ING2 Human

What is ING2 and what are its primary functions in human cells?

ING2 (Inhibitor of Growth Family Member 2) is a plant homeodomain (PHD)-containing protein that functions as a putative tumor suppressor. It plays pivotal roles in regulating cellular senescence, apoptosis, DNA damage repair, gene transcription, and chromatin modification. ING2 serves as a subunit of the mSin3A-HDAC1 (histone deacetylase 1) complex and specifically binds to tri-methylated lysine 4 of histone H3 (H3K4me3) via its PHD finger domain .

Methodologically, ING2 functions can be investigated through:

• Chromatin immunoprecipitation (ChIP) to identify genomic binding sites

• Co-immunoprecipitation to detect protein-protein interactions (particularly with p53 and HDAC1)

• Gene expression analysis following ING2 modulation using quantitative PCR or RNA-sequencing

• Cell cycle analysis using flow cytometry in ING2-deficient or overexpressing cells

How does ING2 interact with tumor suppressor pathways?

ING2 functionally interplays with the p53 tumor suppressor protein in two distinct manners:

• Endogenous ING2 inhibits senescence, and p53-mediated transcriptional repression of ING2 abrogates this inhibition

• Overexpressed ING2 enhances p53 acetylation and stability to induce senescence

In experimental settings, researchers can study this interaction by:

• Using reporter gene assays with p53-responsive elements

• Employing site-directed mutagenesis to determine critical interaction domains

• Performing acetylation assays to measure p53 post-translational modifications

• Conducting cellular senescence assays using β-galactosidase staining

What is known about ING2 expression patterns in human tissues?

ING2 shows tissue-specific expression patterns with particularly abundant expression in human testes. Decreased ING2 expression has been associated with male infertility and defective spermatogenesis in humans . Public datasets reveal that ING2 expression is significantly reduced (>7-fold) in testes from patients with Sertoli-cell only Syndrome and in teratozoospermic sperm (>3-fold reduction) .

Methodological approaches to study expression include:

• RNA-seq analysis of different human tissues

• Immunohistochemistry to visualize protein localization

• Western blotting for protein quantification

• Single-cell RNA sequencing to identify cell-type specific expression

How does ING2 contribute to TGF-β signaling pathways?

ING2 functions as a novel mediator of transforming growth factor-β (TGF-β) dependent transcription and cell cycle arrest. Research findings demonstrate that:

• ING2 enhances TGF-β-induced transcription in multiple cell lines, including augmenting the expression of reporter genes driven by TGF-β-responsive promoters such as PAI-1

• The PHD domain of ING2 is required for its enhancement of TGF-β-dependent transcription

• ING2 promotes the transcriptional activity of TGF-β-regulated Smad2 and Smad3

• ING2 forms a novel interaction with the transcriptional modulator SnoN, which recruits ING2 to Smad2 to induce TGF-β-dependent transcription

Methodologically, researchers investigating ING2's role in TGF-β signaling should:

• Utilize reporter gene assays with TGF-β-responsive elements

• Perform co-immunoprecipitation to detect interactions with Smad proteins

• Examine endogenous target gene expression through RT-qPCR

• Use structure-function analyses with deletion mutants to identify critical domains

What mechanisms underlie ING2-mediated regulation of spermatogenesis?

ING2 regulates spermatogenesis through both p53-dependent and chromatin-mediated mechanisms. Studies in Ing2-deficient mice revealed:

• Male mice lacking Ing2 were infertile, with significantly reduced sperm numbers (~2% of wild type) and motility (~10% of wild type)

• Testicular abnormalities included degeneration of seminiferous tubules and meiotic arrest before pachytene stage

• Molecular analysis showed incomplete meiotic recombination, p53 induction, and enhanced apoptosis

• The phenotype was only partially rescued by concomitant loss of p53, indicating both p53-dependent and p53-independent mechanisms

• Arrest of spermatocytes was characterized by lack of HDAC1 accumulation and deregulated chromatin acetylation

Research methodologies to investigate these mechanisms include:

• Histological analysis of testicular tissue

• TUNEL assays to measure apoptosis

• Immunofluorescence to detect stage-specific markers of spermatogenesis

• ChIP-seq to identify ING2 binding sites in germline chromatin

• RNA-seq to analyze transcriptional changes in ING2-deficient testes

How do the chromatin-binding properties of ING2 influence gene expression?

ING2, as a subunit of the mSin3A-HDAC1 complex, specifically binds to tri-methylated lysine 4 of histone H3 (H3K4me3) via its PHD finger domain . This interaction enables ING2 to:

• Recognize specific chromatin states associated with active transcription

• Recruit histone deacetylase complexes to regulate gene expression

• Modify local chromatin structure in response to DNA damage signals

• Coordinate stage-specific histone modifications essential for processes like spermatogenesis

The chromatin-binding function of ING2 requires:

• An intact PHD finger domain for H3K4me3 recognition

• Association with the mSin3A-HDAC1 complex for effector functions

• Proper nuclear localization for chromatin access

• Stage-specific regulation of chromatin acetylation

Methodological approaches to study ING2 chromatin interactions include:

• ChIP-seq to map genomic binding sites

• ATAC-seq to assess chromatin accessibility changes

• Histone modification profiling in ING2-deficient cells

• Structure-function analyses of PHD domain mutations

• Proximity ligation assays to visualize chromatin interactions in situ

What are the optimal experimental models for studying ING2 function?

