RBBP4 Human

What is RBBP4 and what are its primary functions in human cells?

RBBP4 is a nuclear WD40 motif-containing protein that functions as a histone chaperone and is a core component of multiple chromatin-modifying complexes, including the Nucleosome Remodeling and Deacetylase (NuRD) complex, Polycomb Repressive Complex 2 (PRC2), and DREAM complex . It regulates chromatin structure and gene expression by facilitating histone modifications and nucleosome assembly . RBBP4 plays essential roles in cell cycle regulation, primarily binding to distal regulatory elements and influencing gene expression patterns that control cellular proliferation and differentiation . The protein contains a seven-bladed β-propeller fold that enables interactions with multiple protein partners through two distinct binding sites: a top pocket that interacts with proteins like BCL11A and histone H3, and a side pocket that binds to SUZ12, MTA1, and histone H4 .

How does RBBP4 differ from its homolog RBBP7?

Despite sharing approximately 89% homology in amino acid sequence, RBBP4 and RBBP7 exhibit distinct functional roles in cellular processes . Research demonstrates that RBBP4, but not RBBP7, is indispensable for maintaining the identity of mouse embryonic stem cells (mESCs) . Auxin-induced degradation experiments show that only RBBP4 depletion can reprogram mESCs to totipotent 2C-like cells, while RBBP7 degradation doesn't produce this effect . The functional divergence between these highly similar proteins highlights the specificity of RBBP4's interactions with chromatin and regulatory proteins, particularly its unique role in recruiting specific epigenetic modifiers like G9a and KAP1 to deposit histone marks on transposable elements . These differences are significant when considering therapeutic targeting approaches that aim to modulate RBBP4 without affecting RBBP7 function.

What protein complexes incorporate RBBP4 and how does this affect their function?

RBBP4 serves as a critical component in multiple chromatin-modifying complexes, each with distinct cellular functions:

RBBP4's integration into these complexes enables it to influence diverse cellular processes including differentiation, proliferation, and gene silencing through multiple epigenetic mechanisms . Its role can be either structural, stabilizing complex formation, or functional, directly mediating interactions with histones or DNA .

How does RBBP4 influence neocortical development and neurogenesis?

RBBP4 plays a critical role in regulating neocortical progenitor proliferation and neuronal differentiation during embryonic development . CRISPR/Cas9-mediated knockdown of RBBP4 in mouse neocortical progenitors at embryonic day 12.5 significantly reduces neuronal output, particularly affecting CTIP2-expressing neurons that form deep cortical layers . This indicates RBBP4's essential function during the critical period of deep layer neurogenesis.

Mechanistically, genome-wide occupancy analysis reveals that RBBP4 primarily binds to distal regulatory elements of genes involved in neuron differentiation pathways . A key discovery is that RBBP4 binds to and regulates Cdon, which encodes a receptor protein in the Sonic hedgehog (Shh) signaling pathway . Knockdown of Cdon phenocopies RBBP4 knockdown, resulting in reduced neurogenesis, while CDON overexpression rescues the phenotypes caused by RBBP4 loss . This functional link between RBBP4 and CDON establishes a molecular pathway through which chromatin regulation influences neocortical development, providing insight into how epigenetic factors coordinate neuronal fate specification during brain development.

What is the relationship between RBBP4 dysfunction and neurodevelopmental disorders?

RBBP4 dysfunction has significant implications for neurodevelopmental disorders due to its crucial role in chromatin regulation during brain development . Since RBBP4 regulates the expression of genes essential for proper neurogenesis and neuronal differentiation, its dysregulation can lead to abnormal cortical development .

The reduced neuronal output, particularly of CTIP2-expressing deep layer neurons, observed upon RBBP4 knockdown suggests that RBBP4 mutations or expression changes could contribute to cortical malformations and associated neurodevelopmental conditions . Furthermore, RBBP4's interaction with the Shh signaling pathway through Cdon is particularly significant, as Shh signaling defects are already implicated in various neurodevelopmental disorders, including holoprosencephaly and certain forms of intellectual disability .

