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Histone methylation reader domains are protein modules that recognize specific histone methylation marks, such as methylated or unmethylated lysine or arginine residues on histones. These reader proteins play crucial roles in the epigenetic regulation of gene expression, chromatin structure, and DNA damage repair. Dysregulation of these proteins has been linked to various diseases, including cancer, neurodegenerative diseases, and developmental disorders. Therefore, targeting these proteins with chemical inhibitors has emerged as an attractive approach for therapeutic intervention, and significant progress has been made in this area. In this review, we will summarize the development of inhibitors targeting histone methylation readers, including MBT domains, chromodomains, Tudor domains, PWWP domains, PHD fingers, and WD40 repeat domains. For each domain, we will briefly discuss its identification and biological/biochemical functions, and then focus on the discovery of inhibitors tailored to target this domain, summarizing the property and potential application of most inhibitors. We will also discuss the structural basis for the potency and selectivity of these inhibitors, which will aid in further lead generation and optimization. Finally, we will also address the challenges and strategies involved in the development of these inhibitors. It should facilitate the rational design and development of novel chemical scaffolds and new targeting strategies for histone methylation reader domains with the help of this body of data.

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WD40-repeat (WDR) domain proteins play a crucial role in mediating protein-protein interactions that sustain oncogenesis in human cancers. One prominent example is the interaction between the transcription factor MYC and its chromatin co-factor, WD40-repeat domain protein 5 (WDR5), which is essential for oncogenic processes. The MYC family of proteins is frequently overexpressed in various cancers and has been validated as a promising target for anticancer therapies. The recruitment of MYC to chromatin is facilitated by WDR5, highlighting the significance of their interaction. Consequently, inhibiting the MYC-WDR5 interaction has been shown to induce the regression of malignant tumors, offering an alternative approach to targeting MYC in the development of anticancer drugs. WDR5 has two protein interaction sites, the "WDR5-binding motif" (WBM) site for MYC interaction and the histone methyltransferases SET1 recognition motif "WDR5-interacting" (WIN) site forming MLL complex. Significant efforts have been dedicated to the discovery of inhibitors that target the WDR5 protein. More recently, the successful application of targeted protein degradation technology has enabled the removal of WDR5. This breakthrough has opened up new avenues for inhibiting the interaction between WDR5 and the binding partners. In this review, we address the recent progress made in targeting WDR5 to inhibit MDR5-MYC and MDR5-MLL1 interactions, including its targeted protein degradation and their potential impact on anticancer drug discovery.

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The mitogen-activated protein kinase organizer 1 (MORG1) is a scaffold molecule for the ERK signaling pathway, but also binds to prolyl-hydroxylase 3 and modulates HIFα expression. To obtain further insight into the role of MORG1, knockout-mice were generated by homologous recombination. While Morg1+/- mice developed normally without any apparent phenotype, there were no live-born Morg1-/- knockout offspring, indicating embryonic lethality. The intrauterine death of Morg1-/- embryos is caused by a severe failure to develop brain and other neuronal structures such as the spinal cord and a failure of chorioallantoic fusion. On E8.5, Morg1-/- embryos showed severe underdevelopment and proliferative arrest as indicated by absence of Ki67 expression, impaired placental vascularization and altered phenotype of trophoblast giant cells. On E9.5, the malformed Morg1-/- embryos showed defective turning into the final fetal position and widespread apoptosis in many structures. In the subsequent days, apoptosis and decomposition of embryonic tissue progressed, accompanied by a massive infiltration of inflammatory cells. Developmental aberrancies were accompanied by altered expression of HIF-1/2α and VEGF-A and caspase-3 activation in embryos and extraembryonic tissues. In conclusion, the results suggest a multifactorial process that causes embryonic death in homozygous Morg1 mutant mice, described here, to the best of our knowledge, for the first time.

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WD40 repeat-containing proteins play a key role in many cellular functions including signal transduction, protein degradation, and apoptosis. The WD40 domain is highly conserved, and its typical structure is a ß-propeller consisting of 4-8 blades which probably serves as a scaffold for protein-protein interaction. Some WD40 repeat-containing proteins form part of the corepressor complex of nuclear hormone receptors, a family of ligand-dependent transcription factors that play a central role in the regulation of gene transcription. This explains their involvement in endocrine physiology and pathology. In the present review, we first touch upon the structure of WD40 repeat-containing proteins. Next, we describe our current understanding of the role of WD40 domain-containing proteins in nuclear receptor signaling, e.g., as corepressor or coactivator. In the final part of this review, we focus on WD40 domain-containing proteins that are associated with endocrine pathologies. These pathologies vary from isolated dysfunction of one endocrine axis, e.g., congenital isolated central hypothyroidism, to more complex congenital syndromes comprising endocrine phenotypes, such as the Triple-A syndrome.

