Reciprocal antagonism of PIN1-APC/CCDH1 governs mitotic protein stability and cell cycle entry.

Induced oncoproteins degradation provides an attractive anti-cancer modality. Activation of anaphase-promoting complex (APC/CCDH1) prevents cell-cycle entry by targeting crucial mitotic proteins for degradation. Phosphorylation of its co-activator CDH1 modulates the E3 ligase activity, but little is known about its regulation after phosphorylation and how to effectively harness APC/CCDH1 activity to treat cancer. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1)-catalyzed phosphorylation-dependent cis-trans prolyl isomerization drives tumor malignancy. However, the mechanisms controlling its protein turnover remain elusive. Through proteomic screens and structural characterizations, we identify a reciprocal antagonism of PIN1-APC/CCDH1 mediated by domain-oriented phosphorylation-dependent dual interactions as a fundamental mechanism governing mitotic protein stability and cell-cycle entry. Remarkably, combined PIN1 and cyclin-dependent protein kinases (CDKs) inhibition creates a positive feedback loop of PIN1 inhibition and APC/CCDH1 activation to irreversibly degrade PIN1 and other crucial mitotic proteins, which force permanent cell-cycle exit and trigger anti-tumor immunity, translating into synergistic efficacy against triple-negative breast cancer.

Transcriptional elongation control of hypoxic response.

The release of paused RNA polymerase II (RNAPII) from promoter-proximal regions is tightly controlled to ensure proper regulation of gene expression. The elongation factor PTEF-b is known to release paused RNAPII via phosphorylation of the RNAPII C-terminal domain by its cyclin-dependent kinase component, CDK9. However, the signal and stress-specific roles of the various RNAPII-associated macromolecular complexes containing PTEF-b/CDK9 are not yet clear. Here, we identify and characterize the CDK9 complex required for transcriptional response to hypoxia. Contrary to previous reports, our data indicate that a CDK9 complex containing BRD4 but not AFF1/4 is essential for this hypoxic stress response. We demonstrate that BRD4 bromodomains (BET) are dispensable for the release of paused RNAPII at hypoxia-activated genes and that BET inhibition by JQ1 is insufficient to impair hypoxic gene response. Mechanistically, we demonstrate that the C-terminal region of BRD4 is required for Polymerase-Associated Factor-1 Complex (PAF1C) recruitment to establish an elongation-competent RNAPII complex at hypoxia-responsive genes. PAF1C disruption using a small-molecule inhibitor (iPAF1C) impairs hypoxia-induced, BRD4-mediated RNAPII release. Together, our results provide insight into potentially targetable mechanisms that control the hypoxia-responsive transcriptional elongation.

Targeting BRD4: Potential therapeutic strategy for head and neck squamous cell carcinoma (Review).

As a member of BET (bromodomain and extra-terminal) protein family, BRD4 (bromodomain­containing protein 4) is a chromatin­associated protein that interacts with acetylated histones and actively recruits regulatory proteins, leading to the modulation of gene expression and chromatin remodeling. The cellular and epigenetic functions of BRD4 implicate normal development, fibrosis and inflammation. BRD4 has been suggested as a potential therapeutic target as it is often overexpressed and plays a critical role in regulating gene expression programs that drive tumor cell proliferation, survival, migration and drug resistance. To address the roles of BRD4 in cancer, several drugs that specifically target BRD4 have been developed. Inhibition of BRD4 has shown promising results in preclinical models, with several BRD4 inhibitors undergoing clinical trials for the treatment of various cancers. Head and neck squamous cell carcinoma (HNSCC), a heterogeneous group of cancers, remains a health challenge with a high incidence rate and poor prognosis. Conventional therapies for HNSCC often cause adverse effects to the patients. Targeting BRD4, therefore, represents a promising strategy to sensitize HNSCC to chemo­ and radiotherapy allowing de­intensification of the current therapeutic regime and subsequent reduced side effects. However, further studies are required to fully understand the underlying mechanisms of action of BRD4 in HNSCC in order to determine the optimal dosing and administration of BRD4­targeted drugs for the treatment of patients with HNSCC.

Synthetic Lethality between Cohesin and WNT Signaling Pathways in Diverse Cancer Contexts.

