DPPA3 Human

What is DPPA3 and what is its role in human cells?

DPPA3 (Developmental pluripotency-associated protein 3), also known as Stella or PGC7, is a mammalian-specific protein involved in DNA methylation regulation. In humans, DPPA3 interacts with UHRF1, a key component of the DNA maintenance methylation machinery, potentially influencing DNA methylation patterns during early development. This interaction occurs specifically through binding to the PHD finger domain of UHRF1, though with less efficiency than its mouse ortholog . DPPA3 serves as a marker for naïve pluripotency and is expressed in primordial germ cells, oocytes, and preimplantation embryos .

How does human DPPA3 regulate DNA methylation?

Human DPPA3 regulates DNA methylation through a mechanism involving its interaction with UHRF1 and DNMT1:

• DPPA3 binds to the PHD finger domain of UHRF1 primarily via its conserved VRT motif (residues 85-87)

• This binding can potentially displace UHRF1 from chromatin, inhibiting maintenance DNA methylation

• By affecting UHRF1 activity, DPPA3 indirectly impacts global DNA methylation maintenance during DNA replication

When and where is DPPA3 expressed in humans?

In humans, DPPA3 expression follows a highly specific pattern:

• Primordial germ cells

• Oocytes

• Preimplantation embryos

• Naïve pluripotent stem cells

It is highly expressed in naïve embryonic stem cells (ESCs) and is downregulated upon differentiation . DPPA3 expression is sensitive to DNA methylation, with promoter demethylation leading to increased expression levels .

What is the structure of human DPPA3 when bound to UHRF1?

The crystal structure of human DPPA3 bound to the UHRF1 PHD finger reveals several key features:

• The conserved VRT motif (residues 85-87) binds to the acidic surface of the UHRF1 PHD finger

• Residues 88-101 form a four-turn single α-helix following the VRT motif

• This α-helix is not kinked, unlike mouse DPPA3's structure

• The contact area between human PHD and human DPPA3 is approximately 449 Ų, significantly smaller than that of mouse proteins (approximately 1360 Ų)

This structural configuration results in weaker binding affinity compared to mouse DPPA3, potentially explaining functional differences between species .

How does human DPPA3 structure differ from mouse DPPA3?

Key structural differences between human and mouse DPPA3 include:

These structural differences appear to be taxonomically distributed, with the two α-helix configuration largely restricted to Rodentia (mice, rats), while primates and other mammals display the single α-helix arrangement .

What domains or motifs are functionally important in human DPPA3?

The functionally important domains and motifs in human DPPA3 include:

These structural elements collectively determine DPPA3's interaction specificity and binding affinity with UHRF1.

How evolutionarily conserved is DPPA3 across species?

DPPA3 displays a distinct evolutionary pattern:

• It is uniquely found in mammals, unlike core DNA methylation machinery components (DNMTs, UHRF1, TETs), which are conserved throughout metazoa

• DPPA3 appears to be a relatively recent evolutionary innovation specific to mammals

• Despite this recent evolution, DPPA3 is capable of inducing global DNA demethylation when introduced into non-mammalian vertebrates (Xenopus and medaka)

This evolutionary pattern suggests that DPPA3 emergence may have facilitated the development of mammal-specific epigenetic reprogramming mechanisms, particularly the genome-wide erasure of DNA methylation in early development .

What structural differences exist between human DPPA3 and other mammalian orthologs?

Structural analysis and predictions reveal taxonomic patterns in DPPA3 structure:

• Single α-helix configuration (found in):

  • Homo sapiens

  • Bos taurus

  • Gorilla gorilla gorilla

  • Saimiri boliviensis

  • Puma concolor

  • Nomascus leucogenys

  • Several other non-rodent mammals

• Two α-helices configuration (found in):

  • Mus musculus

  • Rattus norvegicus

  • Cricetulus griseus

  • Primarily restricted to Rodentia

These structural differences are not determined by a single amino acid substitution. The K95P mutation in human DPPA3 did not enhance its binding affinity for human PHD or induce the formation of two α-helices, suggesting more complex evolutionary mechanisms at work .

How did DPPA3 contribute to mammalian evolution?

The evolution of DPPA3 appears to have facilitated significant innovations in mammalian development:

• DPPA3 may have enabled the emergence of global DNA demethylation in mammals, which is a unique feature of mammalian development and naïve pluripotent stem cells

• By coupling active and passive demethylation mechanisms, DPPA3 allows for genome-wide erasure of DNA methylation during preimplantation development

• TET activity works in concert with DPPA3, where TET-mediated active demethylation drives DPPA3 expression, which then causes global passive demethylation by inhibiting maintenance methylation

This evolutionary innovation potentially contributed to the unique epigenetic reprogramming capabilities of mammals, with implications for developmental plasticity and totipotency.

What techniques are used to study human DPPA3-UHRF1 interactions?

