DPY30 Human

What is DPY30 and what are its primary functions in human cellular processes?

DPY30 is a subunit of mammalian COMPASS-like complexes (Complex of Proteins Associated with Set1) that regulates global histone H3 lysine-4 trimethylation (H3K4me3) . Through direct binding to ASH2L (another COMPASS subunit), DPY30 facilitates genome-wide H3K4 methylation patterns . This epigenetic function affects numerous cellular processes including:

• Cell differentiation and lineage commitment

• Cellular metabolism, particularly glycolysis in cancer cells

• Migration and invasion capabilities in malignant cells

• Cell cycle progression and proliferation

DPY30 contains a dimerization/docking (D/D) module that enables its incorporation into protein complexes through hydrophobic interactions with the C-terminal amphipathic α-helix of ASH2L . This structural arrangement is essential for proper functioning of the methyltransferase activity of Set1/Mll complexes.

How evolutionarily conserved is DPY30 and what does this suggest about its biological significance?

DPY30 demonstrates remarkable evolutionary conservation across species, indicating its fundamental importance in chromatin regulation:

• C. elegans: First identified as an essential component of dosage compensation machinery where loss of dpy-30 activity results in XX-specific lethality

• Yeast: The homolog Saf19p (also known as Cps25 or Sdc1) functions within the histone H3 lysine 4 methylation complex

• Zebrafish: DPY30-depleted zebrafish show reduced global H3K4 methylation and disrupted hematopoiesis

• Mammals: Functions as a core component of all Set1/Mll complexes

This high degree of conservation suggests DPY30 plays a critical role in basic epigenetic mechanisms that have been maintained throughout evolution, particularly in regulating development and cell fate determination.

How does DPY30 contribute to histone methylation patterns and what are the downstream effects?

DPY30 enables efficient H3K4 methylation through several mechanisms:

• Directly binds to ASH2L through specific hydrophobic interactions between ASH2L's C-terminal amphipathic α-helix and the inner surface of DPY30's dimerization/docking module

• Facilitates the H3K4 methylation activities of all Set1/Mll complexes

• Preferentially controls H3K4 methylation at developmental and lineage-specific genes

Downstream effects of DPY30-mediated H3K4 methylation include:

• Establishment of active chromatin states associated with gene expression

• Regulation of bivalent promoters (marked by both H3K4me3 and H3K27me3) that poise genes for activation during differentiation

• Maintenance of cell identity through regulation of cell type-specific gene expression programs

When DPY30 function is impaired, significant reductions in H3K4me3 occur at specific genomic regions. For example, mutations affecting the ASH2L-DPY30 interaction result in loss of H3K4me3 at the β locus control region, causing delayed erythroid cell terminal differentiation .

What methodologies are most effective for studying DPY30's impact on the epigenome?

For comprehensive analysis of DPY30's epigenetic functions, researchers should consider these complementary approaches:

• Genome-wide profiling techniques:

  • ChIP-seq for H3K4me1/2/3 to assess methylation patterns across the genome

  • RNA-seq to correlate methylation changes with gene expression

  • ATAC-seq to determine changes in chromatin accessibility

  • CUT&RUN or CUT&Tag as alternatives to traditional ChIP with improved signal-to-noise ratios

• Locus-specific analyses:

  • ChIP-qPCR targeting specific genomic regions of interest

  • Reporter assays to assess promoter/enhancer activity

• Protein-level analyses:

  • Western blotting with antibodies specific for H3K4me1/2/3

  • Mass spectrometry to quantify histone modifications

• Functional validation:

  • Site-directed mutagenesis of key residues in the DPY30-ASH2L interaction interface

  • Domain swap experiments to determine functional regions

The combination of these approaches provides robust evidence for DPY30's specific role in epigenetic regulation beyond correlative observations.

How does DPY30 regulate hematopoietic stem cell (HSC) function and differentiation?

DPY30 plays a critical role in both maintaining HSC identity and enabling proper differentiation:

HSC maintenance effects:

• DPY30 directly controls H3K4 methylation and expression of key hematopoietic stem cell signature genes

• Loss of DPY30 in hematopoietic cells results in significant down-regulation of HSC identity genes

• DPY30 is essential for long-term HSC maintenance, as demonstrated by secondary transplantation experiments showing severe loss of HSCs with DPY30 knockout

Differentiation effects:

• Conditional knockout of DPY30 in the adult hematopoietic system causes severe pancytopenia but paradoxically leads to accumulation of HSCs and early hematopoietic progenitor cells (HPCs)

• DPY30-deficient HSCs show defective multilineage reconstitution with a differentiation block

• In mixed bone marrow chimeras, DPY30-deficient HSCs fail to differentiate or efficiently upregulate lineage-regulatory genes

These findings reveal that DPY30 functions as a master regulator of hematopoiesis by controlling both HSC maintenance and differentiation capacities through epigenetic mechanisms.

What are the experimental approaches for studying DPY30 in development using mouse models?