Cohesin is a highly conserved ring-shaped complex involved in topologically embracing chromatids, gene expression regulation, genome compartmentalization, and genome stability maintenance. Genomic analyses have detected mutations in the cohesin complex in a wide array of human tumors. These findings have led to increased interest in cohesin as a potential target in cancer therapy. Synthetic lethality has been suggested as an approach to exploit genetic differences in cancer cells to influence their selective killing. In this study, we show that mutations in ESCO1, NIPBL, PDS5B, RAD21, SMC1A, SMC3, STAG2, and WAPL genes are synthetically lethal with stimulation of WNT signaling obtained following LY2090314 treatment, a GSK3 inhibitor, in several cancer cell lines. Moreover, treatment led to the stabilization of ß-catenin and affected the expression of c-MYC, probably due to the occupancy decrease in cohesin at the c-MYC promoter. Finally, LY2090314 caused gene expression dysregulation mainly involving pathways related to transcription regulation, cell proliferation, and chromatin remodeling. For the first time, our work provides the underlying molecular basis for synthetic lethality due to cohesin mutations and suggests that targeting the WNT may be a promising therapeutic approach for tumors carrying mutated cohesin.

Identification of ASF1A and HJURP by global H3-H4 histone chaperone analysis as a prognostic two-gene model in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a malignancy with poor prognosis. Abnormal expression of H3-H4 histone chaperones has been identified in many cancers and holds promise as a biomarker for diagnosis and prognosis. However, systemic analysis of H3-H4 histone chaperones in HCC is still lacking. Here, we investigated the expression of 19 known H3-H4 histone chaperones in HCC. Integrated analysis of multiple public databases indicated that these chaperones are highly expressed in HCC tumor tissues, which was further verified by immunohistochemistry (IHC) staining in offline samples. Additionally, survival analysis suggested that HCC patients with upregulated H3-H4 histone chaperones have poor prognosis. Using LASSO and Cox regression, we constructed a two-gene model (ASF1A, HJURP) that accurately predicts prognosis in ICGC-LIRI and GEO HCC data, which was further validated in HCC tissue microarrays with follow-up information. GSEA revealed that HCCs in the high-risk group were associated with enhanced cell cycle progression and DNA replication. Intriguingly, HCCs in the high-risk group exhibited increased immune infiltration and sensitivity to immune checkpoint therapy (ICT). In summary, H3-H4 histone chaperones play a critical role in HCC progression, and the two-gene (ASF1A, HJURP) risk model is effective for predicting survival outcomes and sensitivity to immunotherapy for HCC patients.

CDC45 promotes the stemness and metastasis in lung adenocarcinoma by affecting the cell cycle.

OBJECTIVE: This study aimed to assess the functions of cell division cycle protein 45 (CDC45) in Non-small cell lung cancer (NSCLC) cancer and its effects on stemness and metastasis. METHODS: Firstly, differentially expressed genes related to lung cancer metastasis and stemness were screened by differential analysis and lasso regression. Then, in vitro, experiments such as colony formation assay, scratch assay, and transwell assay were conducted to evaluate the impact of CDC45 knockdown on the proliferation and migration abilities of lung cancer cells. Western blotting was used to measure the expression levels of related proteins and investigate the regulation of CDC45 on the cell cycle. Finally, in vivo model with subcutaneous injection of lung cancer cells was performed to verify the effect of CDC45 on tumor growth. RESULTS: This study identified CDC45 as a key gene potentially influencing tumor stemness and lymph node metastasis. Knockdown of CDC45 not only suppressed the proliferation and migration abilities of lung cancer cells but also caused cell cycle arrest at the G2/M phase. Further analysis revealed a negative correlation between CDC45 and cell cycle-related proteins, stemness-related markers, and tumor mutations. Mouse experiments confirmed that CDC45 knockdown inhibited tumor growth. CONCLUSION: As a novel regulator of stemness, CDC45 plays a role in regulating lung cancer cell proliferation, migration, and cell cycle. Therefore, CDC45 may serve as a potential target for lung cancer treatment and provide a reference for further mechanistic research and therapeutic development.

Phospho-KNL-1 recognition by a TPR domain targets the BUB-1-BUB-3 complex to C. elegans kinetochores.