Several complementary methods can be used to study DPPA3-UHRF1 interactions:

• Structural studies:

  • X-ray crystallography: Successfully used to determine the structure of human DPPA3 bound to the UHRF1 PHD finger

  • Nuclear Magnetic Resonance (NMR): Useful for studying intrinsically disordered proteins like DPPA3

  • AlphaFold2 (AF2) predictions: Provides insights into structural characteristics across species

• Binding assays:

  • Immunoprecipitation (IP): To confirm protein interactions in cellular contexts

  • Pull-down assays: Using recombinant proteins to study direct interactions

• Functional assays:

  • Xenopus egg extracts: To assess the ability of DPPA3 to inhibit UHRF1 chromatin binding and DNA remethylation

  • DPPA3 knockout experiments: To observe effects on DNA methylation patterns

These methods provide complementary information about both the structural basis and functional consequences of DPPA3-UHRF1 interactions.

How can researchers analyze DPPA3's effect on DNA methylation patterns?

Researchers can analyze DPPA3's effect on DNA methylation using several approaches:

• Genome-wide methylation profiling:

  • Reduced Representation Bisulfite Sequencing (RRBS): Detects global methylation changes in DPPA3 knockout models

  • Analysis of specific genomic features affected by DPPA3 loss (promoters, repetitive sequences, imprinting control regions)

• Locus-specific methylation analysis:

  • Bisulfite sequencing of specific loci such as the DPPA3 promoter itself

• Functional genomic approaches:

  • RNA-seq combined with methylation profiling: To correlate changes in gene expression with alterations in DNA methylation

  • Mass spectrometry to identify DPPA3 protein interaction partners

• Heterologous systems:

  • Introduction of DPPA3 into non-mammalian systems (Xenopus, medaka) to assess its capacity to induce DNA demethylation

  • Xenopus egg extracts to evaluate effects on UHRF1 chromatin binding

These methods collectively provide a comprehensive view of how DPPA3 influences DNA methylation at both global and locus-specific levels.

What experimental systems are appropriate for studying human DPPA3 function?

Several model systems can be used to study human DPPA3 function:

• Human cell models:

  • Human embryonic stem cells (hESCs): Express DPPA3 and represent the naïve pluripotent state

  • DPPA3 knockout ESCs: To assess the functional consequences of DPPA3 loss

• Heterologous systems:

  • Xenopus egg extracts: Have been used to assess the ability of human DPPA3 to inhibit UHRF1 chromatin binding

  • Non-mammalian vertebrates (Xenopus, medaka): Reveal the capacity of human DPPA3 to induce global demethylation across species

• Biochemical systems:

  • Recombinant protein studies: To analyze direct interactions between DPPA3 and components of the DNA methylation machinery

  • Crystal structures: To determine precise binding interfaces

Each system offers distinct advantages for understanding different aspects of DPPA3 function.

Why does human DPPA3 bind UHRF1 more weakly than mouse DPPA3?

The weaker binding of human DPPA3 to UHRF1 compared to mouse DPPA3 is explained by several structural factors:

• Different α-helical structures:

  • Human DPPA3 forms a single α-helix after the VRT motif

  • Mouse DPPA3 forms two α-helices in an L-like shape that provide additional contact surfaces

• Reduced contact area:

  • Human DPPA3-UHRF1 interface: ~449 Ų

  • Mouse DPPA3-UHRF1 interface: ~1360 Ų

• Complex sequence determinants:

  • Simple amino acid substitutions (K95P) do not convert human DPPA3 to mouse-like binding

  • The differences likely depend on more complex sequence and structural features

Despite this weaker binding, human DPPA3 still binds UHRF1 with approximately 1.7-fold stronger affinity than histone H3, suggesting it could compete with histone H3 for UHRF1 binding under certain conditions .

How might human DPPA3 function despite its weaker UHRF1 binding?

Several mechanisms might explain how human DPPA3 functions despite weaker UHRF1 binding:

• Concentration-dependent effects:

  • High local concentrations of DPPA3 could compensate for lower binding affinity

  • In specific cellular compartments, DPPA3 levels might be sufficient to compete with histone H3

• Phase separation potential:

  • Human DPPA3 has higher predicted potential for liquid-liquid phase separation than mouse DPPA3

  • Condensed DPPA3 within droplets may increase effective local concentration

• Histone modification context:

  • Post-translational modifications of histone H3 (e.g., methylation of Arg2, phosphorylation of Thr3) reduce its binding to UHRF1

  • In contexts where these modifications are present, even lower-affinity DPPA3 might effectively compete for UHRF1 binding

• Alternative mechanisms:

  • Human DPPA3 might function through additional pathways beyond direct UHRF1 binding

  • Other factors like NLRP14 might complement DPPA3's role in regulating UHRF1 localization

These hypotheses suggest multiple adaptations that could enable human DPPA3 to fulfill its developmental functions despite altered biochemical properties.

What are the major unresolved questions in human DPPA3 research?

Several critical questions remain unanswered regarding human DPPA3:

• Functional significance of species differences:

  • How do structural and binding differences between human and mouse DPPA3 affect their respective roles in development?

  • Has human DPPA3 evolved additional or alternative functions?

• Mechanism in human embryonic development:

  • What is the precise role of DPPA3 in human preimplantation development?

  • How does human DPPA3 contribute to epigenetic reprogramming in the human germline?

