WD Repeat-containing Protein Research Articles

LncRNA VELRP Modulates Pulmonary Arterial Smooth Muscle Cell Proliferation and Promotes Vascular Remodeling in Pulmonary Hypertension.

Pulmonary hypertension is a devastating vascular disorder characterized by extensive pulmonary vascular remodeling, ultimately leading to right ventricular failure and death. Activation of PDGF (platelet-derived growth factor) signaling promotes the hyperproliferation of pulmonary arterial smooth muscle cells (PASMCs), thus contributing to the pulmonary vascular remodeling. However, the molecular mechanisms that govern hyperproliferation of PASMCs induced by PDGF remain largely unknown, including the contribution of long noncoding RNAs (lncRNAs). In this study, we aimed to identify a novel lncRNA regulated by PDGF implicated in PASMC proliferation in pulmonary vascular remodeling. RNA-sequencing analysis was conducted to identify a novel lncRNA named vessel-enriched lncRNA regulated by PDGF-BB (VELRP). Functional investigations of VELRP were performed using knockdown and overexpression strategies along with RNA sequencing. Validation of the function and potential mechanisms of VELRP were performed through Western blot, RNA immunoprecipitation, and chromatin immunoprecipitation assays. We identified a novel vessel-enriched lncRNA with an increased response to PDGF-BB stimulus. VELRP was identified as an evolutionarily conserved RNA molecules. Modulation of VELRP in PASMCs significantly altered cell proliferation. Mechanistically, VELRP enhances trimethylation of H3K4 by interacting with WDR5 (WD repeat-containing protein 5), leading to increased expression of CDK (cyclin-dependent kinase) 1, CDK2, and CDK4 and consequent hyperproliferation of PASMCs. The pathological relevance of VELRP upregulation in pulmonary artery was confirmed using rat pulmonary hypertension models in vivo, as well as in PASMCs from patients with idiopathic pulmonary arterial hypertension patients. Specific knockdown of VELRP in smooth muscle cells using adeno-associated virus type 9 SM22α (smooth muscle protein 22α) promoter-shRNA-mediated silencing of VELRP resulted in a significant decrease in right ventricular systolic pressure and vascular remodeling in rat pulmonary hypertension model. VELRP, as an lncRNA upregulated by PDGF-BB, mediates PASMC proliferation via WDR5/CDK signaling. In vivo studies demonstrate that targeted intervention of VELRP in smooth muscle cells can prevent the development of pulmonary hypertension.

Insights into the distinct membrane targeting mechanisms of WDR91 family proteins.

WDR91 and SORF1, members of the WD repeat-containing protein 91 family, control phosphoinositide conversion by inhibiting phosphatidylinositol 3-kinase activity on endosomes, which promotes endosome maturation. Here, we report the crystal structure of the human WDR91 WD40 domain complexed with Rab7 that has an unusual interface at the C-terminus of the Rab7 switch II region. WDR91 is highly selective for Rab7 among the tested GTPases. A LIS1 homology (LisH) motif within the WDR91 N-terminal domain (NTD) mediates self-association and may contribute partly to the augmented interaction between full-length WDR91 and Rab7. Both the Rab7 binding site and the LisH motif are indispensable for WDR91 function in endocytic trafficking. For the WDR91 orthologue SORF1 lacking the C-terminal WD40 domain, a C-terminal amphipathic helix (AH) mediates strong interactions with liposomes containing acidic lipids. During evolution the human WDR91 ancestor gene might have acquired a WD40 domain to replace the AH for endosomal membrane targeting.

Host WD repeat-containing protein 5 inhibits protein kinase R-mediated integrated stress response during measles virus infection.

MeV is a pathogen that remains a global concern, with an estimated 9 million measles cases and 128,000 measles deaths in 2022 according to the World Health Organization. A large population of the world still has inadequate access to the effective vaccine against the exceptionally transmissible MeV. Measles disease is characterized by a high morbidity in children and in immunocompromised individuals. An important area of research for negative-sense RNA viruses, including MeV, is the characterization of the complex interactome between virus and host occurring at cytoplasmic IBs where viral replication occurs. Despite the progress made in understanding IB structures, little is known regarding the virus-host interactions within IBs and the role of these interactions in promoting viral replication and antagonizing host innate immunity. Herein we provide evidence suggesting a model by which MeV IBs utilize the host protein WDR5 to suppress the protein kinase R-integrated stress response pathway.

