Recombinant Xenopus tropicalis Myb/SANT-like DNA-binding domain-containing protein 4 (msantd4)

What is msantd4 and what are its known molecular characteristics?

Msantd4 (Myb/SANT-like DNA-binding domain-containing protein 4 with coiled-coils) is a nuclear protein that contains DNA-binding domains similar to those found in Myb and SANT proteins. The protein has been identified in various species with high conservation, with Xenopus MSANTD4 showing more than 72% sequence identity to human and mouse MSANTD4 . In humans, this gene is also known as KIAA1826 and is located on chromosome 11q22.3 . The full-length Xenopus tropicalis MSANTD4 protein consists of 408 amino acids and contains distinctive structural features including DNA-binding domains and coiled-coil regions that are likely important for its nuclear localization and function .

What is the genomic organization of msantd4?

The msantd4 gene in humans is located on chromosome 11q22.3 and contains 4 exons . It is encoded on the complement strand with genomic coordinates NC_000011.10 (106007899..106022287) according to NCBI's Gene database . While the specific genomic organization in Xenopus tropicalis has not been fully detailed in the search results, the high conservation between species suggests similar organization. Researchers studying this gene should consider the exon-intron structure when designing primers for expression studies or when planning gene modification experiments.

What is the expression pattern of msantd4 during development?

Semi-quantitative RT-PCR analysis has demonstrated that msantd4 transcripts are detected at all stages of development in Xenopus laevis and in numerous adult tissues . Whole-mount in situ hybridization revealed that msantd4 is expressed at the animal pole from stage 2 onward . This broad developmental expression pattern suggests msantd4 may play fundamental roles in early embryonic development and continue to function in various tissues throughout adulthood.

What are the known molecular functions of msantd4?

Msantd4 has been revealed to function as a repressor of HSPB1 and is indirectly involved in the cold shock response . Its Myb/SANT-like DNA-binding domain suggests it may function as a transcription factor or regulator of gene expression. Studies in Xenopus laevis have shown that both knockdown and overexpression of msantd4 resulted in inhibition of cell proliferation and pro-apoptotic effects in embryos . This suggests msantd4 plays critical roles in regulating cell cycle progression and cell survival during development, with precise expression levels being important for normal development.

How does msantd4 regulate cell proliferation and apoptosis?

The regulatory mechanisms through which msantd4 impacts cell proliferation and apoptosis have been studied using TUNEL and Phospho-Histone 3 (PH3) staining analyses in Xenopus embryos. These studies revealed that both knockdown and overexpression of msantd4 resulted in inhibition of cell proliferation and pro-apoptotic effects . This bidirectional effect suggests that msantd4 likely maintains a delicate balance in cellular homeostasis, where precise levels are required for normal development. The molecular pathways through which msantd4 exerts these effects likely involve its DNA-binding capabilities and potential interactions with other regulatory factors, though the specific downstream targets and signaling cascades require further investigation.

What is the relationship between msantd4 and Pick disease?

Gene-disease retrieval analyses have implied that the msantd4 gene may be attributed to Pick disease . Supporting this connection, inhibition of msantd4 in Xenopus embryos using antisense morpholino oligonucleotides resulted in atrophy of brain and body segments resembling Pick disease symptoms . Pick disease is a rare neurodegenerative disorder characterized by frontotemporal lobar degeneration, and the link to msantd4 suggests this gene may play roles in neuronal maintenance or protection. The molecular mechanisms underlying this connection likely involve msantd4's influence on cell cycle regulation and apoptosis, as aberrant control of these processes could contribute to neurodegeneration.

What techniques are recommended for studying msantd4 expression in Xenopus?

For studying msantd4 expression in Xenopus, several complementary techniques have proven effective:

• Semi-quantitative RT-PCR: This technique has successfully demonstrated msantd4 transcript presence across developmental stages and in adult tissues .

• Whole-mount in situ hybridization: This method effectively revealed spatial expression patterns, showing msantd4 expression at the animal pole from stage 2 onward .

• Quantitative PCR (qPCR): For more precise measurement of expression levels.

• Western blotting: Using specific antibodies against MSANTD4 to detect protein levels, with recombinant proteins serving as positive controls .

When designing primers for these techniques, researchers should consider the conserved regions between species while ensuring specificity for the Xenopus variant.

What are effective approaches for msantd4 loss-of-function studies in Xenopus?

