Recombinant Human Histone H2B type W-T (H2BFWT)

What is the expression pattern of H2BFWT during spermatogenesis?

H2BFWT is exclusively expressed in testis, particularly during the mid-late spermatogonia stages of spermatogenesis . It is preferentially localized in the sub-telomeric regions and promoters of genes highly expressed in testis, from differentiated spermatogonia to early spermatocytes . In mature sperm, immunofluorescence studies have shown that H2BFWT is concentrated in spots located at the basal nuclear area of a subpopulation (approximately 20%) of cells .

What is the genomic location of the human H2BFWT gene?

The H2BFWT gene (officially designated as H2BW1) is located on Chromosome X in humans . This X-chromosomal location has implications for inheritance patterns of H2BFWT-related fertility issues.

How does H2BFWT incorporation affect nucleosome dynamics?

Single-molecule optical tweezers experiments have demonstrated that H2BFWT nucleosomes exhibit a diminished rewrapping rate and an increased unwrapping rate compared to conventional nucleosomes. This translates to approximately 40% destabilization in the H2A/H2B-DNA interaction region . Despite these alterations in stability, H2BFWT nucleosomes can be efficiently remodeled and mobilized by chromatin remodeling complexes such as SWI/SNF, unlike some other histone variants that interfere with remodeling .

What H2BFWT polymorphisms are associated with male infertility?

Two common single nucleotide polymorphisms (SNPs) in the H2BFWT gene, -9C>T and 368A>G, have been significantly associated with male infertility, particularly with idiopathic azoospermia or oligozoospermia . The -9C>T polymorphism is located in the promoter region and may affect transcriptional regulation, while the 368A>G transition results in a histidine to arginine substitution at position 100 (H100R) in the protein .

What is the prevalence of H2BFWT polymorphisms in infertile populations?

Studies have shown significant differences in allele frequencies between infertile men and fertile controls. In a Chinese population study, the frequencies of the -9T allele (52.8% vs. 41.6%, p=0.009) and 368G allele (43.0% vs. 32.5%, p=0.012) were significantly higher in infertile patients than in controls . After stratifying patients, the -9T allele was significantly more frequent in men with azoospermia (57.4% vs. 41.6%, p=0.001), while the 368G allele was more common in men with oligozoospermia (45.4% vs. 32.5%, p=0.007) .

How do H2BFWT polymorphisms affect protein function?

The 368A>G (H100R) polymorphism significantly impacts protein structure and function. Structural analysis using prediction tools indicates that this SNP is likely deleterious (SIFT prediction: deleterious, score: -2.55) . Mechanistically, H2BFWTH100R further destabilizes nucleosomes by changing the surface electrostatic property to a positive charge, which repulses the nearby positively charged H4K91/R92, interfering with these interactions and increasing nucleosome unwrapping rates .

What haplotype patterns of H2BFWT SNPs are relevant to male infertility?

Haplotype analysis has revealed significant patterns associated with infertility risk. The haplotype CA (containing the wild-type alleles at both positions) was significantly decreased in infertile patients compared to controls (22.8% vs. 33.0%, p=0.006), whereas the TG haplotype (containing both variant alleles) was significantly increased (18.3% vs. 7.2%, p<0.001) . This suggests that the combination of these polymorphisms may have a synergistic effect on infertility risk.

What methods are optimal for expressing and purifying recombinant H2BFWT?

For in vitro studies, recombinant H2BFWT can be expressed in bacterial systems and purified to homogeneity using standard histone purification protocols. Studies have successfully used purified recombinant H2BFWT along with canonical histones (H2A, H3, and H4) to reconstitute positioned nucleosomes on DNA fragments, such as the 152-bp fragment containing the X. borealis 5S rRNA gene . Quality control should include SDS-PAGE analysis to confirm protein purity and proper incorporation into nucleosomes.

How can researchers analyze H2BFWT nucleosome stability?

Multiple complementary approaches can assess H2BFWT nucleosome stability:

Single-molecule optical tweezers experiments have been particularly valuable in demonstrating that H2BFWT nucleosomes exhibit approximately 40% destabilization in the H2A/H2B-DNA interaction region .

What experimental approaches can determine the genomic distribution of H2BFWT?

ChIP-seq (Chromatin Immunoprecipitation followed by sequencing) using H2BFWT-specific antibodies is the primary method for mapping the genome-wide distribution of this histone variant. Studies have shown that H2BFWT is preferentially localized in sub-telomeric regions and promoters of genes highly expressed in testis . For immunofluorescence microscopy, HA-tagged H2BFWT can be expressed in cells and visualized using anti-HA antibodies to determine subcellular localization throughout the cell cycle .