Cilia defects upon loss of WDR4 are linked to proteasomal hyperactivity and ubiquitin shortage

The WD repeat-containing protein 4 (WDR4) has repeatedly been associated with primary microcephaly, a condition of impaired brain and skull growth. Often, faulty centrosomes cause microcephaly, yet aberrant cilia may also be involved. Here, we show using a combination of approaches in human fibroblasts, zebrafish embryos and patient-derived cells that WDR4 facilitates cilium formation. Molecularly, we associated WDR4 loss-of-function with increased protein synthesis and concomitant upregulation of proteasomal activity, while ubiquitin precursor pools are reduced. Inhibition of proteasomal activity as well as supplementation with free ubiquitin restored normal ciliogenesis. Proteasome inhibition ameliorated microcephaly phenotypes. Thus, we propose that WDR4 loss-of-function impairs head growth and neurogenesis via aberrant cilia formation, initially caused by disturbed protein and ubiquitin homeostasis.

WDR77 in Pan-Cancer: Revealing expression patterns, genetic insights, and functional roles across diverse tumor types, with a spotlight on colorectal cancer

ObjectiveDespite its involvement in regulating various cellular functions, the expression and role of WD repeat-containing protein 77 (WDR77) in cancer remain elusive. This study aims to explore the expression and potential roles of WDR77 across multiple cancers, with a particular focus on its relevance in colorectal cancer (CRC). MethodsWe obtained WDR77 RNA-seq data, mutations, CNVs, and DNA methylation data from the TCGA, GTEx, and GEO databases to investigate its expression patterns and prognostic value. Additionally, we examined the correlation between WDR77 expression and somatic mutations, copy number variations, DNA methylation, and mRNA modifications. We utilized GSVA, GSEA algorithms, and CRISPR KO data from the Dependency Map database to explore WDR77′s potential biological functions. The association between WDR77 and the tumor immune microenvironment was investigated using ESTIMATE and IOBR algorithms. Finally, we assessed WDR77 expression in CRC and its impact on cell proliferation through qRT-PCR, Western blotting, immunohistochemistry, CCK8, colony formation, and EdU assays. ResultsWDR77 was upregulated in various tumors and correlated with poor patient prognosis. Its high expression positively correlated with pathways related to cell proliferation and negatively correlated with immune-related pathways. In CRC, WDR77 expression was associated with specific clinical features, genomic alterations, and immune microenvironment characteristics. Experimental validation confirmed upregulated WDR77 expression in CRC tissues and cells, with WDR77 knockdown significantly inhibiting CRC cell proliferation. ConclusionWDR77 holds potential as an oncogene and biological marker in various cancers, particularly CRC.

USP36 regulates the proliferation, survival, and differentiation of hFOB1.19 osteoblast

BackgroundEffective bone formation relies on osteoblast differentiation, a process subject to intricate post-translational regulation. Ubiquitin-specific proteases (USPs) repress protein degradation mediated by the ubiquitin-proteasome pathway. Several USPs have been documented to regulate osteoblast differentiation, but whether other USPs are involved in this process remains elusive.MethodsIn this study, we conducted a comparative analysis of 48 USPs in differentiated and undifferentiated hFOB1.19 osteoblasts, identifying significantly upregulated USPs. Subsequently, we generated USP knockdown hFOB1.19 cells and evaluated their osteogenic differentiation using Alizarin red staining. We also assessed cell viability, cell cycle progression, and apoptosis through MTT, 7-aminoactinomycin D staining, and Annexin V/PI staining assays, respectively. Quantitative PCR and Western blotting were employed to measure the expression levels of osteogenic differentiation markers. Additionally, we investigated the interaction between the USP and its target protein using co-immunoprecipitation (co-IP). Furthermore, we depleted the USP in hFOB1.19 cells to examine its effect on the ubiquitination and stability of the target protein using immunoprecipitation (IP) and Western blotting. Finally, we overexpressed the target protein in USP-deficient hFOB1.19 cells and evaluated its impact on their osteogenic differentiation using Alizarin red staining.ResultsUSP36 is the most markedly upregulated USP in differentiated hFOB1.19 osteoblasts. Knockdown of USP36 leads to reduced viability, cell cycle arrest, heightened apoptosis, and impaired osteogenic differentiation in hFOB1.19 cells. USP36 interacts with WD repeat-containing protein 5 (WDR5), and the knockdown of USP36 causes an increased level of WDR5 ubiquitination and accelerated degradation of WDR5. Excessive WDR5 improved the impaired osteogenic differentiation of USP36-deficient hFOB1.19 cells.ConclusionsThese observations suggested that USP36 may function as a key regulator of osteoblast differentiation, and its regulatory mechanism may be related to the stabilization of WDR5.