The most well-documented approach for msantd4 loss-of-function studies in Xenopus involves antisense morpholino oligonucleotides (MOs) . These MOs are designed to bind to msantd4 mRNA and block translation or interfere with splicing. When implementing this technique:

• Design MOs targeting the translation start site or splice junctions

• Include appropriate controls such as mismatch MOs or standard control MOs

• Validate knockdown efficiency via Western blot or RT-PCR

• Consider rescue experiments by co-injecting MO-resistant msantd4 mRNA

The phenotypic outcomes of msantd4 inhibition using MOs included atrophy of brain and body segments, inhibition of cell proliferation, and induction of apoptosis . These effects can be assessed using TUNEL staining for apoptosis and Phospho-Histone 3 (PH3) staining for cell proliferation.

How can recombinant msantd4 protein be produced and purified for biochemical studies?

Recombinant Xenopus tropicalis MSANTD4 protein can be produced using several expression systems:

• Yeast expression system: This has been successfully used to produce full-length (AA 1-408) MSANTD4 with a His tag . Yeast systems can provide eukaryotic post-translational modifications.

• Wheat germ cell-free system: While not specifically mentioned for Xenopus MSANTD4, this system has been used for human MSANTD4 expression with GST tags .

For purification, affinity chromatography using the appropriate tag (His or GST) is recommended, followed by size exclusion chromatography for higher purity. The purified protein can be validated using SDS-PAGE and Western blotting with anti-tag antibodies. Based on commercial preparations, purity greater than 90% can be achieved . The recombinant protein can be used for various applications including ELISA, protein-protein interaction studies, and as antigens for antibody production.

How might msantd4 interact with other transcription factors in development?

Given that msantd4 contains Myb/SANT-like DNA-binding domains, it likely functions within transcriptional regulatory networks. While specific interaction partners have not been fully characterized in the search results, its nuclear localization and DNA-binding capacity suggest it may function similarly to other transcription factors studied in Xenopus development, such as the Mix family proteins described in result .

A methodological approach to investigate msantd4 interactions would include:

• Co-immunoprecipitation: Using tagged msantd4 to pull down interacting proteins, followed by mass spectrometry identification

• ChIP-seq: To identify genomic binding regions of msantd4

• Yeast two-hybrid screening: To identify direct protein-protein interactions

• Comparison with other DNA-binding proteins: Particularly those with similar domains like the Myb/SANT family

Understanding these interactions would provide insight into how msantd4 contributes to developmental gene regulation networks and potentially its role in Pick disease pathogenesis.

What is the evolutionary conservation of msantd4 and what can this tell us about its function?

The high sequence identity (>72%) between Xenopus, human, and mouse MSANTD4 proteins suggests strong evolutionary conservation . This conservation likely reflects important functional constraints on the protein. To further investigate evolutionary aspects:

• Phylogenetic analysis: Construct phylogenetic trees using msantd4 sequences from diverse vertebrate and invertebrate species

• Domain conservation analysis: Determine which domains show highest conservation

• Synteny analysis: Examine conservation of genomic context around msantd4 across species

Comparative functional studies in different model organisms could reveal conserved versus species-specific roles. The high conservation of this protein suggests it likely plays fundamental roles in cellular processes that have been maintained throughout vertebrate evolution.

What are the most promising experimental approaches to elucidate msantd4's role in Pick disease pathogenesis?

Given the potential link between msantd4 and Pick disease , several experimental approaches could help elucidate this connection:

• Patient-derived samples: Analyze msantd4 expression and mutation status in Pick disease patient samples compared to controls

• Animal models: Develop and characterize conditional knockout or transgenic mouse models with altered msantd4 expression specifically in neuronal tissues

• Cellular models: Using iPSC-derived neurons from Pick disease patients or CRISPR-modified cell lines with msantd4 mutations to study cellular phenotypes

• Biochemical studies: Investigate if msantd4 interacts with tau or other proteins implicated in Pick disease pathology

• Transcriptomic analysis: Compare gene expression profiles between msantd4-depleted neurons and Pick disease samples to identify common pathways

The observation that msantd4 inhibition in Xenopus resulted in brain atrophy resembling Pick disease symptoms provides a foundation for these investigations . Researchers should employ multiple approaches and consider that the mechanisms might differ between amphibians and mammals.

How should researchers interpret phenotypic data from msantd4 manipulation experiments?

When interpreting phenotypic data from msantd4 manipulation experiments, researchers should consider several important factors:

• Dose-dependency: Both knockdown and overexpression of msantd4 resulted in inhibition of cell proliferation and pro-apoptosis in embryos , suggesting a potential "Goldilocks effect" where precise levels are required for normal development.

• Temporal considerations: The timing of msantd4 manipulation relative to developmental stages is critical, as its expression begins at the animal pole from stage 2 onward .

• Tissue specificity: While msantd4 is expressed in numerous adult tissues , phenotypic effects may vary by tissue type due to different molecular contexts.

• Compensatory mechanisms: Consider the potential for genetic compensation by related genes following msantd4 manipulation.