During mitosis, the Bub1-Bub3 complex concentrates at kinetochores, the microtubule-coupling interfaces on chromosomes, where it contributes to spindle checkpoint activation, kinetochore-spindle microtubule interactions, and protection of centromeric cohesion. Bub1 has a conserved N-terminal tetratricopeptide repeat (TPR) domain followed by a binding motif for its conserved interactor Bub3. The current model for Bub1-Bub3 localization to kinetochores is that Bub3, along with its bound motif from Bub1, recognizes phosphorylated "MELT" motifs in the kinetochore scaffold protein Knl1. Motivated by the greater phenotypic severity of BUB-1 versus BUB-3 loss in C. elegans, we show that the BUB-1 TPR domain directly recognizes a distinct class of phosphorylated motifs in KNL-1 and that this interaction is essential for BUB-1-BUB-3 localization and function. BUB-3 recognition of phospho-MELT motifs additively contributes to drive super-stoichiometric accumulation of BUB-1-BUB-3 on its KNL-1 scaffold during mitotic entry. Bub1's TPR domain interacts with Knl1 in other species, suggesting that collaboration of TPR-dependent and Bub3-dependent interfaces in Bub1-Bub3 localization and functions may be conserved.

ZNF554 Inhibits Endometrial Cancer Progression via Regulating RBM5 and Inactivating WNT/ß-Catenin Signaling Pathway.

OBJECTIVE: Uterine corpus endometrial carcinoma (UCEC), a kind of gynecologic malignancy, poses a significant risk to women's health. The precise mechanism underlying the development of UCEC remains elusive. Zinc finger protein 554 (ZNF554), a member of the Krüppel-associated box domain zinc finger protein superfamily, was reported to be dysregulated in various illnesses, including malignant tumors. This study aimed to examine the involvement of ZNF554 in the development of UCEC. METHODS: The expression of ZNF554 in UCEC tissues and cell lines were examined by qRT-PCR and Western blot assay. Cells with stably overexpressed or knocked-down ZNF554 were established through lentivirus infection. CCK-8, wound healing, and Transwell invasion assays were employed to assess cell proliferation, migration, and invasion. Propidium iodide (PI) staining combined with fluorescence-activated cell sorting (FACS) flow cytometer was utilized to detect cell cycle distribution. qRT-PCR and Western blotting were conducted to examine relative mRNA and protein levels. Chromatin immunoprecipitation assay and luciferase reporter assay were used to explore the regulatory role of ZNF554 in RNA binding motif 5 (RBM5). RESULTS: The expression of ZNF554 was found to be reduced in both UCEC samples and cell lines. Decreased expression of ZNF554 was associated with higher tumor stage, decreased overall survival, and reduced disease-free survival in UCEC. ZNF554 overexpression suppressed cell proliferation, migration, and invasion, while also inducing cell cycle arrest. In contrast, a decrease in ZNF554 expression resulted in the opposite effect. Mechanistically, ZNF554 transcriptionally regulated RBM5, leading to the deactivation of the Wingless (WNT)/ß-catenin signaling pathway. Moreover, the findings from rescue studies demonstrated that the inhibition of RBM5 negated the impact of ZNF554 overexpression on ß-catenin and p-glycogen synthase kinase-3ß (p-GSK-3ß). Similarly, the deliberate activation of RBM5 reduced the increase in ß-catenin and p-GSK-3ß caused by the suppression of ZNF554. In vitro experiments showed that ZNF554 overexpression-induced decreases in cell proliferation and migration were counteracted by RBM5 knockdown. Additionally, when RBM5 was overexpressed, it hindered the improvements in cell proliferation and migration caused by reducing the ZNF554 levels. CONCLUSION: ZNF554 functions as a tumor suppressor in UCEC. Furthermore, ZNF554 regulates UCEC progression through the RBM5/WNT/ß-catenin signaling pathway. ZNF554 shows a promise as both a prognostic biomarker and a therapeutic target for UCEC.

Induction of the human CDC45 gene promoter activity by natural compound trans­resveratrol.

GGAA motifs in the human TP53 and HELB gene promoters play a part in responding to trans­resveratrol (Rsv) in HeLa S3 cells. This sequence is also present in the 5'­upstream region of the human CDC45 gene, which encodes a component of CMG DNA helicase protein complex. The cells were treated with Rsv (20 µM), then transcripts and the translated protein were analyzed by quantitative RT­PCR and western blotting, respectively. The results showed that the CDC45 gene and protein expression levels were induced after the treatment. To examine whether they were due to the activation of transcription, a 5'­upstream 556­bp of the CDC45 gene was cloned and inserted into a multi­cloning site of the Luciferase (Luc) expression vector. In the present study, various deletion/point mutation­introduced Luc expression plasmids were constructed and they were used for the transient transfection assay. The results showed that the GGAA motif, which is included in a putative RELB protein recognizing sequence, plays a part in the promoter activity with response to Rsv in HeLa S3 cells.