Male-derived phospholipase A2 enhances WD46 expression and increases fertility in Ophraella communa.

Successful bisexual reproduction requires interactions between males and females. Male-derived seminal fluid proteins (SFPs) transferred to females during mating profoundly affect females from pre- to post-mating, and the subsequent shift in female physiology enhances their fertility. SFPs have important evolutionary implications for the fitness of many insects. However, little is known about how females respond to male SFPs. In this study, we identified a male-derived SFP-phospholipase A2 (PLA2) in Ophraella communa. PLA2 is a vital enzyme in eicosanoid biosynthesis; however, it has not been identified as an insect SFP. We found that OcPLA2 is specifically expressed in males, especially in the male accessory glands (MAGs); it is transferred to the female during mating and functions as an SFP to enhance fertility. The expression of a female-derived gene encoding the WD repeat-containing protein 46 (WD46) was upregulated when OcPLA2 entered the female reproductive tract, and this contributed to female egg production by increasing triacylglycerol lipase (TGL) gene expression and the triglyceride (TG) content. This is the first study to identify PLA2 as an SFP in insects. Our findings also shed light on the regulatory role of OcPLA2 in beetle reproduction; the expression of OcPLA2 is initially correlated with female WD46 expression and later with the decline in TGL gene expression and the TG content. This represents a unique mechanism of reproductive regulation by an SFP.

Rab11a Controls Cell Shape via C9orf72 Protein: Possible Relationships to Frontotemporal Dementia/Amyotrophic Lateral Sclerosis (FTDALS) Type 1.

Abnormal nucleotide insertions of C9orf72, which forms a complex with Smith-Magenis syndrome chromosomal region candidate gene 8 (SMCR8) protein and WD repeat-containing protein 41 (WDR41) protein, are associated with an autosomal-dominant neurodegenerative frontotemporal dementia and/or amyotrophic lateral sclerosis type 1 (FTDALS1). The differentially expressed in normal and neoplastic cells (DENN) domain-containing C9orf72 and its complex with SMCR8 and WDR41 function as a guanine-nucleotide exchange factor for Rab GTP/GDP-binding proteins (Rab GEF, also called Rab activator). Among Rab proteins serving as major effectors, there exists Rab11a. However, it remains to be established which Rab protein is related to promoting or sustaining neuronal morphogenesis or homeostasis. In this study, we describe that the knockdown of Rab11a decreases the expression levels of neuronal differentiation marker proteins, as well as the elongation of neurite-like processes, using N1E-115 cells, a well-utilized neuronal differentiation model. Similar results were obtained in primary cortical neurons. In contrast, the knockdown of Rab11b, a Rab11a homolog, did not significantly affect their cell morphological changes. It is of note that treatment with hesperetin, a citrus flavonoid (also known as Vitamin P), recovered the neuronal morphological phenotypes induced by Rab11a knockdown. Also, the knockdown of Rab11a or Rab11b led to a decrease in glial marker expression levels and in morphological changes in FBD-102b cells, which serve as the oligodendroglial differentiation model. Rab11a is specifically involved in the regulation of neuronal morphological differentiation. The knockdown effect mimicking the loss of function of C9orf72 is reversed by treatment with hesperetin. These findings may reveal a clue for identifying one of the potential molecular and cellular phenotypes underlying FTDALS1.

A virtual library of small molecules mimicking dipeptides.

Virtual combinatorial libraries are prevalent in drug discovery due to improvements in the prediction of synthetic reactions that can be performed. This has gone hand in hand with the development of virtual screening capabilities to effectively screen the large chemical spaces spanned by exhaustive enumeration of reaction products. In this study, we generated a small-molecule dipeptide mimic library to target proteins binding small peptides. The library was created based on the general idea of peptide synthesis, that is, amino acid mimics were reacted in silico to form the dipeptide mimics, yielding 2,036,819 unique compounds. After docking calculations, two compounds from the library were synthesized and tested against WD repeat-containing protein 5 (WDR5) and histamine receptors H1-H4 to evaluate whether these molecules are viable in assays. The compounds showed the highest potency at the histamine H3 receptor, with Ki values in the two-digit micromolar range.