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Fruit color is one of the most important traits of jujube (Ziziphus jujuba Mill.). However, the differences in the pigments of different varieties of Jujube are not well studied. In addition, the genes responsible for fruit color and their underlying molecular mechanisms remain unclear. In this study, two jujube varieties, namely "Fengmiguan" (FMG) and "Tailihong" (TLH), were considered. The metabolites from jujube fruits were investigated using ultra-high-performance liquid chromatography/tandem mass spectrometry. Transcriptome was used to screen anthocyanin regulatory genes. The gene function was confirmed by overexpression and transient expression experiments. The gene expression was analyzed by quantitative reverse transcription polymerase chain reaction analyses and subcellular localization. Yeast-two-hybrid and bimolecular fluorescence complementation were used to screen and identify the interacting protein. These cultivars differed in color owing to their respective anthocyanin accumulation patterns. Three and seven types of anthocyanins were found in FMG and TLH, respectively, which played a key role in the process of fruit coloration. ZjFAS2 positively regulates anthocyanin accumulation. The expression profile of ZjFAS2 exhibited its different expression trends in different tissues and varieties. Subcellular localization experiments showed that ZjFAS2 was localized to the nucleus and membrane. A total of 36 interacting proteins were identified, and the possibility of ZjFAS2 interacting with ZjSHV3 to regulate jujube fruit coloration was studied. Herein, we investigated the role of anthocyanins in the different coloring patterns of the jujube fruits and provided a foundation for elucidating the molecular mechanism underlying jujube fruit coloration.

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Cilia are conserved organelles found in many cell types in eukaryotes, and their dysfunction causes defects in environmental sensing and signaling transduction; such defects are termed ciliopathies. Distinct cilia have cell-specific morphologies and exert distinct functions. However, the underlying mechanisms of cell-specific ciliogenesis and regulation are unclear. Here, we identified a WD40-repeat (WDR) protein, NMTN-1 (the homolog of mammalian WDR47), and show that it is specifically required for ciliogenesis of AWB chemosensory neurons in C. elegans. NMTN-1 is expressed in the AWB chemosensory neuron pair, and is enriched at the basal body (BB) of the AWB cilia. Knockout of nmtn-1 causes abnormal AWB neuron cilia morphology, structural integrity, and induces aberrant AWB-mediated aversive behaviors. We further demonstrate that nmtn-1 deletion affects movement of intraflagellar transport (IFT) particles and their cargo delivery in AWB neurons. Our results indicate that NMTN-1 is essential for AWB neuron ciliary morphology and function, which reveal a novel mechanism for cell-specific ciliogenesis. Given that WDR47/NMTN-1 is conserved in mammals, our findings may help understanding of the process of cell-specific ciliogenesis and provide insights for treating ciliopathies.

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Members of the WD40-repeat protein family can be found in all eukaryotic proteomes where they usually serve as interaction platforms for the assembly of large protein complexes and are therefore essential for the integrity of these complexes. In the malaria parasite Plasmodium falciparum, the WD40-repeat protein PfWLP1 has been shown to interact with members of distinct adhesion protein complexes in the asexual blood stages and gametocyte stages. In this study, we demonstrate that the presence of PfWLP1 is crucial for both the stability of these gametocyte-specific adhesion complexes as well as for gametocyte maturation and gametogenesis. Using reverse genetics, we generated a PfWLP1-knockdown parasite line for functional characterization of the protein. Knockdown of PfWLP1 resulted in a slight reduction of gametocyte numbers and significantly the impaired ability of the gametocytes to exflagellate. PfWLP1-knockdown further led to reduced protein levels of the Limulus coagulation factor C-like (LCCL)-domain proteins PfCCp1 and PfCCp2, which are key components of the adhesion complexes. These findings suggest that the interaction of PfWLP1 with members of the PfCCp-based adhesion complex ensures complex stability and thereby contributes to gametocyte viability and exflagellation.

