Recombinant Pan troglodytes WD repeat-containing protein 13 (WDR13)

How conserved is WDR13 across primate species and what does this suggest about its evolutionary importance?

WDR13 demonstrates a highly conserved coding sequence across primate species, suggesting significant evolutionary constraint and functional importance . This conservation is particularly relevant when considering that chimpanzees share approximately 98% of their DNA with humans . The preservation of WDR13 across species indicates it likely serves critical cellular functions that have been maintained throughout primate evolution.

Comparative genomic studies between human and chimpanzee WDR13 could reveal subtle species-specific adaptations while maintaining core functionality. The high conservation may also indicate that experimental findings from human or mouse WDR13 studies might be translatable to chimpanzee research, providing valuable insights into protein function across primates. This conservation makes WDR13 an excellent candidate for evolutionary studies examining functional divergence or conservation of regulatory mechanisms.

What cellular localization and expression patterns does WDR13 exhibit?

WDR13 protein primarily localizes to the nucleus, suggesting it plays a regulatory role in nuclear function in addition to mediating protein-protein interactions . Expression studies have detected WDR13 in all tissues analyzed, though with significantly varied expression levels among different tissues .

In experimental settings, researchers should anticipate differential expression across tissue types when working with recombinant Pan troglodytes WDR13. This variability may reflect tissue-specific functions that could be important when designing experiments targeting particular physiological systems. Subcellular fractionation techniques followed by Western blotting would be the recommended methodology for confirming localization patterns in chimpanzee cells, similar to protocols established for human WDR13 localization studies.

What evidence suggests WDR13 plays a role in cognitive function?

Multiple lines of evidence suggest WDR13 involvement in cognitive function. Studies with Wdr13 knockout mice have revealed alterations in learning and memory capabilities . Specifically, Wdr13-deficient mice demonstrated better performance in spatial memory tasks such as the Morris water maze test, where they spent significantly more time in target quadrants during probe trials (p < 0.005) . Additionally, these knockout mice showed enhanced long-term memory retention when subjected to probe trials 20 days after the learning phase (p < 0.005) .

The molecular basis for these cognitive effects appears to involve altered expression of key synaptic proteins. Wdr13 knockout mice showed increased expression of synaptic genes, including Syn1 (synapsin1), Rab3a, and Camk2a, alongside changes in immediate early genes like Arc and c-Fos when exposed to novel environments . These proteins are critically involved in synaptic plasticity, which underlies learning and memory processes.

For researchers working with recombinant Pan troglodytes WDR13, these findings suggest that cognitive function assessments should be a primary focus area, particularly in comparative studies examining evolutionary adaptations in cognition between humans and chimpanzees.

How is WDR13 implicated in intellectual disability and neurological disorders?

WDR13 has been directly implicated in intellectual disability (ID) in humans. Clinical studies have identified a nonsense variant (c.757C>T, p.Arg253Ter) in the WDR13 gene in a male patient with intellectual disability, with family history suggesting X-linked inheritance . Gene expression analysis showed that patient fibroblasts with this WDR13 mutation exhibited dysregulation of several neural genes involved in intellectual disability development .

The table below summarizes the dysregulated genes found in WDR13-deficient human fibroblasts:

These findings suggest that WDR13 may function as a transcriptional regulator that normally suppresses these genes. When WDR13 is absent or non-functional, the dysregulation of these critical neural genes likely contributes to cognitive impairment .

For researchers interested in Pan troglodytes WDR13, these human findings provide a valuable framework for investigating potential cognitive impacts of WDR13 variants in chimpanzees and could inform studies comparing neurological development between the species.

What behavioral phenotypes are associated with WDR13 deficiency?

Studies of Wdr13 knockout mice have revealed several distinct behavioral phenotypes. These mice exhibited mild anxiety and hyperactivity in open field tests, spending less time in central areas and traversing more distance compared to wild-type controls (p < 0.05) . This behavioral pattern was consistent across different genetic backgrounds (CD1 and C57Bl/6J) .