Unveiling Distinct Proteomic Signatures in Complicated Crohn’s Disease That Could Predict the Disease Course

Crohn’s disease (CD) is characterized by a chronic, progressive inflammation of the gastrointestinal tract often leading to complications, such as strictures and fistulae. Currently, there are no validated tools anticipating short- and long-term outcomes at an early stage. This investigation aims to elucidate variations in protein abundance across distinct CD phenotypes with the objective of uncovering potential biomarkers implicated in disease advancement. Serum samples collected from 30 CD patients and 15 healthy age-matched controls (HC) were subjected to depletion of highly abundant proteins and to a label-free mass spectrometry analysis. Twenty-four proteins were shown to be significantly different when comparing CD with HC. Of these, WD repeat-containing protein 31 (WDR31), and proteins involved in the acute inflammatory response, leucine-rich alpha-2-glycoprotein (LRG1) and serum amyloid A1 (SAA1), were more abundant in the aggressive subgroup. Against standard biomarkers, a positive correlation between SAA1 and WDR31 and C-reactive protein (CRP) was found. In this study, a unique serum biomarker panel for aggressive CD was identified, which could aid in predicting the disease course.

PBRM1 mutation and WDR72 expression as potential combinatorial biomarker for predicting the response to Nivolumab in patients with ccRCC: a tumor marker prognostic study.

Immune checkpoint therapy (ICT) provides a new idea for the treatment of advanced clear cell renal cell carcinoma (ccRCC), which can bring significant benefits to patients. However, the clinical application of ICT is limited because of the lack of predictive biomarkers to select potential responders. This study aims to propose a new biomarker to predict the response to Nivolumab in patients with ccRCC. The genes that significantly improve the prognosis of ccRCC were retrieved from The Cancer Genome Atlas (TCGA) database. The genomic and clinical data were from patients that had been registered in prospective clinical trials (CheckMate 009, CheckMate 010 and CheckMate 025). TCGA, Gene Expression Omnibus (GEO), and The Human Protein Atlas database were used to analyze the gene and protein expression of WD repeat-containing protein 72 (WDR72) in ccRCC. Gene Ontology (GO) & The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) were performed to dig relevant mechanisms of WDR72. Single sample gene set enrichment analysis (ssGSEA) was conducted to evaluate the role of WDR72 in immune infiltration. Cell proliferation assay, FAO and ATP quantification were used to explore and verify the molecular mechanisms. The expression of WDR72, FOXP3, CD8, and CPT1A was examined by IHC in 20 advanced ccRCC tissue samples at the Urology Department of our hospital. The MethSurv was used to identify PBRM1 and WDR72 gene methylation and its effect on prognosis of ccRCC. WDR72 is the most significant gene for improving overall survival (OS) in ccRCC. In all three checkmates, OS and progression free survival (PFS) were found to be significantly higher in WDR72 high expression group than that in WDR72 low expression group (P=0.040 and P=0.012, respectively), and similar conclusions could be drawn from the PBRM1-mutation (MUT) compared with the PBRM1-wildtype (WT) (P=0.007 and P=0.006, respectively). What's more, high expression of WDR72 plus PBRM1-MUT as a combinatorial biomarker showed improved OS (HR=0.388, P=0.0026) and PFS (HR=0.39, P=0.0066) compared to low expression of WDR72 plus PBRM1-WT. Functional enrichment analysis showed that WDR72 was closely positively related to fatty acid degradation and fatty acid beta oxidation pathway in ccRCC. In vitro experiments showed that high expression of WDR72 can promote fatty acids oxidation and inhibit the proliferation of ccRCC cells. Immune analysis revealed that WDR72 high expression was associated with decreased infiltration of Treg cells and low ssGSEA score of check-point. IHC results showed that WDR72 was negatively correlated with FOXP3 expression (r=-0.506, P=0.023) and positively correlated with CPT1A expression (r=0.529, P=0.017). The present study indicated that high expression of WDR72 may indicate a good prognosis of patients treated with Nivolumab and WDR72 expression combined with PBRM1 mutation could be more persuasive to predict the response for ICT in ccRCC patients.

