Recombinant Human Doublesex- and mab-3-related transcription factor B1 (DMRTB1)

What is the basic structure and function of human DMRTB1?

DMRTB1 is a transcription factor belonging to the DMRT family characterized by the DM domain DNA binding motif. Unlike its better-studied family member DMRT1, DMRTB1 contains a proline-rich C-terminal region that may confer unique functional properties. DMRTB1 plays a pivotal role in coordinating the transition between mitosis and meiosis in germ cells, particularly in spermatogenesis. The protein functions through its ability to bind specific DNA sequences and regulate transcription of target genes involved in cellular differentiation .

What expression pattern does DMRTB1 show in normal human testicular tissue?

In men with normal spermatogenesis, DMRTB1 shows strong immunoreactivity in a specific subset of spermatogonia (38.34 ± 2.14%). Immunohistochemical analysis reveals that DMRTB1 is predominantly expressed in A pale and B spermatogonia, as well as in primary spermatocytes in (pre-)leptotene, zygotene, and pachytene stages, which show weaker immunostaining. Adjacent Sertoli cells are consistently immunonegative for DMRTB1 .

How do expression patterns of DMRTB1 differ in pathological conditions?

In patients with spermatogenic arrest at the spermatogonial level, an altered DMRTB1 staining pattern is observed compared to normal spermatogenesis. No DMRTB1 immunoreactivity is detected in Sertoli cells in Sertoli cell-only syndrome. In germ cell neoplasia in situ (GCNIS) tubules, pre-invasive tumor cells are predominantly immunonegative for DMRTB1, with only approximately 0.4 ± 0.03% showing any immunostaining. Seminoma cells consistently show no DMRTB1 immunostaining .

What are the recommended methods for producing recombinant human DMRTB1 for research purposes?

Recombinant human DMRTB1 can be expressed and purified from various host systems, each with distinct advantages:

• E. coli and yeast expression systems offer the highest yields and shortest turnaround times, making them suitable for structural studies and antibody production.

• HEK-293 cells provide a mammalian expression system that can introduce appropriate post-translational modifications. This system is recommended when studying protein-protein interactions or functional assays requiring properly folded protein.

• Insect cells with baculovirus represent an intermediate option that balances yield with post-translational modifications.

The choice of expression system should be guided by the intended experimental application. For functional studies, mammalian or insect cell expression is preferable to ensure proper folding and modifications .

What immunohistochemical approaches are most effective for detecting DMRTB1 in human tissue samples?

For optimal DMRTB1 detection in human testicular tissue, the following protocol has proven effective:

• Use 5 μm sections of paraffin-embedded testicular biopsies fixed in Bouin's solution.

• Perform antigen retrieval using citrate buffer (pH 6.0).

• Apply a commercial rabbit polyclonal anti-DMRTB1 primary antibody.

• Use biotinylated secondary antibodies and streptavidin-peroxidase detection systems.

• Counterstain with hematoxylin for cell identification.

To further characterize DMRTB1-positive cells, consider co-staining with markers such as Ki-67 (proliferation), PLAP (tumor marker), or DMRT1 (related transcription factor). Complementing immunohistochemistry with RT-PCR provides validation of expression at the mRNA level .

How can one establish specificity controls when studying DMRTB1 in experimental models?

To establish specificity of DMRTB1 detection and function:

• Antibody validation: Perform Western blot analysis using recombinant DMRTB1 protein alongside tissue lysates.

• Knockdown/knockout controls: Use siRNA/shRNA against DMRTB1 or CRISPR/Cas9-mediated knockout models to confirm antibody specificity and validate functional findings.

• Cross-reactivity assessment: Test for potential cross-reactivity with other DMRT family members, particularly DMRT1, which shares structural similarities.

• Peptide competition assays: Pre-incubate antibodies with purified DMRTB1 peptide to confirm binding specificity.

• Multiple antibody approach: Use antibodies targeting different epitopes of DMRTB1 to confirm localization patterns.

These controls are essential for establishing the reliability of DMRTB1-focused research and distinguishing its function from other DMRT family members .

How does DMRTB1 coordinate the transition between mitosis and meiosis in human germ cells?

DMRTB1 appears to function as a critical regulator of the mitosis-meiosis transition in male germ cells, though the exact mechanisms in humans remain incompletely characterized. Based on studies of DMRT family proteins and limited DMRTB1-specific research:

• DMRTB1 likely regulates expression of cell cycle proteins that control the exit from mitotic proliferation.

• It may interact with or regulate key meiotic initiators, potentially including STRA8, which is known to be regulated by DMRT1.

• The altered DMRTB1 expression pattern observed in spermatogenic arrest at the spermatogonial level suggests it may be crucial for the transformation of A spermatogonia into B spermatogonia.