Additionally, Wdr13-deficient mice showed impaired performance in novel object recognition tests, spending less time exploring novel objects (p < 0.05) . This suggests potential deficits in certain types of recognition memory, contrasting with their enhanced performance in spatial memory tasks.

These seemingly contradictory findings—improved spatial memory alongside anxiety and certain memory deficits—highlight the complex role WDR13 plays in different aspects of cognition and behavior. For researchers working with recombinant Pan troglodytes WDR13, these observations suggest the importance of comprehensive behavioral assessments when evaluating WDR13 function in primates, as effects may differ across cognitive domains.

What are the recommended protocols for expressing and purifying recombinant Pan troglodytes WDR13?

For efficient expression and purification of recombinant Pan troglodytes WDR13, researchers should consider a bacterial expression system using E. coli BL21(DE3) with a codon-optimized construct. Since WDR13 is a nuclear protein with multiple WD repeats, attention to proper protein folding is critical. The following protocol outline is recommended:

• Clone the Pan troglodytes WDR13 coding sequence into a pET vector system with an N-terminal His-tag to facilitate purification.

• Transform into E. coli BL21(DE3) cells and induce expression using 0.5 mM IPTG at 18°C overnight to enhance proper folding.

• Lyse cells in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, 10% glycerol, and protease inhibitors.

• Purify using nickel affinity chromatography followed by size exclusion chromatography to ensure homogeneity.

• Verify protein integrity using SDS-PAGE and Western blotting with antibodies specific to WDR13.

For functional studies, researchers should consider the nuclear localization of the protein and ensure that any recombinant protein maintains the structural integrity of the WD repeat domains, which are crucial for protein-protein interactions . If studying the regulatory effects of WDR13, in vitro transcription assays using promoter-reporter constructs, similar to those used for demonstrating WDR13's repression of ERE-containing promoters, would be appropriate .

What techniques are most effective for studying WDR13 protein-protein interactions?

Given WDR13's role in protein-protein interactions through its WD repeat domains, several complementary techniques are recommended:

• Co-immunoprecipitation (Co-IP): This remains the gold standard for confirming protein interactions in cellular contexts. For recombinant Pan troglodytes WDR13, epitope-tagged constructs should be expressed in relevant cell lines, followed by immunoprecipitation and Western blotting to identify interacting partners.

• Yeast two-hybrid screening: This can identify novel interaction partners. The WDR13 gene should be cloned as a bait construct, with screening performed against a primate brain cDNA library to identify neurologically relevant interactions.

• Proximity-dependent biotin identification (BioID): This newer technique involves fusing WDR13 to a biotin ligase, allowing biotinylation of proteins in close proximity, which can then be purified and identified by mass spectrometry.

• Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC): These provide quantitative measurements of binding affinities between purified recombinant WDR13 and candidate interacting proteins.

For studying interactions specifically implicated in neurological function, researchers should focus on potential interactions with proteins encoded by genes showing altered expression in WDR13-deficient conditions, particularly CAMK2A and FMR1, which showed the most dramatic expression changes in WDR13-deficient cells (11.4-fold and 6-fold increases, respectively) .

How can researchers effectively measure WDR13 gene expression across different tissues?

For comprehensive analysis of WDR13 expression patterns across tissues, a multi-method approach is recommended:

• Quantitative RT-PCR: Design primers spanning exon junctions to detect specific WDR13 transcripts. Reference genes should be carefully selected based on the tissues being compared, with GAPDH and TBN being suitable options as used in previous WDR13 studies . The table below outlines recommended primer design:

• RNA-Seq: This provides a comprehensive view of transcript variants and expression levels. Deep sequencing (>30 million reads per sample) is recommended for detecting lowly expressed transcripts or splice variants. Analysis should incorporate normalization methods appropriate for cross-tissue comparisons.

• In situ hybridization: For spatial resolution of expression patterns within tissues, particularly useful for brain samples where regional expression differences may be significant.