Research demonstrates that chromatin modifiers like RBBP4 integrate environmental signals with developmental gene expression programs, and mutations in such chromatin regulators are increasingly linked to neurodevelopmental disorders including autism spectrum disorders, intellectual disability, and epilepsy . Understanding RBBP4's specific contributions to these conditions requires further investigation of its target genes and the consequences of its dysregulation in human neural development.

How is RBBP4 implicated in non-small cell lung cancer (NSCLC) progression and prognosis?

RBBP4 is significantly upregulated in non-small cell lung cancer (NSCLC) tissues compared to normal lung tissue, and its overexpression strongly correlates with poor clinical outcomes and reduced survival rates . Analysis of TCGA and GSE30219 datasets reveals RBBP4 as a potential diagnostic and prognostic biomarker for NSCLC .

Mechanistically, RBBP4 appears to modulate tumor immunity and autophagy pathways in NSCLC cells . Eighteen types of immune cells show significant enrichment in tumors with low RBBP4 expression compared to those with high expression, suggesting RBBP4 influences the tumor immune microenvironment . Transcriptomic analysis identified differentially expressed genes (DEGs) associated with RBBP4 that are enriched in autophagy pathways .

Experimental validation demonstrates that RBBP4 knockdown induces autophagy and apoptosis in NSCLC cells, promoting cell death that can be inhibited by the autophagy inhibitor 3-MA . In vivo studies show that targeted siRNA against RBBP4 significantly reduces tumor development in mouse xenograft models, elevating autophagy-related protein levels and inducing apoptosis and necrosis in tumor tissues . These findings position RBBP4 as both a potential diagnostic marker and therapeutic target in NSCLC through its modulation of autophagic cell death.

What experimental approaches are most effective for studying RBBP4's role in cancer progression?

Effective investigation of RBBP4's role in cancer progression requires a multi-faceted experimental approach:

• Expression analysis: Quantitative assessment of RBBP4 levels in cancer tissues versus normal tissues using immunohistochemistry, RT-qPCR, and western blotting, complemented by bioinformatic analysis of cancer databases like TCGA and GEO .

• Genetic manipulation: CRISPR/Cas9-mediated knockout, siRNA knockdown, or inducible degradation systems (e.g., auxin-inducible degron) to reduce RBBP4 expression in cancer cell lines, alongside overexpression studies to examine gain-of-function effects .

• Pathway analysis: Transcriptome sequencing of RBBP4-manipulated cells followed by Gene Ontology, KEGG pathway enrichment, and protein-protein interaction network analyses to identify downstream effectors and biological processes .

• Functional assays: Assessment of cell proliferation, migration, invasion, apoptosis, and specific processes like autophagy using appropriate assays (e.g., MTT, wound healing, transwell, Annexin V/PI staining, LC3 puncta formation) .

• In vivo models: Xenograft models using RBBP4-manipulated cancer cells to evaluate tumor growth, metastasis, and response to therapies .

• ChIP-seq and epigenetic profiling: Genome-wide occupancy analysis to identify direct RBBP4 targets and associated epigenetic modifications in cancer cells .

• Small molecule antagonist testing: Screening and validation of compounds targeting RBBP4 interactions, particularly those disrupting its binding with partner proteins at the top pocket .

This comprehensive approach enables researchers to elucidate both the mechanistic basis of RBBP4's contribution to cancer and its potential as a therapeutic target.

How does RBBP4 regulate pluripotency and cell fate transitions in stem cells?

RBBP4 functions as an epigenetic barrier that maintains pluripotency and prevents spontaneous differentiation of stem cells toward totipotent states . In mouse embryonic stem cells (mESCs), RBBP4 is indispensable for maintaining cellular identity and preventing unscheduled activation of totipotency programs .

Auxin-induced degradation experiments demonstrate that selective removal of RBBP4, but not its homolog RBBP7, reprograms mESCs to totipotent 2C-like cells . Additionally, RBBP4 depletion enhances the transition from mESCs to trophoblast cells, indicating its role in restricting lineage plasticity .

