HSF2BP Human

What is HSF2BP and what is its normal function in human cells?

HSF2BP (Heat Shock Factor 2 Binding Protein) is primarily a meiosis-specific protein that plays essential roles in homologous recombination (HR) during gametogenesis. It was initially identified as an interactor of the heat-shock response transcription factor HSF2 . The protein's primary function involves facilitating proper meiotic homologous recombination through its interaction with the tumor suppressor protein BRCA2 .

In normal human physiology, HSF2BP expression is largely restricted to germline tissues, particularly in the testis . The protein forms a complex with BRME1 (C19ORF57) and interacts with several key DNA repair proteins including BRCA2, RAD51, RPA, and PALB2 . This complex is essential for the proper orchestration of meiotic recombination events, including the processing of DNA double-strand breaks (DSBs) and the loading of recombinases to recombination nodules .

How does HSF2BP structurally interact with BRCA2?

The interaction between HSF2BP and BRCA2 involves a specific structural arrangement where HSF2BP binds to BRCA2 through a repeated motif. Crystal structure analysis has revealed that BRCA2 contains a repeated 23 amino acid motif, with each repeat binding to the same conserved surface of one armadillo (ARM) domain of HSF2BP .

In this complex, two BRCA2 fragments hold together two ARM dimers through a large interface. This arrangement creates an exceptionally strong interaction - the strongest interaction involving BRCA2 measured to date, with nanomolar affinity . The dimeric armadillo domain of HSF2BP is critical for this binding, establishing a structural foundation for the protein's function in meiotic homologous recombination .

What phenotypes are associated with HSF2BP mutations in humans?

Mutations in HSF2BP are associated with fertility disorders in humans, most notably Primary Ovarian Insufficiency (POI). A missense variant S167L in HSF2BP has been identified in a consanguineous family with three cases of POI . Another variant, G224*, was found to affect recombination rate in males, with homozygous individuals appearing healthy but potentially infertile .

Functional studies of these variants have demonstrated that they impair the nuclear localization of HSF2BP and affect its DNA repair capacity . The S167L variant specifically acts as a hypomorphic allele that reduces protein expression and/or stability of the HSF2BP/BRME1 complex, leading to defects in meiotic recombination .

Notably, HSF2BP joins other meiotic recombination genes with recently identified variants in POI patients, many of which encode BRCA2-interacting factors involved in the repair of induced DSBs at early stages of meiosis .

How does HSF2BP negatively regulate homologous recombination in DNA interstrand crosslink repair?

HSF2BP exhibits a fascinating dual role - while essential for meiotic HR, its ectopic expression in somatic cells actually disrupts HR in the context of interstrand crosslink (ICL) repair. The mechanism involves several steps:

• When overexpressed in non-germline cells, HSF2BP triggers the removal of BRCA2 from ICL sites

• This removal is followed by proteasomal degradation of BRCA2

• The depleted BRCA2 leads to reduced RAD51 accumulation at ICL damage sites

• This inhibits the HR step of the Fanconi anemia (FA) pathway of ICL repair

Experimentally, this phenomenon has been demonstrated using clonogenic survival assays in cell lines with ectopic HSF2BP expression, showing hypersensitization to ICL-inducing agents like MMC and cisplatin, as well as PARP inhibitors . The specificity of this effect is evidenced by several observations:

• HSF2BP overexpression does not affect sensitivity to ionizing radiation

• It does not change BRCA2 concentration or nuclear localization

• It does not affect HR at restriction enzyme-induced DSBs (as measured by direct repeat GFP gene conversion)

• It induces chromosomal aberrations, including radial chromosome formation after MMC treatment - a hallmark of FA patient cells

The direct inhibitory effect on ICL repair has been biochemically recapitulated in the Xenopus egg extract-based ICL repair system, where addition of recombinant human or Xenopus HSF2BP reduced ICL repair efficiency by 70-80% .

What is the molecular mechanism by which HSF2BP-S167L variant impacts fertility?

The HSF2BP-S167L variant, identified in POI patients, acts as a hypomorphic allele compared to complete loss-of-function mutations. Functional analysis in mouse models has revealed the following molecular mechanisms:

• Decreased protein stability: The S167L variant leads to reduced loading of both HSF2BP and its partner BRME1 at recombination nodules

• Reduced recombinase activity: Meiocytes carrying this variant show a reduced number of foci formed by the recombinases RAD51/DMC1

• Lower crossover frequency: The diminished recombinase activity results in a decreased frequency of crossovers during meiosis

• Reproductive consequences: Female mice homozygous for the S167L variant (Hsf2bp S167L/S167L) show reduced fertility with smaller litter sizes

These molecular defects are less severe than those observed in complete knockout models, explaining the hypomorphic nature of this variant. The data suggest that meiotic progression requires a critical threshold level of HSF2BP/BRME1 for the proper loading of recombinases to the recombination nodules .

What is the significance of HSF2BP amplification in cancer cells?

HSF2BP, while normally restricted to germline tissues, shows aberrant expression in human cancer cell lines . Analysis of the cBioPortal Cancer Genomics database revealed that the HSF2BP gene is frequently amplified in certain cancers, reaching 1-2% in breast and ovarian cancers .