• Western blotting: To confirm protein expression levels, using antibodies verified for cross-reactivity with Pan troglodytes WDR13.

When analyzing expression data, researchers should be aware that previous studies have identified splice variants of WDR13 , necessitating careful primer design and data analysis to distinguish between different transcript forms.

How might WDR13 function differ between humans and chimpanzees, and what experimental approaches could detect these differences?

Despite the high genetic similarity between humans and chimpanzees (approximately 98%) , subtle differences in WDR13 function could contribute to species-specific neurological traits. To investigate these differences, researchers should consider:

• Comparative genomic analysis of the WDR13 locus, including promoter regions and regulatory elements, to identify sequence variations that might affect expression patterns or regulation.

• Cross-species complementation experiments: Express human WDR13 in chimpanzee cells with suppressed endogenous WDR13 (and vice versa) to determine if the proteins are functionally interchangeable or exhibit species-specific effects on downstream gene expression.

• Chromatin immunoprecipitation sequencing (ChIP-seq) using recombinant WDR13 from both species to identify potential differences in DNA binding patterns or target genes.

• Comparative transcriptomics of neural cells expressing either human or chimpanzee WDR13 to identify differentially regulated genes that might contribute to species-specific neurological traits.

These approaches could reveal subtle functional differences in WDR13 that might contribute to cognitive divergence between humans and great apes, particularly given WDR13's implicated role in intellectual disability pathways and cognitive function .

What molecular mechanisms explain the seemingly contradictory effects of WDR13 deficiency on different aspects of cognition?

The paradoxical findings that Wdr13 knockout mice show enhanced spatial memory yet exhibit anxiety and recognition memory deficits suggest complex, context-dependent functions of WDR13. To elucidate these mechanisms, researchers should consider:

• Cell-type specific conditional knockout studies to determine if WDR13 functions differently across neuronal populations.

• Time-controlled inducible knockout systems to distinguish between developmental and acute effects of WDR13 deficiency.

• Pathway-specific analyses focusing on the PI3K/Akt signaling pathway, which has been implicated in WDR13 function . Studies in pancreatic cells showed that WDR13 deficiency activates this pathway, which could lead to increased expression of CAMK2A and FMRP—proteins that interact with each other and play roles in synaptic plasticity and cell survival .

• Electrophysiological studies examining long-term potentiation (LTP) and long-term depression (LTD) in Wdr13-deficient neural networks, as these synaptic plasticity mechanisms underlie different forms of learning and memory.

Understanding these mechanisms in detail would provide insights into how a single protein can exert apparently opposing effects on different cognitive domains, which has implications for both evolutionary neurobiology and potential therapeutic interventions for conditions involving WDR13 dysfunction.

How does WDR13 regulate gene expression, and what are its direct transcriptional targets?

WDR13 appears to function as a transcriptional regulator, as evidenced by its nuclear localization and the dysregulation of multiple genes in its absence . To further characterize its regulatory mechanisms:

• Chromatin immunoprecipitation followed by sequencing (ChIP-seq) using recombinant Pan troglodytes WDR13 would identify direct binding sites across the genome, revealing potential direct transcriptional targets.

• RNA-seq analysis comparing wild-type and WDR13-knockdown conditions in relevant cell types, particularly neural cells, would provide a comprehensive view of genes under WDR13 regulation.

• Assays testing WDR13 interaction with known transcriptional regulators would clarify whether it functions as a co-repressor or co-activator. Previous research has shown that WDR13 represses luciferase transcription from a promoter containing an estrogen response element (ERE) in the presence of estradiol, and co-expression of WDR13 with c-JUN decreased AP1 promoter activity , suggesting a potential role as a transcriptional repressor.

• Investigation of post-translational modifications of WDR13 and how these might affect its regulatory function would provide insights into context-dependent activity.

Understanding the direct transcriptional targets and regulatory mechanisms of WDR13 would illuminate how this protein influences diverse cellular processes, particularly those related to neurological function and development.