Several experimental models have proven valuable for investigating ING2 function:

• Cellular Models:

  • Human cell lines (HepG2, Mv1Lu) for studying TGF-β signaling

  • Primary testicular cells for spermatogenesis studies

  • Inducible expression systems to control ING2 levels temporally

• Animal Models:

  • Ing2 knockout mice provide insights into developmental functions

  • Conditional knockout models to study tissue-specific effects

  • Double knockout models (e.g., Ing2/p53) to dissect pathway interactions

• Molecular Tools:

  • Expression vectors for wild-type and mutant ING2 proteins

  • Reporter constructs with TGF-β or p53-responsive elements

  • Domain deletion constructs to map functional regions

When selecting an experimental model, researchers should consider:

• The specific aspect of ING2 function under investigation

• The evolutionary conservation of pathways between model organisms and humans

• The availability of reagents and genetic tools

• The physiological relevance to human disease processes

How can researchers effectively investigate ING2's role in transcriptional regulation?

To study ING2's transcriptional regulatory functions, researchers should employ a combination of approaches:

• Transcriptomic Analysis:

  • RNA-seq to identify differentially expressed genes

  • ChIP-seq to map ING2 binding sites genome-wide

  • ATAC-seq to assess changes in chromatin accessibility

  • CUT&RUN for high-resolution mapping of chromatin interactions

• Reporter Assays:

  • Luciferase reporter systems with specific promoters (e.g., PAI-1)

  • Gal4-fusion heterologous transcription assays to measure Smad2/3 activity

  • Mutational analysis of response elements

• Protein-Protein Interactions:

  • Co-immunoprecipitation to identify transcription factor complexes

  • Proximity ligation assays for in situ visualization

  • Domain mapping through deletion mutants

  • Mass spectrometry to identify novel interaction partners

• Chromatin Modifications:

  • ChIP for specific histone marks (e.g., H3K4me3, acetylation)

  • Sequential ChIP to identify co-occupancy with other factors

  • Functional assays for HDAC activity in ING2-containing complexes

What strategies can be employed to investigate the therapeutic potential of targeting ING2?

Though the search results don't directly address therapeutic targeting of ING2, researchers interested in this question could consider these methodological approaches:

• Target Validation:

  • Analyze ING2 expression in patient samples versus healthy controls

  • Correlate expression with disease progression and outcomes

  • Perform CRISPR-Cas9 knockout/knockdown in disease models

  • Utilize patient-derived xenografts to assess relevance

• Intervention Approaches:

  • Develop small molecule inhibitors targeting the PHD domain

  • Design peptide inhibitors of ING2-protein interactions

  • Explore antisense oligonucleotides or siRNA for expression modulation

  • Consider degrader technologies (PROTACs) for protein depletion

• Phenotypic Screening:

  • Establish cellular assays based on ING2-dependent processes

  • Screen compound libraries for modulators of these processes

  • Validate hits through target engagement studies

  • Assess effects on downstream pathways (e.g., TGF-β signaling)

• Biomarker Development:

  • Identify gene expression signatures associated with ING2 activity

  • Develop assays to measure ING2 complex formation

  • Correlate chromatin modifications with ING2 function

  • Establish patient stratification criteria for clinical studies

Research challenges in targeting ING2 would include:

• Achieving specificity among PHD domain-containing proteins

• Determining appropriate disease indications

• Understanding potential side effects (especially on fertility)

• Developing appropriate drug delivery methods

How does ING2 function compare to other ING family members?

While the search results focus specifically on ING2, researchers should note that ING2 belongs to a family of proteins with related but distinct functions:

• Structural Comparison:

  • All ING family members contain a conserved PHD finger domain

  • ING proteins differ in their N-terminal domains

  • Different family members associate with distinct protein complexes

• Functional Overlap and Distinction:

  • Different ING proteins may interact with p53 through distinct mechanisms

  • Family members may show tissue-specific expression patterns

  • Knockout phenotypes vary between family members

• Research Methodology:

  • Comparative expression analysis across tissues

  • Domain-swapping experiments to identify functional determinants

  • Cross-complementation studies in knockout models

  • Comparative ChIP-seq to map binding site specificities

Research approaches should include:

• Side-by-side functional assays with multiple ING family members

• Careful antibody validation to ensure specificity

• Consideration of compensatory mechanisms in knockout models

• Evolutionary analysis to understand functional divergence

What contradictions or unresolved questions exist in ING2 research?

Based on the search results, several areas of ING2 research contain apparent contradictions or knowledge gaps that represent opportunities for further investigation:

• Dual Role in p53 Signaling:

  • Endogenous ING2 inhibits senescence, yet overexpressed ING2 enhances p53-driven senescence

  • This apparent contradiction suggests context-dependent functions requiring further elucidation

• Role in Cancer:

  • Despite functioning as a putative tumor suppressor, "spontaneous tumor incidence and spectrum were similar in wild-type and Ing2-deficient mice"

  • This unexpected finding requires reconciliation with in vitro studies

• Mechanistic Questions:

  • How does ING2 coordinate with SnoN to activate TGF-β responses when SnoN is commonly thought to act as a transcriptional corepressor?

  • What determines whether ING2 functions in gene activation versus repression?

• Therapeutic Implications:

  • Could targeting ING2 have unintended consequences on fertility?

  • How can specificity be achieved given ING2's involvement in fundamental cellular processes?

Research methodologies to address these contradictions include:

• Careful dose-response studies to evaluate concentration-dependent effects

• Tissue-specific and inducible knockout models to resolve temporal effects

• Single-cell approaches to identify cell-type specific functions

• Detailed structure-function analyses to map interaction domains