Important role and underlying mechanism of non­SMC condensin I complex subunit G in tumours (Review).

At present, the incidence of tumours is increasing on a yearly basis, and tumourigenesis is usually associated with chromosomal instability and cell cycle dysregulation. Moreover, abnormalities in the chromosomal structure often lead to DNA damage, further exacerbating gene mutations and chromosomal rearrangements. However, the non­SMC condensin I complex subunit G (NCAPG) of the structural maintenance of chromosomes family is known to exert a key role in tumour development. It has been shown that high expression of NCAPG is closely associated with tumour development and progression. Overexpression of NCAPG variously affects chromosome condensation and segregation during cell mitosis, influences cell cycle regulation, promotes tumour cell proliferation and invasion, and inhibits apoptosis. In addition, NCAPG has been associated with tumour cell stemness, tumour resistance and recurrence. The aim of the present review was to explore the underlying mechanisms of NCAPG during tumour development, with a view towards providing novel targets and strategies for tumour therapy, and through the elucidation of the mechanisms involved, to lay the foundation for future developments in health.

Acetylation of Rec8 cohesin complexes regulates reductional chromosome segregation in meiosis.

For establishing sister chromatid cohesion and proper chromosome segregation in mitosis in fission yeast, the acetyltransferase Eso1 plays a key role. Eso1 acetylates cohesin complexes, at two conserved lysine residues K105 and K106 of the cohesin subunit Psm3. Although Eso1 also contributes to reductional chromosome segregation in meiosis, the underlying molecular mechanisms have remained elusive. Here, we purified meiosis-specific Rec8 cohesin complexes localized at centromeres and identified a new acetylation at Psm3-K1013, which largely depends on the meiotic kinetochore factor meikin (Moa1). Our molecular genetic analyses indicate that Psm3-K1013 acetylation cooperates with canonical acetylation at Psm3-K105 and K106, and plays a crucial role in establishing reductional chromosome segregation in meiosis.

Validation of CDC45 as a novel biomarker for diagnosis and prognosis of gastric cancer.

Background: Cell division cycle protein 45 (CDC45) has been demonstrated to play vital roles in the progression of various malignancies. However, the clinical significance of CDC45 in gastric cancer (GC) remains unreported. Method: In this study, we employed the TCGA database and the TCGA & GTEx dataset to compare the mRNA expression levels of CDC45 between gastric cancer tissues and adjacent or normal tissues (p < 0.05 was considered statistically significant), which was further validated in multiple datasets including GSE13911, GSE29272, GSE118916, GSE66229, as well as RT-qPCR. Furthermore, we harnessed the Human Protein Atlas (HPA) to evaluate the protein expression of CDC45, which was subsequently verified through immunohistochemistry (IHC). To ascertain the diagnostic utility of CDC45, receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC) were calculated in TCGA database, and further validated it in TCGA & GTEx and GSE66229 datasets. The Kaplan-Meier method was used to reveal the prognostic importance of CDC45 in The Cancer Genome Atlas (TCGA) database and authenticated through the GSE66229, GSE84433, and GSE84437 datasets. Through cBioPortal, we identified co-expressed genes of CDC45, and pursued enrichment analysis. Additionally, we availed gene set enrichment analysis (GSEA) to annotate the biological functions of CDC45. Results: Differential expression analysis revealed that CDC45 was significantly upregulated at both the mRNA and protein levels in GC (all p < 0.05). Remarkably, CDC45 emerged as a promising prognostic indicator and a novel diagnostic biomarker for GC. In a comprehensive the drug susceptibility analysis, we found that patients with high expression of CDC45 had high sensitivity to various chemotherapeutic agents, among which 5-fluorouracil, docetaxel, cisplatin, and elesclomol were most evident. Furthermore, our findings suggested a plausible association between CDC45 and immune cell infiltration. Enrichment analysis revealed that CDC45 and its associated genes may play crucial roles in muscle biofunction, whereas GSEA demonstrated significant enrichment of gene sets pertaining to G protein-coupled receptor ligand binding and G alpha (i) signaling events. Conclusion: Our study elucidates that upregulation of CDC45 is intricately associated with immune cell infiltration and holds promising potential as a favorable prognostic marker and a novel diagnostic biomarker for GC.