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The MYC gene, one of the most common dysregulated driver genes in human cancers, is composed of three paralogous genes C-MYC, N-MYC and L-MYC. It is abnormally activated in more than half of cancer types. Since MYC plays an important role in the formation, maintenance and progression of cancer, targeting MYC is an effective strategy for cancer treatment. As a potential anti-cancer target, MYC is considered "undruggable" because it lacks a suitable pocket for accommodating small molecule inhibitors. Recently, under the guidance of protein structure information and many computational tools, many indirect strategies to inhibit MYC have emerged and shown favorable anti-cancer effects in tumor models. In this paper, the recent small molecules that indirectly target MYC are divided into inhibitors acting on the protein-protein interaction (PPI) among MYC and other proteins, and targeting inhibitors regulating MYC action. Additionally, the introduction and assessment towards compounds with different mechanisms are summarized to provide reference for the further research of MYC inhibitors.

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SCF-type E3 ubiquitin ligases provide specificity to numerous selective protein degradation events in plants, including those that enable survival under environmental stress. SCF complexes use F-box (FBX) proteins as interchangeable substrate adaptors to recruit protein targets for ubiquitylation. FBX proteins almost universally have structure with two domains: A conserved N-terminal F-box domain interacts with a SKP protein and connects the FBX protein to the core SCF complex, while a C-terminal domain interacts with the protein target and facilitates recruitment. The F-BOX STRESS INDUCED (FBS) subfamily of plant FBX proteins has an atypical structure, however, with a centrally located F-box domain and additional conserved regions at both the N- and C-termini. FBS proteins have been linked to environmental stress networks, but no ubiquitylation target(s) or biological function has been established for this subfamily. We have identified two WD40 repeat-like proteins in Arabidopsis that are highly conserved in plants and interact with FBS proteins, which we have named FBS INTERACTING PROTEINs (FBIPs). FBIPs interact exclusively with the N-terminus of FBS proteins, and this interaction occurs in the nucleus. FBS1 destabilizes FBIP1, consistent with FBIPs being ubiquitylation targets SCFFBS1 complexes. This work indicates that FBS proteins may function in stress-responsive nuclear events, and it identifies two WD40 repeat-like proteins as new tools with which to probe how an atypical SCF complex, SCFFBS, functions via FBX protein N-terminal interaction events.

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Microtubules are polarized intracellular polymers that play key roles in the cell, including in transport, polarity, and cell division. Across eukaryotic cell types, microtubules adopt diverse intracellular organization to accommodate these distinct functions coordinated by specific cellular sites called microtubule-organizing centers (MTOCs). Over 50 years of research on MTOC biology has focused mainly on the centrosome; however, most differentiated cells employ non-centrosomal MTOCs (ncMTOCs) to organize their microtubules into diverse arrays, which are critical to cell function. To identify essential ncMTOC components, we developed the biotin ligase-based, proximity-labeling approach TurboID for use in C. elegans. We identified proteins proximal to the microtubule minus end protein PTRN-1/Patronin at the apical ncMTOC of intestinal epithelial cells, focusing on two conserved proteins: spectraplakin protein VAB-10B/MACF1 and WDR-62, a protein we identify as homologous to vertebrate primary microcephaly disease protein WDR62. VAB-10B and WDR-62 do not associate with the centrosome and instead specifically regulate non-centrosomal microtubules and the apical targeting of microtubule minus-end proteins. Depletion of VAB-10B resulted in microtubule mislocalization and delayed localization of a microtubule nucleation complex É£-tubulin ring complex (γ-TuRC), while loss of WDR-62 decreased the number of dynamic microtubules and abolished γ-TuRC localization. This regulation occurs downstream of cell polarity and in conjunction with actin. As this is the first report for non-centrosomal roles of WDR62 family proteins, we expand the basic cell biological roles of this important disease protein. Our studies identify essential ncMTOC components and suggest a division of labor where microtubule growth and localization are distinctly regulated.

