly higher in CRC tissue than in adjacent non-cancerous tissue (2.61 ± 0.07 vs 1.90 ± 0.03, P < 0.001) .

• Prostate cancer: Higher BTF3 expression correlates with higher Gleason scores and advanced tumor stages as shown in Table 1 .

• Multiple cancers: Overexpression has been documented in pancreatic, gastric, and breast cancers .

Patients with high expression of the ESC signature involving BTF3 exhibited poor outcomes, with this signature strongly predicting metastasis and death in diverse epithelial cancers .

What molecular mechanisms underlie BTF3's oncogenic activity?

BTF3 contributes to oncogenesis through multiple mechanisms:

• Transcriptional regulation of oncogenes:

  • In colorectal cancer, BTF3 transcriptionally activates CHD1L (chromodomain helicase DNA binding protein 1 Like), a known oncogene .

  • Regulates MAD2L2, MCM3, and PLK1, affecting cell viability, apoptosis, and cell cycle progression .

• Cell cycle and apoptosis regulation:

  • BTF3 knockdown results in cell cycle arrest at S and G2/M phases .

  • Silencing BTF3 increases apoptosis in cancer cells .

• Epithelial-Mesenchymal Transition (EMT) promotion:

  • BTF3 knockdown increases E-cadherin expression while decreasing N-cadherin and ZEB2, indicating reduced EMT .

  • Regulates JAK2/STAT3 signaling pathway, critical for EMT and metastasis .

• Cancer stem cell maintenance:

  • BTF3 is highly expressed in CD49f^hi Trop2^hi cells, which mark prostate progenitor basal stem cells and aggressive prostate cancer cells .

How does BTF3 influence DNA damage repair and chemotherapy response?

BTF3 plays a significant role in DNA damage repair mechanisms and consequently affects chemotherapy response:

• DNA repair regulation: BTF3b regulates the transcriptional expression of Replication Factor C (RFC) family genes involved in DNA replication and damage repair processes .

• Cisplatin sensitivity: Enforced BTF3 overexpression in prostate cancer cells induces substantial accumulation of cisplatin-DNA adducts and renders cells more sensitive to cisplatin treatment both in vitro and in vivo .

• Replication and repair balance: BTF3 knockdown results in decreased expression of RFC genes, leading to attenuated DNA replication, deficient DNA damage repair, and increased G2/M arrest .

This relationship between BTF3 expression and cisplatin sensitivity suggests BTF3 expression levels may serve as a potential biomarker to predict cisplatin treatment response in certain cancers.

What techniques are most effective for BTF3 expression analysis in tissue samples?

Multiple techniques can be employed for analyzing BTF3 expression in tissues:

• Immunohistochemistry (IHC):

  • Allows visualization and quantification of BTF3 protein in tissue sections.

  • Can be automated using analysis protocols in ImageJ software for unbiased quantification .

  • Sensitivity for BTF3 as a cancer biomarker ranges from 0.68 to 0.74 .

• Real-time PCR (qRT-PCR):

  • Primer sequences for BTF3: 5′-AGCTTGGTGCGGATAGTCTGA-3′ (forward) and 5′-GTGCTTTTCCATCCACAGATTG-3′ (reverse) .

  • Protocol typically involves initial denaturation at 95°C for 10 min followed by 30 cycles at 95°C for 1 min, annealing at 53°C for 1 min, extension at 72°C for 1 min, and final extension at 72°C for 5 min .

• Triple-labeled immunofluorescence:

  • Used to study co-localization of BTF3 with other proteins.

  • Has shown significant changes in co-localization coefficients for BTF3 and NDRG1 co-expression in biochemical relapse vs non-relapse cancer epithelium (p<0.02) .

• RNA sequencing:

  • For comprehensive transcriptome analysis following BTF3 manipulation.

  • Requires proper normalization algorithms and filtering with threshold p-value <0.05 and fold change >2 .

How can BTF3 function be effectively studied through gene manipulation approaches?

Several approaches are used to manipulate BTF3 expression and study functional consequences:

• RNA interference approaches:

  • siRNA transfection: For short-term knockdown studies .