Upregulation of POC1A in lung adenocarcinoma promotes tumour progression and predicts poor prognosis.

Lung adenocarcinoma (LUAD) is characterized by a high incidence rate and mortality. Recently, POC1 centriolar protein A (POC1A) has emerged as a potential biomarker for various cancers, contributing to cancer onset and development. However, the association between POC1A and LUAD remains unexplored. We extracted The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) data sets to analyse the differential expression of POC1A and its relationship with clinical stage. Additionally, we performed diagnostic receiver operator characteristic (ROC) curve analysis and Kaplan-Meier (KM) survival analysis to assess the diagnostic and prognostic value of POC1A in LUAD. Furthermore, we investigated the correlation between POC1A expression and immune infiltration, tumour mutation burden (TMB), immune checkpoint expression and drug sensitivity. Finally, we verified POC1A expression using real-time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC). Cell experiments were conducted to validate the effect of POC1A expression on the proliferation, migration and invasion of lung cancer cells. POC1A exhibited overexpression in most tumour tissues, and its overexpression in LUAD was significantly correlated with late-stage presentation and poor prognosis. The high POC1A expression group showed lower levels of immune infiltration but higher levels of immune checkpoint expression and TMB. Moreover, the high POC1A expression group demonstrated sensitivity to multiple drugs. In vitro experiments confirmed that POC1A knockdown led to decreased proliferation, migration, and invasion of lung cancer cells. Our findings suggest that POC1A may contribute to tumour development by modulating the cell cycle and immune cell infiltration. It also represents a potential therapeutic target and marker for the diagnosis and prognosis of LUAD.

Plot twists and cutting corners with atypical SMCs.

In this issue of Molecular Cell, two papers provide insight into atypical structural maintenance of chromosomes protein complexes (SMCs). Jeppsson et al.1 link Smc5/6 to supercoiled DNA, and Roisné-Hamelin et al.2 show how Wadjet SMC bends and cleaves invading DNAs.

Detection of hepatocellular carcinoma methylation markers in salivary DNA.

BACKGROUND: Alterations to DNA methylation have been identified in both hepatocellular carcinoma (HCC) tumor and circulating DNA from affected individuals. These markers have potential utility in HCC screening. Adherence to HCC screening is poor and acceptable HCC screening tests are needed. METHODS: A feasibility study was performed on a subset of case patients and control subjects from a prior study of risk factors for HCC. Case patients (n=12) included adults aged 47-85 years with a first diagnosis of HCC between 2011 and 2016 and without viral hepatitis. Control subjects (n=12) were matched on age, sex, and state of residence. Participants provided saliva samples for DNA genotyping. Log fold change in salivary DNA methylation at 1359 CpG sites representing 25 candidate genes previously associated with HCC was compared across case patients and control subjects. RESULTS: The quantity of DNA ranged from 9.65 to 257.79 µg. The purity of DNA isolates was good, with mean OD260/280 ratio of 1.78 (SD: 0.14). Of 25 candidate genes, 16 had at ≥1 CpG site with detectable differences in methylation across HCC case patients and control subjects. Sites differentially methylated in HCC case patients included genes encoding tumor suppressors (PRDM2, RUNX3, p15/16, and RASSF1/5), regulators of cell cycle progression (DAPK1 and TP73), and DNA repair (MGMT and GSTP1). No associations met the significance threshold 3.7 × 10-5 required for multiple comparisons. CONCLUSIONS: Salivary DNA may be a feasible alternative to blood samples in the era of novel DNA-based screening tests for HCC. The ease of saliva-based testing supports further investigation of its potential.

Structural basis for the phase separation of the chromosome passenger complex.