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Nuclear factor erythroid 2-related factor 2 (Nrf2) is a critical transcription factor that orchestrates cellular responses to oxidative stress. Because the dysregulation of Nrf2 has been implicated in many diseases, precise regulation of its protein level is crucial for maintaining homeostasis. Kelch-like-ECH-associated protein 1 (Keap1) and WD40 repeat protein 23 (WDR23) directly regulate Nrf2 levels via similar but distinct proteasome-dependent pathways. WDR23 forms a part of the WDR23-Cullin 4A-RING ubiquitin ligase complex (CRL4AWDR23), whereas Keap1 serves as a substrate adaptor for the Cullin 3-containing ubiquitin ligase complex. However, the mechanisms underlying crosstalk between these Keap1 and WDR23 pathways for the regulation of Nrf2 levels have not been investigated. Here, we showed that knockdown (KD) of Keap1 upregulated the expression of Cullin4A (CUL4A) in a specificity protein 1 (Sp1)-dependent manner. We also revealed that Sp1 interacted with Keap1, leading to ubiquitination of Sp1. Increases in Sp1 by Keap1 KD triggered Sp1 binding to the fourth Sp1 binding site (Sp1_M4) within the -230/+50 region of the CUL4A gene. We also demonstrated that the overexpression and KD of Sp1 reduced and increased Nrf2 protein levels, respectively. These effects were abrogated by the WDR23 KD, suggesting that Sp1 also regulates Nrf2 levels via the ubiquitin ligase complex CRL4AWDR23. In conclusion, we discovered Sp1 as a novel substrate of Keap1 and provided evidence that Sp1 regulates the expression of CUL4A. We revealed a novel role for Sp1 in mediating crosstalk between two independent regulators of Nrf2 protein levels.

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Missense and frameshift pathogenic variants and microdeletions involving TBL1XR1 gene have been described in patients with intellectual disability, autism, Rett-like features and schizophrenia, some of them with the clinical diagnosis of Pierpont syndrome, a rare pattern of multiple congenital anomalies, but others without dysmorphic findings or with non-specific ones, and also patients with only some of the features associated with Pierpont syndrome. We here present a case with a de novo novel missense variant in TBL1XR1 gene with overlapping features with Pierpont syndrome and autism, a neurobehavioral manifestation not previously reported in Pierpont syndrome. This patient expands the phenotypic spectrum of TBL1XR1 gene pathogenic variants.

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Immune cells are especially dependent on the proper functioning of the actin cytoskeleton, and both innate and adaptive responses rely on it. Leukocytes need to adhere not only to substrates but also to cells in order to form synapses that pass on instructions or kill infected cells. Neutrophils literally squeeze their cell body during blood extravasation and efficiently migrate to the inflammatory focus. Moreover, the development of immune cells requires the remodeling of their cytoskeleton as it depends on, among other processes, adhesive contacts and migration. In recent years, the number of reports describing cytoskeletal defects that compromise the immune system has increased immensely. Furthermore, a new emerging paradigm points toward a role for the cellular actin content as an essential component of the so-called homeostasis-altering molecular processes that induce the activation of innate immune signaling pathways. Here, we review the role of critical actin-cytoskeleton-remodeling proteins, including the Arp2/3 complex, cofilin, coronin and WD40-repeat containing protein 1 (WDR1), in immune pathophysiology, with a special focus on autoimmune and autoinflammatory traits.

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BACKGROUND: The WD40-repeat containing proteins, including DDB1-CUL4-associated factors (DCAFs), are abundant and conserved proteins that play important roles in different cellular processes including spermatogenesis. DCAFs are subset of WD40 family proteins that contain WDxR motif and have been proposed to function as substrate receptor for Cullin4-RING-based E3 ubiquitin ligase complexes to recruit diverse proteins for ubiquitination, a vital process in spermatogenesis. Large number of WD40 genes has been identified in different species including mouse and human. However, a systematic expression profiling of WD40 genes in different tissues of mouse and human has not been investigated. We hypothesize that large number of WD40 genes may express highly or specifically in the testis, where their expression is uniquely regulated during testis development and spermatogenesis. Therefore, the objective of this study is to mine and characterize expression patterns of WD40 genes in different tissues of mouse and human with particular emphasis on DCAF genes expressions during mouse testicular development. RESULTS: Publically available RNA sequencing (RNA seq) data mining identified 347 and 349 WD40 genes in mouse and human, respectively. Hierarchical clustering and heat map analyses of RNA seq datasets revealed differential expression patterns of WD40 genes with around 60-73% of the genes were highly or specifically expressed in testis. Similarly, around 74-83% of DCAF genes were predominantly or specifically expressed in testis. Moreover, WD40 genes showed distinct expression patterns during embryonic and postnatal testis development in mice. Finally, different germ cell populations of testis showed specific patterns of WD40 genes expression. Predicted gene ontology analyses revealed more than 80% of these proteins are implicated in cellular, metabolic, biological regulation and cell localization processes. CONCLUSIONS: We have identified large number of WD40 family genes that are highly or specifically expressed in the testes of mouse and human. Moreover, WD40 genes have distinct expression patterns during embryonic and postnatal development of the testis in mice. Further, different germ cell populations within the testis showed specific patterns of WD40 genes expression. These results provide foundation for further research towards understanding the functional genomics and molecular mechanisms of mammalian testis development and spermatogenesis.