Mechanistically, RBBP4 maintains the epigenetic barriers to totipotency through several coordinated activities:

• It binds to endogenous retroviruses (ERVs) and functions as an upstream regulator by recruiting specific epigenetic modifiers: G9a to deposit H3K9me2 on ERVL elements and KAP1 to deposit H3K9me3 on ERV1/ERVK elements .

• It facilitates maintenance of nucleosome occupancy at ERV sites within heterochromatin regions through interaction with the chromatin remodeler CHD4 .

• RBBP4 depletion leads to loss of heterochromatin marks and subsequent activation of transposable elements (TEs) and 2C-specific genes that drive the transition toward totipotency .

These findings illustrate that RBBP4 is required for heterochromatin assembly at specific genomic loci, particularly transposable elements, which is critical for preventing unscheduled activation of totipotency programs in pluripotent stem cells .

What is the significance of RBBP4's interaction with transposable elements (TEs) in stem cell biology?

RBBP4's interaction with transposable elements (TEs) represents a crucial regulatory mechanism in stem cell biology, particularly in maintaining the balance between pluripotency and totipotency . Transposable elements, especially endogenous retroviruses (ERVs), are normally silenced in most cell types but become activated in totipotent cells during early embryonic development .

The significance of RBBP4's interaction with TEs includes:

• Epigenetic barrier maintenance: RBBP4 binds to three classes of ERVs (ERV1, ERVK, and ERVL) and recruits specific epigenetic modifiers to establish repressive histone modifications, creating an epigenetic barrier that prevents activation of totipotency programs .

• Cell fate regulation: By controlling the epigenetic state of TEs, RBBP4 regulates the expression of nearby 2C-specific genes that are critical for totipotency, thus influencing cell fate decisions .

• Genome stability: Proper silencing of TEs through RBBP4-mediated mechanisms helps maintain genome stability by preventing unscheduled transposition events that could lead to genomic instability .

• Developmental timing control: RBBP4's regulation of TEs ensures that totipotency programs are activated only at appropriate developmental stages, preventing premature or delayed activation that could disrupt normal development .

• Evolutionary significance: The regulatory relationship between RBBP4 and TEs reflects an evolutionary compromise where cells utilize chromatin regulators to control potentially harmful genomic elements while allowing their conditional activation during specific developmental windows .

Understanding RBBP4's role in TE regulation provides insights into the molecular mechanisms underlying cellular plasticity and developmental potential, with implications for regenerative medicine and reprogramming technologies .

What are the most effective knockdown or knockout strategies for studying RBBP4 function?

Several genetic manipulation approaches have proven effective for studying RBBP4 function, each with specific advantages depending on the research question:

• CRISPR/Cas9-mediated knockdown: This approach has been successfully employed to downregulate RBBP4 in neocortical progenitors, allowing for the study of its role in neurogenesis . The advantage of CRISPR/Cas9 is the ability to achieve efficient target specificity while potentially enabling both transient and stable modifications.

• Auxin-inducible degron (AID) system: This approach enables rapid, reversible, and temporally controlled degradation of RBBP4 protein . The AID system is particularly valuable for studying RBBP4's acute functions while avoiding compensatory mechanisms that might arise during conventional knockout strategies. This method revealed RBBP4's role as an epigenetic barrier to totipotency in stem cells .

• siRNA-mediated knockdown: Short-interfering RNA approaches have been successfully used to reduce RBBP4 expression in cancer cells, both in vitro and in vivo . The advantage of siRNA is the relative ease of delivery and the ability to achieve transient knockdown, which is useful for studying immediate cellular responses before compensatory mechanisms develop.

• Conditional knockout models: For in vivo studies, conditional knockout approaches using Cre-loxP systems can be effective for tissue-specific or temporally controlled RBBP4 deletion, allowing for the examination of its role in specific developmental contexts or adult tissues.