  • shRNA stable expression: Using lentiviral vectors for long-term studies .

• In vitro functional assays:

  • Cell proliferation: MTT assays show decreased cell viability after BTF3 knockdown .

  • Apoptosis: Flow cytometry with Annexin V/PI staining demonstrates increased apoptosis after BTF3 silencing .

  • Cell cycle: BTF3 knockdown induces S and G2/M cell cycle arrest .

  • Migration and invasion: Wound healing assays (protocol details in reference ) show decreased migration after BTF3 knockdown .

• In vivo xenograft models:

  • Protocol: 2×10^6 cancer cells (control or BTF3-manipulated) are subcutaneously injected into nude mice. Tumor size is measured weekly, and mice are typically sacrificed after 3 weeks .

  • BTF3 knockdown in HT29 cells resulted in reduced tumor size (42.3%, p<0.05, N=6) compared to control .

What genomic and proteomic methods reveal BTF3's interaction network?

Several advanced methods help elucidate BTF3's interaction network:

• RNA-Seq and ChIP-Seq combined analysis:

  • RNA-Seq identifies differentially expressed genes after BTF3 knockdown (292 DEGs identified in one study) .

  • ChIP-Seq reveals DNA binding sites of BTF3 (149 genes with differential peaks identified) .

  • Combined analysis identifies direct transcriptional targets of BTF3, such as CHD1L in colorectal cancer .

• Immunoprecipitation and mass spectrometry (IP-MS):

  • Protocol: Cells are lysed with IP buffer, and beads coupled with BTF3 antibody are incubated with lysates overnight at 4°C. Eluted proteins are analyzed by LC-MS/MS .

  • Identified 542 proteins specifically interacting with BTF3, including NACA .

• Gene Ontology (GO) and pathway analysis:

  • GO analysis of BTF3-interacting proteins revealed enrichment in:

    • Biological process: Protein targeting to ER

    • Cellular component: Cytosolic ribosome and nuclear proteins

    • Molecular function: RNA binding and DNA binding

  • KEGG pathway analysis identified neuroactive ligand-receptor interaction, ErbB2, and PPAR as significant pathways affected by BTF3 .

How does BTF3 function as a component of the Nascent-polypeptide Associated Complex?

As the β-subunit of NAC (βNAC), BTF3 plays crucial roles in protein processing:

• Co-translational protein folding:

  • Binds to nascent peptides emerging from ribosomes

  • Prevents misfolding and aggregation of polypeptide chains

• Protein targeting regulation:

  • GO analysis of BTF3-interacting proteins shows "protein targeting to ER" as the most significant biological process

  • Regulates peptide translocation to the endoplasmic reticulum and mitochondria

• Proteolysis involvement:

  • NAC complexes participate in protein ubiquitination and proteolysis

  • BTF3 inhibits ubiquitin-mediated degradation of specific proteins

• Stress response:

  • Functions in both biotic and abiotic stress responses

  • Helps maintain protein homeostasis under stress conditions

This NAC-related role is distinct from BTF3's transcription factor function and represents a cytoplasmic activity complementing its nuclear role.

What is the relationship between BTF3 and microRNA regulation?

BTF3 expression is regulated by specific microRNAs, establishing an important post-transcriptional control mechanism:

• miR-497-5p regulation:

  • Computational prediction using miRwalk, Targetscan, and miRDB identified miR-497-5p as a potential regulator of BTF3

  • miR-497-5p shows consistently decreased expression in colorectal cancer datasets (GSE128446, GSE81581, and GSE35982)

  • Transfection of miR-497-5p mimics in HT29 cells significantly decreased BTF3 expression

  • Dual luciferase assay confirmed direct binding of miR-497-5p to BTF3 3'UTR

• Clinical correlations:

  • miR-497-5p expression correlates significantly with lymphatic invasion, pathologic M, pathologic N, and pathological stage in colorectal cancer

  • Correlation analysis showed a significantly negative coefficient between miR-497-5p and BTF3 expression

• Other miRNA regulators:

  • miR-802 has been reported to downregulate BTF3 in ovarian cancer

Understanding these miRNA-BTF3 interactions provides potential therapeutic targets and diagnostic markers for cancer.