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Ubiquitination is a reversible post-translational modification that has emerged as a critical regulator of synapse development and function. However, the mechanisms that regulate the deubiquitinating enzymes (DUBs) responsible for the removal of ubiquitin from target proteins are poorly understood. We have previously shown that the DUB ubiquitin-specific protease 46 (USP-46) removes ubiquitin from the glutamate receptor GLR-1 and regulates its trafficking and degradation in Caenorhabditis elegans We found that the WD40-repeat proteins WDR-20 and WDR-48 bind and stimulate the catalytic activity of USP-46. Here, we identified another mechanism by which WDR-48 regulates USP-46. We found that increased expression of WDR-48, but not WDR-20, promotes USP-46 abundance in mammalian cells in culture and in C. elegans neurons in vivo Inhibition of the proteasome increased USP-46 abundance, and this effect was nonadditive with increased WDR-48 expression. We found that USP-46 is ubiquitinated and that expression of WDR-48 reduces the levels of ubiquitin-USP-46 conjugates and increases the t1/2 of USP-46. A point-mutated WDR-48 variant that disrupts binding to USP-46 was unable to promote USP-46 abundance in vivo Finally, siRNA-mediated knockdown of wdr48 destabilizes USP46 in mammalian cells. Together, these results support a model in which WDR-48 binds and stabilizes USP-46 protein levels by preventing the ubiquitination and degradation of USP-46 in the proteasome. Given that a large number of USPs interact with WDR proteins, we propose that stabilization of DUBs by their interacting WDR proteins may be a conserved and widely used mechanism that controls DUB availability and function.

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MAIN CONCLUSION: MiMYB1 and MibHLH2 play key roles in anthocyanin biosynthesis in Matthiola incana flowers. We established a transient expression system using Turnip mosaic virus vector in M. incana. Garden stock (Matthiola incana (L.) R. Br.) is a popular flowering plant observed from winter to spring in Japan. Here we observed that anthocyanin accumulation in 'Vintage Lavender' increased with flower development, whereas flavonol accumulation remained constant throughout flower development. We obtained five transcription factor genes, MiMYB1, MibHLH1, MibHLH2, MiWDR1, and MiWDR2, from M. incana floral cDNA contigs. Yeast two-hybrid analyses revealed that MiMYB1 interacted with MibHLH1, MibHLH2, and MiWDR1, but MiWDR2 did not interact with any transcription factor. Expression levels of MiMYB1 and MibHLH2 increased in petals during floral bud development. Their expression profiles correlated well with the temporal profiles of MiF3'H, MiDFR, MiANS, and Mi3GT transcripts and anthocyanin accumulation profile. On the other hand, MibHLH1 was expressed weakly in all organs of 'Vintage Lavender'. However, high expression levels of MibHLH1 were detected in petals of other cultivars with higher levels of anthocyanin accumulation than 'Vintage Lavender'. MiWDR1 and MiWDR2 maintained constant expression levels in petals during flower development and vegetative organs. Transient MiMYB1 expression in 1-month-old M. incana seedlings using a Turnip mosaic virus vector activated transcription of the endogenous anthocyanin biosynthetic genes MiF3'H, MiDFR, and MiANS and induced ectopic anthocyanin accumulation in leaves. Therefore, MiMYB1 possibly interacts with MibHLH2 and MiWDR1, and this trimeric protein complex activates the transcription of anthocyanin biosynthetic genes in M. incana flowers. Moreover, MibHLH1 acts as an enhancer of anthocyanin biosynthesis with the MiMYB1-MibHLH2-MiWDR1 complex. This study revealed the molecular mechanism involved in the regulation of anthocyanin accumulation levels in M. incana flowers.