When selecting an approach, researchers should consider several factors: the desired degree and duration of RBBP4 depletion, potential compensatory mechanisms (particularly from RBBP7), cell type-specific efficiency, and whether acute versus chronic loss is more relevant to the biological question. Control experiments should include rescue conditions through RBBP4 re-expression and careful monitoring of off-target effects.

How can researchers effectively study RBBP4 binding partners and genomic targets?

To comprehensively characterize RBBP4's binding partners and genomic targets, researchers should employ a combination of techniques:

• Chromatin Immunoprecipitation followed by sequencing (ChIP-seq): This technique has been successfully used to map genome-wide occupancy of RBBP4, revealing its binding to distal regulatory elements and specific targets like Cdon . For optimal results, researchers should use validated RBBP4-specific antibodies and appropriate controls, including input DNA and IgG pulldowns.

• Co-immunoprecipitation (Co-IP) followed by mass spectrometry: This approach identifies protein-protein interactions of RBBP4 within macromolecular complexes like NuRD, PRC2, and DREAM . Crosslinking conditions should be optimized to capture both stable and transient interactions.

• Proximity-dependent biotinylation (BioID or TurboID): These methods involve fusing RBBP4 to a biotin ligase to identify proximal proteins in living cells, capturing both direct and indirect interactions within the native cellular environment.

• Immunofluorescence co-localization: This technique visualizes the spatial relationship between RBBP4 and potential partners or chromatin marks, providing context for biochemical interaction data.

• CUT&RUN or CUT&Tag: These techniques offer high resolution and sensitivity for mapping genomic binding sites with lower background than traditional ChIP-seq, particularly valuable for studying RBBP4's association with repetitive elements like ERVs .

• Protein-fragment complementation assays: These assays validate specific binary interactions between RBBP4 and candidate partners identified through proteomics approaches.

• CRISPR activation/interference at RBBP4 binding sites: This approach tests the functional significance of RBBP4 binding at specific genomic loci.

For comprehensive analysis, researchers should integrate datasets from multiple techniques and perform computational analyses to identify enriched sequence motifs, chromatin states, and functional pathways associated with RBBP4 binding . This multi-omics approach provides mechanistic insights into how RBBP4 coordinates gene expression programs through its diverse interactions.

How can small molecule antagonists of RBBP4 be developed and optimized for research applications?

Development of effective RBBP4 small molecule antagonists involves several key steps and considerations:

• Target site identification: Crystal structure analysis has revealed that RBBP4 contains two distinct binding pockets: a top pocket that interacts with proteins like BCL11A and histone H3, and a side pocket that binds SUZ12, MTA1, and histone H4 . The top pocket has been successfully targeted by the small molecule OICR17251, providing a foundation for further development .

• Structure-guided design: The crystal structure of RBBP4 in complex with OICR17251 offers valuable insights for structure-based drug design approaches . Molecular docking, molecular dynamics simulations, and structure-activity relationship (SAR) studies can guide rational optimization of lead compounds.

• High-throughput screening: Employing biochemical assays that measure disruption of RBBP4-peptide interactions, such as fluorescence polarization or AlphaScreen, can identify novel chemical scaffolds with activity against RBBP4 . Virtual screening approaches can complement physical screening to expand the chemical diversity of potential antagonists.

• Selectivity optimization: Given the high homology between RBBP4 and RBBP7 (89% amino acid identity), achieving selectivity is challenging but critical . Computational approaches that identify subtle structural differences between binding pockets and careful SAR studies can help develop compounds that preferentially target RBBP4.

• Cellular validation: Compounds should be evaluated for their ability to disrupt RBBP4 interactions in cellular contexts, using techniques such as proximity ligation assays, co-immunoprecipitation, or phenotypic readouts like changes in gene expression or cell fate .

• Target engagement studies: Cellular thermal shift assays (CETSA) or related techniques can confirm direct binding of compounds to RBBP4 in cells, providing evidence of on-target activity.

• Probe optimization: For research applications, optimizing properties like cell permeability, stability, and minimal off-target effects is essential, even if other drug-like properties are less critical.

The successful development of OICR17251 demonstrates the feasibility of targeting RBBP4 and provides a foundation for developing more potent and selective antagonists for research applications .