This amplification has significant implications:

• Genomic instability: Ectopic HSF2BP expression promotes genomic instability that could be beneficial for tumor evolution

• Mutual exclusivity with BRCA2 mutations: Amplification of HSF2BP rarely overlaps with mutations in BRCA2, indicating these events are likely mutually exclusive

• Therapeutic vulnerability: Cancer cells with elevated HSF2BP are hypersensitive to ICL-inducing agents (mitomycin C and cisplatin) and PARP inhibitors

• FA-like phenotype: HSF2BP overexpression creates a phenotype characteristic of cells from Fanconi anemia patients, establishing a new mechanism that can lead to an FA-like cellular phenotype without protein-coding mutations

This discovery provides a novel perspective on cancer pathogenesis, showing that ectopic expression of a wild-type meiotic protein can contribute to genomic instability in cancer. More importantly, it suggests that HSF2BP could be exploited as a targetable vulnerability in cancer therapy .

What experimental models are available to study HSF2BP function in meiosis?

Several experimental models have been developed to study HSF2BP function:

• Mouse models:

  • Knockout models: Complete deletion of Hsf2bp demonstrates essential roles in meiotic recombination

  • Knock-in models: Hsf2bp S167L/S167L mice replicate the hypomorphic phenotype observed in human patients

  • Exon deletion models: Deletion of exon 12 (encoding the first BRCA2-binding repeat) provides insights into the structural requirements for HSF2BP function

• Cell line models:

  • Overexpression systems: Stable expression of GFP-tagged or untagged HSF2BP in HeLa and U2OS cells enables study of its effects on DNA repair

  • RNAi knockdown: Silencing HSF2BP in cells that express it allows assessment of its necessity in various contexts

• In vitro biochemical systems:

  • Xenopus egg extract-based ICL repair system: This cell-free system recapitulates replication-dependent ICL repair and allows testing the direct effects of recombinant HSF2BP on repair efficiency

  • Protein interaction studies: Co-immunoprecipitation experiments with tagged HSF2BP reveal its interaction partners in testis extracts

• Structural biology approaches:

  • Crystal structures of HSF2BP fragments in complex with BRCA2 have revealed the molecular details of their interaction

These complementary approaches enable comprehensive investigation of HSF2BP function from molecular to organismal levels.

How can researchers detect and quantify HSF2BP expression in different tissues and cell types?

Detection and quantification of HSF2BP can be accomplished through several methods:

• Transcript analysis:

  • RT-PCR and qPCR can detect HSF2BP mRNA levels in different tissues

  • RNA-seq provides comprehensive transcriptome analysis, allowing detection of alternatively spliced forms

  • In situ hybridization can localize expression in tissue sections

• Protein detection:

  • Western blotting using anti-HSF2BP antibodies detects protein levels in total protein extracts

  • Immunohistochemistry or immunofluorescence visualizes HSF2BP in tissue sections or cells

  • For low expression levels, overexpression of tagged versions (GFP-HSF2BP) may facilitate detection

• Subcellular localization:

  • Immunofluorescence microscopy reveals the nuclear localization pattern of HSF2BP

  • Co-localization with DNA damage markers (like γH2AX) or other recombination proteins at recombination nodules can be assessed

• Protein complex analysis:

  • Co-immunoprecipitation with partners like BRME1, BRCA2, RAD51, or PALB2 helps determine whether HSF2BP is in functional complexes

  • Size exclusion chromatography can separate complexes containing HSF2BP based on molecular size

As HSF2BP expression is typically restricted to germline tissues but can be aberrantly expressed in cancer cells, careful selection of appropriate detection methods based on expected expression levels is crucial.

What approaches can be used to study the impact of HSF2BP variants on protein function?

Several complementary approaches can assess the functional impact of HSF2BP variants:

• Mouse model generation:

  • CRISPR/Cas9-mediated knock-in of specific variants (such as S167L) allows assessment of their effects on fertility and meiotic recombination in vivo

  • Comparison with knockout models differentiates hypomorphic from complete loss-of-function effects

• Cellular localization and stability:

  • Immunofluorescence microscopy to assess variant proteins' nuclear localization

  • Cycloheximide chase assays to measure protein stability and turnover rates

  • Western blotting to quantify steady-state protein levels

• Protein-protein interaction analysis:

  • Co-immunoprecipitation to assess if variants affect binding to partners like BRME1, BRCA2, and other recombination proteins

  • Yeast two-hybrid or mammalian two-hybrid assays to quantify interaction strengths

  • In vitro binding assays with purified components to measure direct binding affinities

• DNA repair capacity assays:

  • Sensitivity to DNA-damaging agents (MMC, cisplatin, PARP inhibitors) in cells expressing variant HSF2BP

  • Homologous recombination reporter assays to measure HR efficiency

  • Immunofluorescence for RAD51/DMC1 foci formation to assess recombinase loading

  • Crossover counting in meiotic spreads from model organisms expressing variants

• Structural analysis:

  • X-ray crystallography or cryo-EM of variant proteins in complex with partners to determine structural perturbations

  • Molecular dynamics simulations to predict variant effects on protein stability and interactions

These multifaceted approaches provide comprehensive insights into how specific variants affect HSF2BP function, from molecular interactions to physiological consequences.