• The protein may function through pioneer transcription factor activity similar to DMRT1, binding to closed chromatin and facilitating access for other transcriptional regulators.

Further research using ChIP-seq and conditional knockout models is needed to fully elucidate the target genes and molecular mechanisms through which DMRTB1 controls this critical developmental transition .

What is known about the interactions between DMRTB1 and other DMRT family members in reproductive development?

While direct interactions between DMRTB1 and other DMRT family members have not been extensively characterized, several patterns suggest potential functional relationships:

• DMRT family members can potentially form mixed complexes, as demonstrated by in vitro and in vivo studies of other DMRT proteins.

• DMRT1 has been shown to bind its own promoter and those of six other Dmrt genes, suggesting auto- and cross-regulation within this gene family.

• The expression patterns of DMRTB1 and DMRT1 in testicular tissue show some overlap but distinct differences, suggesting complementary rather than redundant functions.

The stoichiometry of DMRT protein binding to DNA may be functionally significant, as demonstrated by studies of DMRT1 where different binding stoichiometries may lead to different regulatory outcomes. Whether DMRTB1 exhibits similar variable stoichiometry binding and potential for heteromeric interactions with other DMRT proteins remains an important research question .

What role might DMRTB1 play in testicular pathologies and germ cell tumors?

The absence of DMRTB1 in germ cell neoplasia in situ (GCNIS) and seminoma cells suggests potential tumor suppressor functions:

• The near-complete absence of DMRTB1 in pre-invasive tumor cells (only 0.4% showing immunoreactivity) contrasts with its expression in normal spermatogonia.

• This absence might be associated with uncontrolled neoplastic cell proliferation and progression into invasive germ cell tumors.

• If DMRTB1 functions similarly to DMRT1, which has demonstrated tumor suppressor properties in mouse models, it may regulate pluripotency factors and cell cycle proteins that prevent malignant transformation.

Research examining DMRTB1 expression in larger cohorts of testicular germ cell tumors and functional studies in cell models are needed to confirm this potential tumor suppressor role and identify the mechanisms involved .

How can ChIP-sequencing be optimized to identify DMRTB1 binding sites and target genes?

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) for DMRTB1 requires careful optimization:

• Antibody selection: Use ChIP-grade antibodies specifically validated for DMRTB1, as cross-reactivity with other DMRT family members could confound results.

• Cross-linking optimization: Due to potential pioneer factor activity (as seen with DMRT1), test multiple formaldehyde concentrations and fixation times to capture both stable and transient interactions.

• Sonication parameters: Optimize sonication conditions to generate DNA fragments of 200-300 bp for highest resolution of binding sites.

• Controls: Include:

  • Input chromatin controls

  • IgG antibody controls

  • DMRTB1 knockdown/knockout samples as negative controls

  • Positive controls targeting known DMRT binding regions

• Motif analysis: Apply computational approaches similar to those used for DMRT1 to identify DMRTB1-specific binding motifs, which may resemble the conserved DM domain recognition sequence.

• Integration with transcriptome data: Combine ChIP-seq with RNA-seq from DMRTB1 knockdown/overexpression experiments to correlate binding with gene regulation .

What are the key considerations when designing conditional knockout models to study DMRTB1 function?

When designing conditional knockout models for DMRTB1 studies:

• Targeting strategy:

  • Design floxed alleles that target critical exons encoding the DM domain to ensure complete loss of function.

  • Consider potential regulatory elements that might affect expression of neighboring genes.

• Cre driver selection:

  • For reproductive studies, consider germ cell-specific drivers (e.g., Ngn3-Cre for undifferentiated spermatogonia).

  • For broader developmental studies, use stage-specific or inducible Cre systems.

• Genetic background considerations:

  • Be aware that genetic background significantly affects phenotypes of DMRT family member knockouts (as demonstrated with DMRT1).

  • Consider using mixed backgrounds with appropriate controls or backcrossing to pure strains.

• Phenotypic analysis pipeline:

  • Establish comprehensive evaluation protocols for spermatogenesis including histology, immunostaining, and fertility assessment.

  • Include molecular analyses (RNA-seq, ChIP-seq) at key developmental timepoints.

• Compound mutant considerations:

  • Design strategies to assess functional redundancy with other DMRT family members through compound mutants.

These considerations will help develop models that accurately reveal DMRTB1's specific functions while accounting for potential compensatory mechanisms .

What cellular models are most appropriate for studying DMRTB1 function in vitro?