How does BTF3 interact with the ubiquitination and proteolysis machinery?

BTF3 plays a sophisticated role in protein ubiquitination and degradation:

• E3 ligase interactions:

  • IP-MS studies identified interactions between BTF3 and various ubiquitination machinery components

  • BTF3 potentially interacts with HERC2, an E3 ubiquitin ligase involved in p53 regulation

• Protein stability regulation:

  • BTF3 may promote p53 ubiquitination and degradation by recruiting HERC2

  • This mechanism could explain BTF3's oncogenic effects by destabilizing tumor suppressors

• Proteasome pathway involvement:

  • BTF3 has been reported to inhibit ubiquitin-mediated BMI1 degradation in other cancer types

  • NAC complexes, of which BTF3 is a component, are directly involved in protein ubiquitination and proteolysis

This function adds another layer to BTF3's complex role in cancer progression, as it could directly affect the stability and activity of key oncoproteins and tumor suppressors.

Can BTF3 function as a biomarker for cancer diagnosis and prognosis?

BTF3 shows considerable promise as a clinical biomarker:

• Diagnostic potential:

  • In prostate cancer tissue arrays, BTF3 showed increased expression in malignant vs. non-malignant prostate (by 2-2.5 fold, p<0.0001)

  • Operating characteristics indicate sensitivity in the range of 0.68 to 0.74

  • Combination with other markers (HINT1, NDRG1, and ODC1) in a logistic regression model demonstrates improved diagnostic power

• Prognostic value:

  • BTF3 expression correlates significantly with Gleason score (p=0.006) and tumor stage (p=0.02) in prostate cancer

  • The ESC signature involving BTF3 strongly predicts metastasis and death in diverse epithelial cancers

• Triple-labeled immunofluorescence:

  • BTF3, HINT1, and NDRG1 co-localization shows significant change in biochemical relapse vs. non-relapse cancer epithelium (p<0.02)

  • This approach could be used for stratification of prostate cancer patients

What therapeutic strategies might target BTF3 or its pathways?

Several therapeutic approaches targeting BTF3 show potential:

• RNA interference:

  • BTF3-siRNA attenuates the tumorigenicity of colorectal cancer cells via MAD2L2, MCM3, and PLK1 activity reduction

  • BTF3-silencing decreases cell viability, induces apoptosis, and blocks cell cycle progression

• Targeting downstream pathways:

  • JAK2/STAT3 pathway: BTF3 knockdown decreases phosphorylation of JAK2/STAT3, suggesting JAK2/STAT3 inhibitors might be effective in BTF3-overexpressing tumors

  • FOXM1 pathway: BTF3 regulates FOXM1, suggesting FOXM1 inhibitors as potential therapeutics

• miRNA-based approaches:

  • miR-497-5p decreases BTF3 expression and could have therapeutic potential in BTF3-overexpressing cancers

• Chemosensitivity prediction:

  • BTF3 expression levels may serve as a biomarker to predict cisplatin treatment response in prostate cancer

  • This could help stratify patients for platinum-based therapies

What methodological challenges exist in translating BTF3 research to clinical applications?

Several challenges must be addressed for clinical translation:

• Tissue-specific effects:

  • BTF3 has dual functions as a transcription factor and NAC component with potentially different roles across tissue types

  • Research must clarify tissue-specific mechanisms before developing targeted therapies

• Standardization of detection methods:

  • Quantitative analysis of BTF3 requires standardized protocols

  • Automated analysis protocols in ImageJ software represent one approach to unbiased quantification

• Functional redundancy:

  • The relationship between BTF3 isoforms (BTF3a and BTF3b) needs further clarification

  • Both isoforms promote cell growth but have distinct transcriptional activities

• Pathway complexity:

  • BTF3 affects multiple signaling pathways simultaneously

  • Identifying the most critical targets for therapeutic intervention requires comprehensive pathway analysis

  • Figure 6 from reference depicts a schematic of BTF3's functional and expression mechanisms in colorectal cancer, illustrating this complexity