Dual Targeting Novel WDR5/ATAD2 Oncogenic Signaling through CK2/Ikaros Axis Demonstrates Synergistic Efficacy in T-ALL

Background WD repeat-containing protein 5 (WDR5) is highly expressed in human cancers and its high expression is linked with adverse prognoses. However, the oncogenic roles of WDR5 and underlying mechanisms are undefined in T-cell acute lymphoblastic leukemia (T-ALL). Casein Kinase II (CK2) is a pro-oncogene, and CK2-mediated IKAROS dysfunction plays critical roles in high-risk ALL, and CK2 inhibitor CX-4945 has the anti-leukemic effect by restoring IKAROS function. This study aims to explore the synergistic anti-leukemic efficacy of dual targeting novel WDR5/ATAD2 oncogenic signaling via the CK2/IKAROS axis in T-ALL. Methods Clinical samples from 38 T-ALL patients and 32 healthy controls were obtained from Zhongda Hospital Southeast University. The expression of WDR5 and ATAD2 were tested by q-PCR and Western blot. RNA-seq was performed after CEM cells were treated with WDR5 inhibitor (OICR-9429), CX-4945 and vehicle control for 72 hours. The human leukemia xenograft mouse model is established by intravenously injected with WDR5 knockdown CEM (CEM-shWDR5) cells and shRNA control (CEM-shNC) into NSG mice. Mice were treated for 25 days in 4 groups [CEM-shNC + vehicle, CEM-sh WDR5 + vehicle; CEM-shNC + CX-4945 (100 mg/kg via gavage), CEM-sh WDR5 + CX-4945]. Leukemia burden in mice spleen and bone marrow was analyzed by flow cytometry. The survival of mice was compared by the Kaplan-Meier method. The synergistic effect of CX-4945 and OICR-9429 was analyzed by CCK8 assay. This study was approved by the Ethics Committee of Zhongda Hospital Southeast University Results WDR5 ishighly expressed in T-ALL patients compared with normal controls( P<0.001) and is higher in the relapsed samples than paired newly-diagnosed controls ( P<0.05) (Fig. 1A-B). WDR5 knockdown in T-ALL cells significantly suppressed cell proliferation and induced cell cycle arrest in vitro ( P<0.01) (Fig. 1C), and in vivo leukemia development (Fig. 1D). Also, WDR5 inhibitor OICR-9429 induces cell proliferation and cell cycle arrest in the cells. RNA-seq revealed that ATAD2 is downregulated upon OICR-9429 treatment and indicated that ATAD2 is the downstream target of WDR5. WDR5 knockdown suppresses ATAD2 expression ( P<0.01) (Fig. 1E). In addition, ATAD2 ishighly expressed in T-ALL patients versus normal controls ( P<0.05) (Fig. 1F), and ATAD2 silencing significantly suppresses cell proliferation and induces cell cycle arrest of T-ALL cells ( P<0.01) (Fig. 1G). These data indicate WDR5/ATAD2 signaling act as a novel potential oncogenic pathway in T-ALL. Furthermore, we found that IKAROS transcriptionally suppresses WDR5 expression, and CK2-mediated hyperphosphorylation of IKAROS is one of the major mechanisms of IKAROS dysfunction in ALL. CX-4945 strongly suppresses WDR5 expression in an IKAROS-dependent manner (Fig. 1H-J). The synergistic effect of CX-4945 with WDR5 disruption on cell proliferation suppression and cell cycle arrest was observed in T-ALL both in in vitro and in vivo xenograft mouse model. The combination of CX-4945 with WDR5 disruption significantly prolonged the survival of mice compared with single CX-4945 [55(52-58) days vs. 38.5(36-41) days， P<0.001] and single WDR5 disruption control [55(52-58) days vs. 41.5(39-44) days, P<0.001] (Fig. 1K). Consistently, the combination significantly reduces the size and weight of the spleen (Fig. 1L), % human CD45+ T-ALL cells in the spleen (Fig. 1M) and bone marrow (data not shown), and the expression of WDR5 and ATAD2 in the spleen versus single treatment controls ( P<0.01) (Fig. 1N-P). Finally, a synergistic effect of CX-4945 with OICR-9429 on cell proliferation suppression and down-regulation of WDR5 and ATAD2 was observed in the primary leukemic cells from T-ALL patients (Fig. 2A-D). Together, these data indicated the synergistic efficacy of CX-4945 with OICR-9429 on T-ALL and the novel model was summarized in Fig.2E. Conclusion In this study, we identified a novel WDR5/ATAD2 oncogenic signaling regulated by CK2/IKAROS axis in T-ALL. Our study reveals a model that dual targeting WDR5/ATAD2 signaling through direct inhibiting oncoproteins and via CK2/IKAROS axis to transcriptionally repress the oncoprotein to achieve synergistic efficacy. Our results further highlight the combination of CX-4945 with WDR5 inhibition is a potential option for the therapy of T-ALL patients.

BRWD1 orchestrates small pre-B cell chromatin topology by converting static to dynamic cohesin.