For in vitro studies of DMRTB1 function, consider the following cellular models:

• Primary human spermatogonial stem cell cultures:

  • Most physiologically relevant but technically challenging

  • Maintain expression of key germ cell markers and DMRTB1

• Testicular cell lines:

  • GC-1 spg and GC-2 spd cell lines (mouse spermatogonial and spermatocyte-like cells)

  • TCam-2 (human seminoma cell line)

  • Check for endogenous DMRTB1 expression before use

• Inducible expression systems:

  • Establish stable cell lines with doxycycline-inducible DMRTB1 expression

  • Useful for studying dose-dependent and temporal effects

• Cell differentiation models:

  • Human induced pluripotent stem cells (hiPSCs) differentiated toward germ cell lineage

  • Can recapitulate aspects of the mitosis-to-meiosis transition

• Three-dimensional organoid cultures:

  • Testicular organoids that better represent the cellular microenvironment

  • Allow for more complex cell-cell interactions

When selecting a model, consider the specific aspect of DMRTB1 function being studied, whether it requires the context of the mitosis-meiosis transition, and the availability of interacting partners identified in vivo .

How can multi-omics approaches advance our understanding of DMRTB1 function?

Integrating multiple omics technologies can provide comprehensive insights into DMRTB1 function:

• Integrated genomics approach:

  • Combine ChIP-seq (binding sites), ATAC-seq (chromatin accessibility), and RNA-seq (expression changes) to identify direct and indirect targets

  • Apply to both DMRTB1 overexpression and knockdown models

• Proteomics integration:

  • Use IP-mass spectrometry to identify DMRTB1 interacting proteins

  • Perform phosphoproteomics to map signaling pathways affected by DMRTB1

• Single-cell multi-omics:

  • Apply scRNA-seq and scATAC-seq to heterogeneous testicular tissue to identify cell-specific functions

  • Track developmental trajectories dependent on DMRTB1 expression

• Computational modeling:

  • Develop gene regulatory network models incorporating DMRTB1

  • Use machine learning to predict cellular responses to DMRTB1 perturbation

• Data visualization and integration:

  • Create comprehensive databases of DMRTB1-dependent events across different experimental conditions

  • Develop computational tools to integrate DMRTB1 data with existing databases on spermatogenesis

This multi-dimensional approach can reveal how DMRTB1 coordinates complex developmental transitions and interacts with other regulatory networks .

What are the potential clinical implications of DMRTB1 research for male infertility diagnosis and treatment?

DMRTB1 research may have significant clinical applications for male infertility:

• Diagnostic biomarker development:

  • The altered expression patterns of DMRTB1 in spermatogenic arrest suggest potential as a diagnostic marker

  • Testicular biopsy immunostaining for DMRTB1 could help classify specific forms of spermatogenic failure

• Genetic testing applications:

  • Screening for DMRTB1 mutations or polymorphisms in infertile men

  • Development of prognostic indicators based on DMRTB1 status

• Therapeutic target exploration:

  • Potential for modulating DMRTB1 activity to overcome specific blocks in spermatogenesis

  • Development of small molecules that might enhance or restore DMRTB1 function

• In vitro gametogenesis implications:

  • Understanding DMRTB1's role in the mitosis-meiosis transition could improve protocols for generating functional gametes in vitro

  • Potential application in fertility preservation strategies

• Comparative DMRT family analysis:

  • Understanding the interrelated functions of DMRT family members could lead to more comprehensive diagnostic and therapeutic approaches

These applications require further validation of DMRTB1's precise mechanisms in human spermatogenesis and careful clinical correlation studies .

How might DMRTB1 function in non-reproductive tissues and what are the implications for broader human biology?

While DMRTB1 is primarily studied in reproductive contexts, potential roles in other tissues warrant investigation:

• Neurological development and function:

  • Other DMRT family members have demonstrated roles in nervous system development

  • Expression patterns in neural tissues should be characterized

• Immune system regulation:

  • DMRT1 has been shown to regulate immune responses by repressing TLR4-dependent inflammatory signaling

  • DMRTB1 may have similar immunomodulatory functions that remain unexplored

• Cancer biology beyond reproductive tissues:

  • The potential tumor suppressor role observed in testicular tissue may extend to other tissues

  • Analysis of DMRTB1 expression in various cancer types could reveal new biomarkers

• Developmental biology:

  • Early embryonic expression patterns outside of gonadal tissues should be systematically characterized

  • Potential roles in cell fate decisions in diverse lineages

• Regenerative medicine:

  • If DMRTB1 regulates cellular transitions in non-reproductive contexts, it could have applications in directing cell differentiation for regenerative medicine

These broader investigations may reveal unexpected functions of DMRTB1 and connect reproductive biology to other areas of human health and disease .

Table 1: DMRTB1 Expression in Human Testicular Cell Types

Data compiled from immunohistochemical studies of human testicular biopsy specimens

Table 2: Comparison of DMRTB1 and DMRT1 Features and Functions

Data compiled from multiple studies on DMRT family proteins

Table 3: Recommended Recombinant DMRTB1 Expression Systems for Different Research Applications