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In plants, the cryptochrome photoreceptors suppress the activity of the COP1/SPA ubiquitin ligase to initiate photomorphogenesis in blue light. Both CRY1 and CRY2 interact with the COP1/SPA complex in a blue light-dependent manner. The mechanisms underlying the inhibition of COP1 activity through direct interactions with photoactivated CRYs are not fully understood. Here we tested the hypothesis that CRY2 inhibits COP1 by displacing the degradation substrates from COP1. To this end, we analyzed the role of a conserved valine-proline (VP) motif in the C-terminal domain of CRY2 (CCT2), which resembles the core COP1-WD40-binding sequences present in the substrates of COP1. We show that the VP motif in CRY2 is essential for the interaction of CRY2 with COP1 in yeast two-hybrid assays and in planta. Mutations in the VP motif of CRY2 abolished the CRY2 activity in photomorphogenesis, indicating the importance of VP. The interaction between COP1 and its VP-containing substrate PAP2 was prevented in the presence of coexpressed CRY2, but not in the presence of CRY2 carrying a VP mutation. Thus, since both PAP2 and CRY2 engage VP motifs to bind to COP1, these results demonstrate that CRY2 outcompetes PAP2 for binding to COP1. We further found that the previously unknown interaction between SPA1-WD and CCT2 occurs via the VP motif in CRY2, suggesting structural similarities in the VP-binding pockets of COP1-WD40 and SPA1-WD40 domains. A VP motif present in CRY1 is also essential for binding to COP1. Thus, CRY1 and CRY2 might share this mechanism of COP1 inactivation.

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The Arabidopsis thaliana WD40-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1) controls epidermis development, playing opposite roles in trichome differentiation and root hair formation. We isolated and characterized LbTTG1 (encoding a WD40-repeat protein with high sequence similarity to TTG1) from the recretohalophyte Limonium bicolor, which actively excretes absorbed salt via a salt gland. The complete open reading frame of LbTTG1 was 1,095 bp, encoding a protein of 364 amino acids, and showed highest expression during the salt gland initiation stage. We heterologously expressed LbTTG1 in wild type and ttg1-13 Arabidopsis plants to verify the protein's function, and the copies of LbTTG1 were identified in transgenic strains using southern blotting. Trichomes were extremely induced on the first true leaves of plants heterologously expressing LbTTG1, whereas no trichomes were produced by ttg1-13 plants. Conversely, plants heterologously expressing LbTTG1 produced fewer root hairs than ttg1-13 plants. In plants heterologously expressing LbTTG1 compared to controls, epidermis differentiation genes (GLABRA1 and GLABRA3) were up-regulated while genes encoding negative regulators of trichome development (TRIPTYCHON and CAPRICE) were down-regulated. Under increased NaCl concentrations, both of the transgenic lines showed enhanced germination and root length, and accumulated less malondialdehyde (MDA) and Na+ and produced more proline, soluble sugar, and higher glutathione S-transferase activity, compared with the ttg1-13 mutant. These results indicate that LbTTG1 participates in epidermis development in Arabidopsis, similarly to other WD40-repeat proteins, and specifically increases salt tolerance of transgenic Arabidopsis by reducing ion accumulation and increasing osmolyte levels.

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G-protein ßγ subunits are key participants in G-protein signaling. These subunits facilitate interactions between receptors and G proteins that are critical for the G protein activation cycle at the plasma membrane. In addition, they play roles in directly transducing signals to an ever expanding range of downstream targets, including integral membrane and cytosolic proteins. Emerging data indicate that Gßγ may play additional roles at intracellular compartments including endosomes, the Golgi apparatus, and the nucleus. Here, we discuss the molecular and structural basis for their ability to coordinate this wide range of cellular activities.

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A hub protein in protein interaction networks will typically have a large number of diverse interactions. Determining the core interactions and the function of such a hub protein remains a significant challenge in the study of networks. Proteins with WD40 repeats represent a large class of proteins that can be hub proteins. WDR76 is a poorly characterized WD40 repeat protein with possible involvement in DNA damage repair, cell-cycle progression, apoptosis, gene expression regulation, and protein quality control. WDR76 has a large and diverse interaction network that has made its study challenging. Here we rigorously carry out a series of affinity purification coupled to mass spectrometry (AP-MS) analyses to map out the WDR76 interactome through different biochemical conditions. We apply AP-MS analysis coupled to size-exclusion chromatography to resolve WDR76-based protein complexes. Furthermore, we also show that WDR76 interacts with the CCT complex via its WD40 repeat domain and with DNA-PK-KU, PARP1, GAN, SIRT1, and histones outside of the WD40 domain. An evaluation of the stability of WDR76 interactions led to focused and streamlined reciprocal analyses that validate the interactions with GAN and SIRT1. Overall, the approaches used to study WDR76 would be valuable to study other proteins containing WD40 repeat domains, which are conserved in a large number of proteins in many organisms.

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