Effective experimental designs for studying DPY30 in mouse development include:

Conditional knockout strategies:

• Generation of mice harboring a DPY30 knockout-first allele that can be converted to a conditional allele via Flp recombination

• Using tissue-specific Cre recombinase expression to delete DPY30 in specific cell lineages

• For hematopoietic studies, using systems like Mx1-Cre activated by polyinosinic-polycytidylic acid (pIpC) injections

Transplantation models:

• Mixed bone marrow chimeras to study cell-autonomous effects of DPY30 loss in a normal microenvironment

• Secondary transplantation using immunophenotypically sorted donor-derived LT-HSCs to assess long-term HSC function

Molecular analyses:

• RNA-seq of purified cell populations to identify DPY30-regulated genes

• Gene Set Enrichment Analysis (GSEA) to determine effects on specific gene programs

• ChIP-seq to map H3K4 methylation changes at specific genomic loci

These approaches have revealed that DPY30 selectively controls developmental gene expression programs and is essential for proper differentiation across multiple lineages.

How does DPY30 contribute to colorectal cancer (CRC) progression?

DPY30 promotes colorectal cancer progression through multiple mechanisms:

Metabolic reprogramming:

• DPY30 enhances glycolysis in CRC cells through two major channels: influencing signaling pathways and regulating gene transcription

• DPY30 knockdown attenuates aerobic glycolysis by repressing H3K4me3 establishment on promoters of key glycolytic enzyme genes (HK1, PFKL, and ALDOA)

• The changes in glycolysis are related to the PI3K-AKT signaling pathway

Cell proliferation and cycle regulation:

• DPY30 is overexpressed in CRC tissues

• DPY30 promotes proliferation and cell cycle progression of CRC cells

These findings indicate that DPY30 acts as an oncogenic factor in CRC by reprogramming cellular metabolism to support rapid proliferation, a hallmark of cancer cells. This makes DPY30 a potential therapeutic target for CRC treatment .

What role does DPY30 play in osteosarcoma progression and metastasis?

In osteosarcoma (OS), DPY30 functions as a promoter of malignant phenotypes:

Migration and invasion:

• DPY30 knockdown impairs migration and invasion capabilities in OS cells

• High DPY30 expression levels correlate with increased distant metastases in OS patients

• DPY30 silencing attenuates the mesenchymal phenotype in OS cells, shown by reduced N-cadherin expression

Signaling pathway modulation:

• DPY30 knockdown suppresses activation of the PI3K/AKT signaling pathway

• The PI3K/AKT pathway promotes cell proliferation and metabolic reprogramming necessary for metastasis

• This pathway can crosstalk with other oncogenic signals such as WNT signaling to drive cellular changes including epithelial-to-mesenchymal transition (EMT)

These findings parallel observations in other cancer types, suggesting DPY30 may function as a general oncogenic factor by regulating both epigenetic landscapes and key signaling pathways that promote cancer progression.

What methodological approaches are most effective for DPY30 knockdown in cancer cell models?

For effective DPY30 manipulation in cancer cell models, researchers have successfully employed these approaches:

Lentiviral vector-based shRNA:

• Multiple validated shRNA sequences targeting human DPY30:

  • 5′-GACCACCAAATCCCATTGAAT-3′ (shDPY30#1)

  • 5′-GCACAGTTTGAAGATCGAAAC-3′ (shDPY30#2)

  • 5′-GACAACCAAATCCCATTGAAT-3′ (shDPY30#3)

• Protocol details:

  • Annealing and insertion of oligonucleotides into pLKO.1 lentiviral vector

  • Transfection of HEK293T cells with the lentiviral vector and packing plasmids using polyethylenimine

  • Collection of viral supernatant by centrifugation

  • Transduction of target cells at MOI of 5

  • Selection with 1.0 μg/mL puromycin for two weeks

Experimental validation:

• Western blotting to confirm protein knockdown

• RT-qPCR to verify reduced transcript levels

• Functional assays to assess phenotypic changes:

  • Proliferation assays (MTT, BrdU incorporation)

  • Migration/invasion assays (wound healing, transwell)

  • Glycolysis measurements (extracellular acidification rate, lactate production)

These methodologies provide robust tools for investigating DPY30's role in cancer progression and potential therapeutic applications.

How does DPY30 structurally interact with COMPASS complex components?

The structural basis for DPY30 incorporation into COMPASS-like complexes has been elucidated:

Key interaction with ASH2L:

• DPY30 incorporates into COMPASS-like complexes through hydrophobic interactions between the amphipathic α-helix on ASH2L's C-terminus and the inner surface of the DPY30 dimerization/docking (D/D) module

• This interaction is critical for proper COMPASS complex assembly and function

• Mutations disrupting this interaction lead to loss of H3K4me3 at specific genomic loci

Structural characteristics:

• Human DPY-30-like protein has been crystallized, diffracting to 2.7 Å resolution

• Crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2 with specific unit-cell parameters

• The asymmetric unit contains two molecules with 49% solvent content

Understanding these structural interactions provides a foundation for developing potential therapeutic agents targeting the DPY30-ASH2L interface, which could modulate COMPASS complex activity in disease states.

What is the relationship between DPY30 and the PI3K/AKT signaling pathway?