Lymphocyte development consists of sequential and mutually exclusive cell states of proliferative selection and antigen receptor gene recombination. Transitions between each state require large, coordinated changes in epigenetic landscapes and transcriptional programs. How this occurs remains unclear. Here we demonstrate that in small pre-B cells, the lineage and stage-specific epigenetic reader bromodomain and WD repeat-containing protein 1 (BRWD1) reorders three-dimensional chromatin topology to affect the transition between proliferative and gene recombination molecular programs. BRWD1 regulated the switch between poised and active enhancers interacting with promoters, and coordinated this switch with Igk locus contraction. BRWD1 did so by converting chromatin-bound static to dynamic cohesin competent to mediate long-range looping. ATP-depletion revealed cohesin conversion to be the main energetic mechanism dictating dynamic chromatin looping. Our findings provide a new mechanism of cohesin regulation and reveal how cohesin function can be dictated by lineage contextual mechanisms to facilitate specific cell fate transitions.

Participation of WD repeat-containing protein 54 (WDR54) in rat sperm-oocyte fusion through interaction with both IZUMO1 and JUNO

Fertilization is a complex process that depends on the fusion of the cell membrane of sperm with that of oocyte, and it involves sperm-oocyte recognition, binding, and fusion, which are mediated by multiple proteins. Among those proteins, IZUMO1 and its receptor JUNO have been identified as essential factors for sperm-oocyte recognition and fusion. However, the interaction between IZUMO1 and JUNO alone does not lead to cell membrane fusion, suggesting the involvement of additional proteins in sperm-oocyte membrane fusion. In this study, we have discovered that a protein called WDR54, which consists of WD-repeat modules, is located on the cell membrane of sperm, as well as on the cell membrane and in the cytoplasm of the oocyte. We have found that WDR54 is involved in sperm-oocyte fertilization. When sperm and oocyte were treated with anti-WDR54 ascites, the in vitro fertilization (IVF) rate significantly decreased. Furthermore, our research has shown that WDR54 interacts with both IZUMO1 and JUNO, and it colocalizes with IZUMO1 on the surface of the sperm head and with JUNO on the oocyte surface. Through structural analysis of the putative complexes of WDR54-IZUMO1 and WDR54-JUNO, we infer that these three proteins could form a complex of JUNO-WDR54-IZUMO1-JUNO (referred to as the “JWIJ complex”) on the oocyte surface. Our findings suggest that WDR54 is an important factor involved in sperm-oocyte adhesion and fusion. This discovery provides new insight into the mechanisms of mammalian sperm-oocyte adhesion and fusion.

Pharmacological inhibition of the mixed lineage leukemia 1-menin interaction aggravates acute kidney injury induced by folic acid and ischemia-reperfusion in mice.

Mixed lineage leukemia 1 (MLL1) is a methyltransferase that induces histone H3 lysine 4 trimethylation (H3K4me3) and partially exerts its untoward functional effects by interacting with multiple subunits including menin and WD repeat-containing protein 5 (WDR5). In this study, we investigated the role and mechanisms of MLL1 in murine models of acute kidney injury induced by folic acid (FA) and ischemia-reperfusion. Injury to the kidney elevated the expression of MLL1, menin, WDR5, and H3K4Me3, which was accompanied by increased serum creatinine and blood urea nitrogen, renal tubular injury, and apoptosis. Pharmacological inhibition of MLL1 activity with MI503 to disrupt the interaction between MLL1 with menin further increased serum creatinine and blood urea nitrogen levels, enhanced expression of neutrophil gelatinase-associated lipocalin and kidney injury molecule-1, and induced more apoptosis in the kidney following FA and ischemia-reperfusion injury. In contrast, MI503 treatment decreased the expression of vimentin and proliferating cell nuclear antigens. Similarly, treatment with MM102 to disrupt the interaction between MLL1 and WDR5 also worsened renal dysfunction, aggravated tubular cell injury, increased apoptosis, and inhibited cellular dedifferentiation and proliferation in mice following FA injection. Moreover, MI503 inhibited FA-induced phosphorylation of epidermal growth factor receptor, signal transducer and activator of transcription 3, and extracellular signal-regulated kinase-1/2 in injured kidneys. Collectively, these data suggest that MLL1 contributes to renal protection and functional recovery and promotes renal regeneration through a mechanism associated with activation of the epidermal growth factor receptor signaling pathway.NEW & NOTEWORTHY Mixed lineage leukemia 1 (MLL1) is a methyltransferase that induces histone H3 lysine 4 trimethylation and exerts its functional roles by interacting with multiple subunits. In this study, we demonstrated that inhibition of MLL1 activity by MI503 or MM102 aggravated renal injury and apoptosis and suppressed renal tubular cell dedifferentiation and proliferation, suggesting that MLL1 activation during acute kidney injury acts as an intrinsic protective mechanism to mediate renal tubular cell survival and regeneration.