How can researchers investigate the functional consequences of H2BFWT incorporation?

In vitro transcription assays have demonstrated that RNA Polymerase II can transcribe through H2BFWT nucleosomes more efficiently than through conventional nucleosomes, suggesting a role in facilitating gene expression . For studying effects on chromosome condensation, mitotic chromosome assembly assays have shown that, unlike conventional H2B, H2BFWT is unable to recruit chromosome condensation factors, a property determined by its divergent N-terminal tail .

What methods can detect H2BFWT in sperm and testis samples?

For detecting H2BFWT in sperm samples, isolated nucleosomes can be purified through a sucrose gradient containing 0.6 M NaCl to remove non-specifically associated proteins, followed by electrophoretic analysis and Western blotting with H2BFWT-specific antibodies . Immunofluorescence microscopy has revealed that H2BFWT is concentrated in spots located at the basal nuclear area in approximately 20% of mature sperm cells .

How does H2BFWT contribute to chromatin remodeling during spermatogenesis?

H2BFWT appears to play a role in regulating spermatogenesis-related gene expression by decreasing transcriptional barriers through its nucleosome-destabilizing effects . Unlike some histone variants that interfere with chromatin remodeling, H2BFWT nucleosomes can be efficiently remodeled and mobilized by SWI/SNF complexes, similar to conventional nucleosomes . This suggests that H2BFWT may facilitate both active remodeling processes and transcription during specific stages of spermatogenesis.

What are the molecular determinants of H2BFWT's unique properties?

The distinctive properties of H2BFWT are largely determined by its amino acid sequence, particularly in the N-terminal tail which shows the lowest homology with conventional H2B. Experimental evidence using chimeric proteins has demonstrated that replacing the N-terminal tail of H2BFWT with that of conventional H2B rescues the ability to recruit chromosome condensation factors and participate in mitotic chromosome assembly . This indicates that the N-terminal domain is critical for the functional specificity of H2BFWT.

How might H2BFWT function as an epigenetic marker?

H2BFWT has been proposed to act as a specific epigenetic marker based on several observations: (1) it is found in sperm nuclei and appears to be associated with telomeric chromatin, (2) it shows a specific distribution pattern in a subpopulation of sperm cells, and (3) it has unique functional properties distinct from conventional H2B . This suggests that H2BFWT may contribute to the epigenetic programming of certain genomic regions during spermatogenesis and potentially influence post-fertilization events.

What is the evolutionary significance of H2BFWT being specific to primates?

Unlike many histone variants that are conserved across species, H2BFWT appears to be specific to primates . This primate-specific expression suggests a relatively recent evolutionary adaptation potentially related to unique aspects of primate reproduction. Comparative genomic studies could provide insights into the evolutionary forces driving the emergence and maintenance of this specialized histone variant.

How do testis-specific histone variants collectively contribute to spermatogenesis?

H2BFWT is part of a broader repertoire of testis-specific histone variants involved in the dynamic chromatin transitions during spermatogenesis. Another testis-specific H2B variant, hTSH2B, has also been identified with 85% homology to somatic H2B . Understanding how these variants work together, potentially in different stages or genomic contexts, is essential for comprehending the complex chromatin reorganization that occurs during sperm development.

How can researchers distinguish between causative effects of H2BFWT variants and mere associations?

To establish causality beyond association studies, researchers should: (1) perform functional studies demonstrating mechanistic links between H2BFWT variants and altered nucleosome stability, (2) correlate specific variants with detailed clinical phenotypes, (3) use bioinformatic predictions to assess functional consequences (as done with SNAP and SIFT for the H100R variant), and (4) develop model systems to test the effects of variants on spermatogenesis . The combination of structural analysis, in vitro functional studies, and genetic association data provides stronger evidence for causative relationships.

What considerations are important when interpreting H2BFWT localization patterns?

When analyzing the genomic distribution of H2BFWT, researchers should consider: (1) the developmental timing of H2BFWT expression during spermatogenesis, (2) co-localization with other chromatin features or histone modifications, (3) correlation with transcriptional activity of associated genes, and (4) changes in distribution patterns in response to cellular signals or stress. The preferential localization of H2BFWT in sub-telomeric regions and promoters of testis-specific genes suggests specific regulatory functions that should be interpreted in the context of spermatogenic gene expression programs .