• Technical validation: Always validate knockdown or overexpression efficiency at both mRNA and protein levels to correlate with observed phenotypes.

A standardized scoring system for phenotypic severity can help quantify results and enable statistical comparisons across experimental conditions.

What tools and databases are available for studying msantd4 variants and their potential clinical significance?

Several resources are available for studying msantd4 variants and their potential clinical significance:

When analyzing variants, researchers should consider both coding and non-coding regions, as regulatory variants may affect expression levels without altering protein sequence. The observation that both increased and decreased msantd4 levels affect development suggests that regulatory variants might be particularly relevant to disease mechanisms.

How can differential expression of msantd4 in normal versus disease states be accurately quantified and interpreted?

For accurate quantification and interpretation of differential msantd4 expression:

• Reference gene selection: When performing qPCR, carefully select stable reference genes appropriate for the specific tissues and conditions being compared.

• Multiple techniques: Validate findings using complementary approaches such as qPCR, Western blotting, and in situ hybridization.

• Single-cell approaches: Consider single-cell RNA-seq to identify cell-type specific changes that might be masked in bulk tissue analysis.

• Protein localization: Assess not only total protein levels but also subcellular localization using immunofluorescence.

• Isoform analysis: Examine potential differential expression of specific msantd4 isoforms, as the gene has 4 exons and may produce multiple transcript variants.

When interpreting results, researchers should recognize that altered expression might be either causative or compensatory in disease states. Correlation with other disease markers and examination of temporal progression can help distinguish these possibilities.

What are the common technical challenges in working with recombinant msantd4 protein and how can they be overcome?

Working with recombinant msantd4 protein presents several technical challenges:

• Protein solubility: MSANTD4 contains DNA-binding domains and coiled-coil regions that may affect solubility. To address this:

  • Optimize expression conditions (temperature, induction time)

  • Test different solubilization buffers with varying salt concentrations

  • Consider fusion tags known to enhance solubility (MBP, SUMO)

  • Express specific domains separately if the full-length protein proves problematic

• Maintaining protein activity: DNA-binding proteins can lose activity during purification. Solutions include:

  • Include DNA-binding competitors or chaperones during purification

  • Avoid freeze-thaw cycles

  • Test activity immediately after purification

• Specificity validation: Confirm the specificity of the recombinant protein:

  • Use Western blotting with specific antibodies

  • Perform functional assays to verify DNA-binding capability

  • Compare activities of wild-type and mutant variants

The available commercial recombinant preparations have achieved >90% purity , suggesting these challenges can be overcome with appropriate optimization.

How can researchers troubleshoot unsuccessful msantd4 knockdown or overexpression experiments?

When troubleshooting unsuccessful msantd4 manipulation experiments:

• For morpholino knockdowns:

  • Verify morpholino binding site conservation in your specific Xenopus population

  • Design and test alternative morpholinos targeting different regions

  • Use Western blotting to confirm protein reduction

  • Consider using a combination of splice-blocking and translation-blocking morpholinos

• For overexpression:

  • Verify mRNA quality by gel electrophoresis before injection

  • Test different doses to find optimal expression levels

  • Confirm protein expression by Western blotting

  • Consider using inducible expression systems for temporal control

• General considerations:

  • Validate injection technique using lineage tracers

  • Include appropriate positive controls known to produce phenotypes

  • Consider potential genetic compensation mechanisms

  • Account for maternal contribution of msantd4 in early developmental stages

For advanced approaches, CRISPR/Cas9-mediated genome editing can provide more complete knockout than morpholinos, though this requires careful design to avoid off-target effects.

What are the best practices for designing experiments to study msantd4 and HSPB1 interaction?

As msantd4 has been revealed to function as a repressor of HSPB1 , investigating this interaction requires careful experimental design:

• Protein-protein interaction studies:

  • Co-immunoprecipitation using antibodies against native proteins or tags

  • Proximity ligation assay for detecting interactions in situ

  • FRET or BiFC for monitoring interactions in living cells

  • In vitro binding assays using purified recombinant proteins

• Transcriptional regulation analysis:

  • Luciferase reporter assays using HSPB1 promoter constructs

  • ChIP assays to detect msantd4 binding to HSPB1 regulatory regions

  • Expression correlation studies across tissues and developmental stages

  • Response element mapping through deletion/mutation analysis

• Functional relationship studies:

  • Rescue experiments combining HSPB1 manipulation with msantd4 knockdown/overexpression

  • Cold shock response assays with varying levels of both proteins

  • Epistasis analysis through sequential and simultaneous manipulation

When designing these experiments, researchers should be mindful of the cellular context, as the interaction may be contingent on specific developmental stages, cell types, or stress conditions.