• Regulatory pathways:

  • What factors control DPPA3 expression in different developmental contexts?

  • How is DPPA3 itself regulated post-translationally?

• Phase separation biology:

  • Does human DPPA3's predicted higher potential for liquid-liquid phase separation contribute to its function?

  • How might phase separation compensate for weaker direct binding to UHRF1?

• Clinical relevance:

  • What role might DPPA3 play in human fertility and developmental disorders?

  • What is the significance of DPPA3 in testicular germ cell tumors?

Addressing these questions will require innovative approaches combining structural biology, biochemistry, developmental biology, and clinical research.

What is DPPA3's role in human embryonic development?

DPPA3's role in human embryonic development appears to involve several processes:

• DNA methylation regulation:

  • May contribute to establishing appropriate DNA methylation patterns during early development

  • Could influence global demethylation during preimplantation development, though potentially with different efficiency compared to mouse DPPA3

• Expression pattern:

  • DPPA3 is expressed in primordial germ cells, oocytes, and preimplantation embryos

  • Serves as a marker for naïve pluripotency

• Species-specific considerations:

  • Human DPPA3's weaker binding to UHRF1 compared to mouse DPPA3 suggests potential differences in its function during early human development

  • May work in concert with other factors like NLRP14 to regulate UHRF1 localization

Understanding DPPA3's precise role in human embryonic development remains challenging due to ethical and technical limitations on human embryo research.

How does TET activity relate to DPPA3 function?

TET enzymes and DPPA3 function in a coordinated pathway:

• TET-mediated activation of DPPA3:

  • TET enzymes catalyze active DNA demethylation at specific loci, including the DPPA3 promoter

  • This demethylation enables DPPA3 expression in naïve pluripotent cells

  • In TET mutant embryonic stem cells, DPPA3 expression is reduced due to hypermethylation of its promoter

• DPPA3-mediated global demethylation:

  • Once expressed, DPPA3 can bind to UHRF1 and potentially displace it from chromatin

  • This inhibits maintenance DNA methylation, causing global passive demethylation

• Coupling of active and passive demethylation:

  • TET activity is required for global demethylation, albeit indirectly

  • TET-mediated active demethylation is locus-specific and activates DPPA3

  • DPPA3 then drives large-scale passive demethylation

This pathway represents a recently evolved mechanism in mammals that couples active and passive demethylation to achieve genome-wide hypomethylation during development .

What do we know about DPPA3 in human disease contexts?

Research on DPPA3 in human disease contexts is still emerging:

The clinical significance of DPPA3 remains an underdeveloped area that warrants further research, particularly given the observed species-specific differences in DPPA3 function that limit direct extrapolation from mouse models.

How has our understanding of human DPPA3 evolved recently?

Recent advances in human DPPA3 research include:

• Structural insights:

  • Crystal structure of human DPPA3 bound to UHRF1 PHD finger revealed unexpected differences from mouse DPPA3

  • Identification of a single α-helix configuration in human DPPA3 versus two α-helices in mouse DPPA3

• Functional differences:

  • Demonstration that human DPPA3 binds UHRF1 more weakly than mouse DPPA3

  • Evidence that human DPPA3 is less effective at inhibiting UHRF1 chromatin binding in Xenopus egg extracts

• Evolutionary perspective:

  • Recognition that DPPA3's structure follows taxonomic patterns, with the two α-helix configuration largely restricted to Rodentia

  • Understanding that DPPA3 is a mammal-specific protein that may have facilitated the evolution of global DNA demethylation

These advances highlight the importance of species-specific studies rather than assuming functional conservation across mammals.

What technological advances are needed for future DPPA3 research?

Several technological advances would enhance future DPPA3 research:

• Improved human developmental models:

  • Advanced in vitro models of human embryonic development

  • Better systems for studying human primordial germ cell development

  • Models that accurately recapitulate human-specific aspects of epigenetic regulation

• Advanced imaging techniques:

  • Methods to visualize DPPA3-UHRF1 interactions in living cells

  • Techniques to monitor DNA methylation dynamics in real-time

  • Approaches to detect potential phase separation of DPPA3 in cellular contexts

• Integrative omics approaches:

  • Combined analysis of DNA methylation, protein interactions, and gene expression

  • Single-cell multi-omics to understand cell-to-cell variation in DPPA3 function

  • Computational methods to predict functional consequences of species differences

These technological advances would help bridge the gap between structural insights and functional understanding of human DPPA3.

How might understanding human DPPA3 impact clinical applications?

Understanding human DPPA3 could have several potential clinical implications:

• Reproductive medicine:

  • Insights into human-specific mechanisms of epigenetic regulation during early development

  • Potential biomarkers for embryo quality or developmental competence

  • Improved understanding of epigenetic reprogramming in assisted reproductive technologies

• Regenerative medicine:

  • Better control of epigenetic states in human pluripotent stem cells

  • Improved protocols for differentiation or reprogramming

  • Enhanced understanding of naïve versus primed pluripotency in humans

• Cancer biology:

  • Insights into the epigenetic basis of testicular germ cell tumors

  • Understanding potential roles of DPPA3 in other cancers that reactivate germline programs