BRWD3 promotes KDM5 degradation to maintain H3K4 methylation levels

Histone modifications are critical for regulating chromatin structure and gene expression. Dysregulation of histone modifications likely contributes to disease states and cancer. Depletion of the chromatin-binding protein BRWD3 (Bromodomain and WD repeat-containing protein 3), a known substrate-specificity factor of the Cul4-DDB1 E3 ubiquitin ligase complex, results in increased H3K4me1 (H3 lysine 4 monomethylation) levels. The underlying mechanism linking BRWD3 and H3K4 methylation, however, has yet to be defined. Here, we show that depleting BRWD3 not only causes an increase in H3K4me1 levels but also causes a decrease in H3K4me3 (H3 lysine 4 trimethylation) levels, indicating that BRWD3 influences H3K4 methylation more broadly. Using immunoprecipitation coupled to quantitative mass spectrometry, we identified an interaction between BRWD3 and the H3K4-specific lysine demethylase 5 (KDM5/Lid), an enzyme that removes tri- and dimethyl marks from H3K4. Moreover, analysis of ChIP-seq (chromatin immunoprecipitation sequencing) data revealed that BRWD3 and KDM5 are significantly colocalized throughout the genome and H3K4me3 are highly enriched at BRWD3 binding sites. We show that BRWD3 promotes K48-linked polyubiquitination and degradation of KDM5 and that KDM5 degradation is dependent on both BRWD3 and Cul4. Critically, depleting KDM5 fully restores altered H3K4me3 levels and partially restores H3K4me1 levels upon BRWD3 depletion. Together, our results demonstrate that BRWD3 regulates KDM5 activity to balance H3K4 methylation levels.

Long non-coding RNA X-Inactive Specific Transcript (XIST) interacting with USF2 promotes osteogenic differentiation of periodontal ligament stem cells through regulation of WDR72 transcription.

Periodontal ligament stem cells (PDLSCs) are the most potential cells in periodontal tissue regeneration and bone tissue regeneration. Our prior work had revealed that WD repeat-containing protein 72 (WDR72) was crucial for osteogenic differentiation of PDLSCs. Here, we further elucidated its underlying mechanism in PDLSC osteogenic differentiation. Human PDLSCs, isolated and identified by flow cytometry, were prepared for osteogenic differentiation induction. Levels of WDR72, long non-coding RNA X-Inactive Specific Transcript (XIST), upstream stimulatory factor 2 (USF2), and osteogenic marker genes (Runx2, Osteocalcin, and Collagen I) in human PDLSCs and clinical specimens were detected by RT-qPCR. Protein expressions of WDR72, Runx2, Osteocalcin, and Colla1 were tested by Western blot. The interactions among the molecules were verified by RIP, RNA pull-down, ChIP, and luciferase reporter assays. Osteogenic differentiation was evaluated by alkaline phosphatase (ALP) and alizarin red staining (ARS). WDR72 was decreased in periodontal tissues of periodontitis patients, and overexpression reversed TNF-α-mediated suppressive effects on PDLSC osteogenic differentiation. Mechanically, XIST recruited the enrichment of USF2 to the WDR72 promoter region, thereby positively regulating WDR72. WDR72 silencing overturned XIST-mediated biological effects in PDLSCs. WDR72, regulated by the XIST/USF2 axis, enhances osteogenic differentiation of PDLSCs, implying a novel strategy for alleviating periodontitis.

The role of antifreeze genes in the tolerance of cold stress in the Nile tilapia (Oreochromis niloticus)