What are the emerging connections between RBBP4 and cellular stress responses or disease states?

Emerging research highlights several important connections between RBBP4 and cellular stress responses or disease states:

• Cancer progression and therapeutic resistance: RBBP4 overexpression in non-small cell lung cancer (NSCLC) correlates with poor prognosis and altered immunity profiles . RBBP4 appears to suppress autophagy-mediated cell death pathways, as its knockdown induces autophagy and apoptosis in NSCLC cells . This suggests RBBP4 may contribute to therapeutic resistance by inhibiting stress-induced cell death mechanisms.

• Cell cycle dysregulation: RBBP4 functions within the DREAM complex to inhibit E2F activity, and its downregulation alongside E2F upregulation is frequently observed in cells with cell cycle abnormalities . This implicates RBBP4 in maintaining proper cell cycle checkpoint functions that prevent uncontrolled proliferation.

• Neurodevelopmental disorders: RBBP4's critical role in neocortical neurogenesis and its regulatory function in the Shh signaling pathway through Cdon suggest its dysregulation may contribute to neurodevelopmental disorders . Mutations in chromatin regulators like RBBP4 are increasingly linked to conditions such as autism spectrum disorders and intellectual disability .

• Cellular reprogramming and differentiation: RBBP4 functions as an epigenetic barrier for the transition of pluripotent stem cells into totipotent states, suggesting it may be involved in maintaining cell identity under stress conditions that might otherwise trigger dedifferentiation .

• Genome stability: Through its role in silencing transposable elements and maintaining heterochromatin structure, RBBP4 likely contributes to genome stability under genotoxic stress conditions . Loss of proper heterochromatin maintenance can lead to genomic instability associated with aging and cancer.

These emerging connections highlight RBBP4 as a central coordinator of cellular responses to various stressors and developmental cues, with its dysregulation potentially contributing to multiple disease states through altered epigenetic regulation of key cellular processes.

What methodological challenges exist in reconciling in vitro and in vivo findings regarding RBBP4 function?

Researchers face several methodological challenges when attempting to reconcile in vitro and in vivo findings regarding RBBP4 function:

• Functional redundancy with RBBP7: Despite clear evidence that RBBP4 and RBBP7 have distinct functions in some contexts (like stem cell totipotency), their high homology (89%) creates challenges for specific targeting . In vivo, compensatory mechanisms involving RBBP7 may mask phenotypes observed in simpler in vitro systems. Researchers must design careful experiments that can distinguish between shared and unique functions.

• Context-dependent activities: RBBP4 functions within multiple protein complexes (NuRD, PRC2, DREAM) whose composition and activity vary across cell types and developmental stages . In vitro studies may not recapitulate the full complexity of these interactions, leading to findings that don't translate to in vivo contexts. Using tissue-specific or inducible approaches in animal models can help address this challenge.

• Temporal considerations: Acute versus chronic loss of RBBP4 may produce different outcomes due to compensatory mechanisms. The auxin-inducible degron system offers advantages for studying immediate effects of RBBP4 loss, but translating these findings to stable knockouts in vivo remains challenging .

• Cellular heterogeneity: In vivo tissues contain heterogeneous cell populations with varying RBBP4 expression and dependency. Advanced techniques like single-cell RNA-seq coupled with spatial transcriptomics can help dissect cell type-specific roles of RBBP4 that might be obscured in bulk analysis.

• Transposable element regulation: RBBP4's regulation of transposable elements like ERVs is a crucial aspect of its function, but these repetitive sequences are challenging to analyze with standard genomics approaches . Advanced techniques like CUT&RUN or CUT&Tag with optimized computational pipelines for repetitive element analysis are needed to accurately assess RBBP4's genomic targets in both in vitro and in vivo contexts.

• Species differences: While mouse models provide valuable insights, species-specific differences in chromatin regulation may affect the translation of findings to human contexts. Complementary studies in human cell systems, including organoids or humanized mouse models, can help address this challenge.