DPY30 positively regulates the PI3K/AKT pathway through mechanisms that are still being elucidated:

Observed effects:

• DPY30 knockdown suppresses activation of the PI3K/AKT signaling pathway in osteosarcoma cells

• The PI3K/AKT pathway promotes cell proliferation and metabolic reprogramming that supports metastasis

• In colorectal cancer, DPY30-mediated glycolysis changes are related to PI3K-AKT signaling

Potential mechanisms:

• Epigenetic regulation of genes encoding PI3K/AKT pathway components

• Indirect effects through intermediary factors whose expression is controlled by DPY30

• Possible non-histone targets of DPY30-containing complexes that directly modulate PI3K/AKT signaling

Researchers suggest that genome-wide approaches such as RNA-seq, ChIP-seq, or ATAC-seq would help elucidate how DPY30, primarily known as a histone H3K4 methyltransferase component, influences oncogenic PI3K/AKT signaling .

How does DPY30 contribute to metabolic reprogramming in cancer cells?

DPY30 drives metabolic reprogramming in cancer cells through both direct and indirect mechanisms:

Epigenetic regulation of metabolic genes:

• In colorectal cancer, DPY30 establishes H3K4me3 on promoters of key glycolytic enzyme genes (HK1, PFKL, and ALDOA)

• This epigenetic modification enhances expression of these glycolytic enzymes, promoting aerobic glycolysis

Signaling pathway modulation:

• DPY30 positively regulates the PI3K/AKT pathway

• PI3K/AKT signaling is a major regulator of cellular metabolism that promotes glycolysis

Functional consequences:

• Enhanced glycolysis (Warburg effect) supports rapid cancer cell proliferation

• Metabolic reprogramming provides building blocks for biosynthesis

• Altered metabolism contributes to cancer cell survival in hypoxic microenvironments

This dual regulation through both direct epigenetic control of metabolic gene expression and modulation of metabolic signaling pathways positions DPY30 as a master regulator of cancer metabolism.

What are the current challenges in developing DPY30 as a therapeutic target?

Developing DPY30-targeted therapies presents several significant challenges:

Essential developmental functions:

• DPY30 is essential for embryonic development (DPY30-/- mouse embryos die between E7.5 and E9.5)

• It plays critical roles in normal hematopoiesis and stem cell function

• Targeting DPY30 might cause significant toxicity, particularly in rapidly renewing tissues

Technical challenges:

• Designing specific inhibitors of protein-protein interactions (such as DPY30-ASH2L) is generally difficult

• Epigenetic regulators often lack enzymatic pockets suitable for small molecule binding

• Achieving tissue-specific targeting to avoid systemic effects

Knowledge gaps:

• Incomplete understanding of tissue-specific functions of DPY30

• Limited information on the full spectrum of DPY30 interaction partners

• Unclear mechanisms linking DPY30 to signaling pathways like PI3K/AKT

Despite these challenges, DPY30 remains a promising target given its overexpression in multiple cancer types and its role in driving malignant phenotypes. Future research should focus on identifying cancer-specific vulnerabilities related to DPY30 function that could be therapeutically exploited with minimal effects on normal tissues.

How might comprehensive genomic approaches advance our understanding of DPY30 function?

Advanced genomic techniques offer powerful opportunities to elucidate DPY30's functions:

Integrated multi-omics approaches:

• Combined ChIP-seq, RNA-seq, and ATAC-seq to correlate H3K4 methylation patterns, gene expression changes, and chromatin accessibility

• Proteomics to identify non-histone targets of Set1/Mll complexes

• Metabolomics to comprehensively profile metabolic changes associated with DPY30 modulation

• Single-cell technologies to reveal cell type-specific functions of DPY30

Key research questions addressable through these approaches:

• Genome-wide mapping of DPY30 binding sites and associated H3K4 methylation patterns in different cell types

• Determination of direct versus indirect targets of DPY30

• Identification of transcription factors that cooperate with DPY30 in different cellular contexts

• Understanding context-dependent functions of DPY30 in normal versus disease states

Researchers have noted that genome-wide approaches would be particularly valuable for understanding how DPY30 impacts the PI3K/AKT signaling pathway , potentially revealing unexpected connections between epigenetic regulation and oncogenic signaling.

What is the significance of DPY30's relationship with BAP18 and the nucleosome remodeling factor complex?

The interaction between DPY30 and BAP18 (a subunit of the nucleosome remodeling factor complex) suggests broader roles for DPY30 beyond histone methylation:

Functional implications:

• Potential coordination between histone modification and nucleosome remodeling activities

• Possible role in regulating chromatin accessibility in concert with structural changes

• Integration of multiple chromatin regulatory mechanisms through DPY30 as a hub protein

Research has defined a consensus sequence for DPY30 binding proteins and found that DPY30 interacts with BAP18 . This interaction may represent a critical link between different chromatin regulatory complexes, potentially explaining how DPY30 can achieve context-specific effects on gene expression.

Further investigation of this relationship could reveal how epigenetic modifications and chromatin structure are coordinately regulated during development and disease progression, potentially identifying novel therapeutic strategies targeting these integrated processes.