BackgroundTilapia is one of the most essential farmed fishes in the world. It is a tropical and subtropical freshwater fish well adapted to warm water but sensitive to cold weather. Extreme cold weather could cause severe stress and mass mortalities in tilapia. The present study was carried out to investigate the effects of cold stress on the up-regulation of antifreeze protein (AFP) genes in Nile tilapia (Oreochromis niloticus). Two treatment groups of fish were investigated (5 replicates of 15 fish for each group in fibreglass tanks/70 L each): 1) a control group; the fish were acclimated to lab conditions for two weeks and the water temperature was maintained at 25 °C during the whole experimental period with feeding on a commercial diet (30% crude protein). 2) Cold stress group; the same conditions as the control group except for the temperature. Initially, the temperature was decreased by one degree every 12 h. The fish started showing death symptoms when the water temperature reached 6–8 °C. In this stage the tissue (muscle) samples were taken from both groups. The immune response of fish exposed to cold stress was detected and characterized using Differential Display-PCR (DD-PCR).ResultsThe results indicated that nine different up-regulation genes were detected in the cold-stressed fish compared to the control group. These genes are Integrin-alpha-2 (ITGA-2), Gap junction gamma-1 protein-like (GJC1), WD repeat-containing protein 59 isoform X2 (WDRP59), NUAK family SNF1-like kinase, G-protein coupled receptor-176 (GPR-176), Actin cytoskeleton-regulatory complex protein pan1-like (PAN-1), Whirlin protein (WHRN), Suppressor of tumorigenicity 7 protein isoform X2 (ST7P) and ATP-binding cassette sub-family A member 1-like isoform X2 (ABCA1). The antifreeze gene type-II amplification using a specific PCR product of 600 bp, followed by cloning and sequencing analysis revealed that the identified gene is antifreeze type-II, with similarity ranging from 70 to 95%. The in-vitro transcribed gene induced an antifreeze protein with a molecular size of 22 kDa. The antifreeze gene, ITGA-2 and the WD repeat protein belong to the lectin family (sugar–protein).ConclusionsIn conclusion, under cold stress, Nile tilapia express many defence genes, an antifreeze gene consisting of one open reading frame of approximately 0.6 kbp.

Pathogenesis underlying hexanucleotide repeat expansions in C9orf72 gene in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and fatal neurodegenerative disorder. Mutations in C9orf72 and the resulting hexanucleotide repeat (GGGGCC) expansion (HRE) has been identified as a major cause of familial ALS, accounting for about 40 % of familial and 6 % of sporadic cases of ALS in Western patients. The pathological outcomes of HRE expansion in ALS have been recognized as the results of two mechanisms that include both the toxic gain-of-function and loss-of-function of C9ORF72. The gain of toxicity results from RNA and dipeptide repeats (DPRs). The HRE can be bidirectionally transcribed into RNA foci, which can bind to and disrupt RNA splicing, transport, and translation. The DPRs that include poly-glycine-alanine, poly-glycine-proline, poly-glycine- arginine, poly-proline-alanine, and poly-proline-arginine can induce toxicity by direct binding and sequestrating other proteins to interfere rRNA synthesis, ribosome biogenesis, translation, and nucleocytoplasmic transport. The C9ORF72 functions through binding to its partners-Smith-Magenis chromosome regions 8 (SMCR8) and WD repeat-containing protein (WDR41). Loss of C9ORF72 function results in impairment of autophagy, deregulation of autoimmunity, increased stress, and disruption of nucleocytoplasmic transport. Further insight into the mechanism in C9ORF72 HRE pathogenesis will facilitate identifying novel and effective therapeutic targets for ALS.

WDR74 serves as a novel therapeutic target by its oncogenic role in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is one of the most refractory human malignancies. WD repeat-containing protein 74 (WDR74) is involved in the tumorigenesis of various cancers, however, its clinical implications and biological function in HCC have yet to be clearly determined. MethodsBioinformatics analysis was conducted using various databases, including The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and UALCAN. The expression of WDR74 was confirmed in HCC tumor samples and the corresponding adjacent nontumor samples by qRT-PCR, western blot and immunohistochemistry. Functional enrichment analysis was used for the biological function prediction. In vitro experiments were performed to determine the effects of WDR74 on HCC cell proliferation. ResultsOur findings revealed that WDR74 was markedly upregulated in HCC tissues. Increased WDR74 expression had an unfavorable overall survival (OS). Multivariate Cox regression analysis demonstrated that WDR74 was an independent prognostic factor for OS in patients with HCC. Functional enrichment analysis suggested a significant correlation with cytokine-cytokine receptor interaction pathway in both TCGA-LIHC and GSE112790 datasets. Gene set enrichment analysis showed that WDR74 is probably involved in several pathways, such as MYC targets, ribosome, translation, and cell cycle. Finally, WDR74 knockdown reduced HCC cell proliferation by restraining the G1/S cell cycle transition and inducing apoptosis. ConclusionsThe current study demonstrates that elevated WDR74 expression is linked to an accelerated rate of tumor cell proliferation and is indicative of a poorer outcome in patients with HCC. Therefore, WDR74 could be used as a reliable prognostic biomarker and is a potential therapeutic target for HCC.