Addressing these challenges requires integrated experimental approaches that combine acute and chronic loss-of-function studies, cell type-specific analyses, and careful consideration of compensatory mechanisms across different biological contexts.

What are the most promising therapeutic applications of RBBP4 research?

Based on current research, several promising therapeutic applications of RBBP4 research are emerging:

• Cancer therapeutics: RBBP4's overexpression in NSCLC and its association with poor prognosis suggest it could be a valuable therapeutic target . Small molecule antagonists that disrupt RBBP4's interactions with partner proteins could potentially restore autophagy-mediated cell death in cancer cells . Combination therapies targeting RBBP4 alongside conventional treatments might overcome resistance mechanisms in aggressive tumors.

• Regenerative medicine: The discovery that RBBP4 degradation can reprogram pluripotent stem cells to totipotent-like states opens possibilities for enhanced cellular reprogramming protocols . Temporary inhibition of RBBP4 could improve the efficiency of generating specialized cell types for therapeutic applications or create more developmentally plastic cells with enhanced differentiation potential.

• Neurodevelopmental disorder treatments: Understanding RBBP4's role in neocortical development and neurogenesis could inform therapeutic strategies for conditions associated with aberrant brain development . Modulating RBBP4 or its downstream targets, like CDON in the Shh pathway, might help correct developmental abnormalities if intervention occurs during critical developmental windows.

• Epigenetic therapy: As a core component of several chromatin-modifying complexes, RBBP4 represents a node through which multiple epigenetic pathways can be influenced . Selective modulation of RBBP4's interactions with specific complexes could enable precise epigenetic reprogramming for various conditions characterized by epigenetic dysregulation.

• Cellular senescence and aging: Through its role in maintaining heterochromatin structure and genome stability, RBBP4 may influence cellular senescence and aging processes . Interventions targeting RBBP4 could potentially address age-related chromatin changes associated with various degenerative conditions.

The development of the first small molecule antagonist of RBBP4 (OICR17251) represents a significant step toward realizing these therapeutic applications . Further optimization of compound specificity, potency, and delivery strategies will be essential for translating these promising research directions into clinical applications.

What are the critical knowledge gaps that need to be addressed in RBBP4 research?

Despite significant advances in understanding RBBP4 function, several critical knowledge gaps remain that require attention from researchers:

• Structural basis of complex-specific functions: While RBBP4 is known to function within multiple complexes (NuRD, PRC2, DREAM), the structural determinants that direct it to specific complexes in different cellular contexts remain poorly understood . Structural studies of RBBP4 within intact complexes are needed to understand these context-dependent functions.

• Differential roles of RBBP4 and RBBP7: Despite their high homology, RBBP4 and RBBP7 clearly have non-redundant functions in some contexts, such as stem cell totipotency . A systematic comparison of their binding partners, post-translational modifications, and genomic targets would help clarify the molecular basis of their functional divergence.

• Tissue-specific requirements: Most studies have focused on RBBP4 in specific contexts like embryonic stem cells or cancer cell lines . Comprehensive analysis of tissue-specific expression, binding patterns, and functional requirements for RBBP4 across different human tissues would provide a more complete understanding of its physiological roles.

• Developmental dynamics: Although RBBP4's role in neocortical development has been established, its functions throughout other aspects of embryonic and postnatal development require further investigation . Temporal analysis of RBBP4 activity across developmental stages would enhance our understanding of when and how it influences cell fate decisions.

• Human disease associations: While RBBP4 overexpression has been linked to poor prognosis in NSCLC, systematic analysis of RBBP4 alterations across human diseases, including genetic disorders, would provide valuable insights into its broader pathological relevance .

• Regulation of RBBP4 itself: Little is known about how RBBP4 expression, stability, and activity are regulated. Understanding the upstream factors that control RBBP4 function would provide additional therapeutic entry points.

• Long-term consequences of RBBP4 modulation: While acute effects of RBBP4 depletion have been studied, the long-term consequences of its inhibition on genome stability, cellular identity, and organism health remain largely unexplored .