The physical basis of phase separation is thought to consist of the same types of bonds that specify conventional macromolecular interactions yet is unsatisfyingly often referred to as 'fuzzy'. Gaining clarity on the biogenesis of membraneless cellular compartments is one of the most demanding challenges in biology. Here, we focus on the chromosome passenger complex (CPC), that forms a chromatin body that regulates chromosome segregation in mitosis. Within the three regulatory subunits of the CPC implicated in phase separation - a heterotrimer of INCENP, Survivin, and Borealin - we identify the contact regions formed upon droplet formation using hydrogen/deuterium exchange mass spectrometry (HXMS). These contact regions correspond to some of the interfaces seen between individual heterotrimers within the crystal lattice they form. A major contribution comes from specific electrostatic interactions that can be broken and reversed through initial and compensatory mutagenesis, respectively. Our findings reveal structural insight for interactions driving liquid-liquid demixing of the CPC. Moreover, we establish HXMS as an approach to define the structural basis for phase separation.

A comprehensive review on role of Aurora kinase inhibitors (AKIs) in cancer therapeutics.

Aurora kinases (AURKs) are a family of serine /threonine protein kinases that have a crucial role in cell cycle process mainly in the event of chromosomal segregation, centrosome maturation and cytokinesis. The family consists of three members including Aurora kinase A (AURK-A), Aurora kinase B (AURK-B) and Aurora kinase C (AURK-C). All AURKs contain a conserved kinase domain for their activity but differ in their cellular localization and functions. AURK-A and AURK-B are expressed mainly in somatic cells while the expression of AURK-C is limited to germ cells. AURK-A promotes G2 to M transition of cell cycle by controlling centrosome maturation and mitotic spindle assembly. AURK-B and AURK-C form the chromosome passenger complex (CPC) that ensures proper chromosomal alignments and segregation. Aberrant expression of AURK-A and AURK-B has been detected in several solid tumours and malignancies. Hence, they have become an attractive therapeutic target against cancer. The first part of this review focuses on AURKs structure, functions, subcellular localization, and their role in tumorigenesis. The review also highlights the functional and clinical impact of selective as well as pan kinase inhibitors. Currently, >60 compounds that target AURKs are in preclinical and clinical studies. The drawbacks of existing inhibitors like selectivity, drug resistance and toxicity have also been addressed. Since, majority of inhibitors are Aurora kinase inhibitor (AKI) type-1 that bind to the active (DFGin and Cin) conformation of the kinase, this information may be utilized to design highly selective kinase inhibitors that can be combined with other therapeutic agents for better clinical outcomes.

MYC activity at enhancers drives prognostic transcriptional programs through an epigenetic switch.

The transcription factor MYC is overexpressed in most cancers, where it drives multiple hallmarks of cancer progression. MYC is known to promote oncogenic transcription by binding to active promoters. In addition, MYC has also been shown to invade distal enhancers when expressed at oncogenic levels, but this enhancer binding has been proposed to have low gene-regulatory potential. Here, we demonstrate that MYC directly regulates enhancer activity to promote cancer type-specific gene programs predictive of poor patient prognosis. MYC induces transcription of enhancer RNA through recruitment of RNA polymerase II (RNAPII), rather than regulating RNAPII pause-release, as is the case at promoters. This process is mediated by MYC-induced H3K9 demethylation and acetylation by GCN5, leading to enhancer-specific BRD4 recruitment through its bromodomains, which facilitates RNAPII recruitment. We propose that MYC drives prognostic cancer type-specific gene programs through induction of an enhancer-specific epigenetic switch, which can be targeted by BET and GCN5 inhibitors.

Overburden of rare ALMS1 deleterious variants in Chinese early-onset type 2 diabetes with severe insulin resistance.

AIMS: Alström syndrome (AS) is a rare recessive disorder characterised by diabetes, obesity, insulin resistance (IR), and visual and hearing impairments. Mutations in the ALMS1 gene have been identified as the causative agents of AS. This study aimed to explore the relationship between rare ALMS1 variants and clinical features in Chinese patients with early-onset type 2 diabetes (age at diagnosis ≤40 years; EOD). MATERIALS AND METHODS: ALMS1 gene sequencing was performed in 611 Chinese individuals with EOD, 36 with postprandial hyperinsulinemia, and 47 with pre-diabetes and fasting IR. In-silico prediction algorithm and the American College of Medical Genetics Guidelines (ACMG) were used to evaluate the deleteriousness and pathogenicity of the variants. RESULTS: Sixty-two rare ALMS1 variants (frequency <0.005) were identified in 82 patients with EOD. Nineteen variants were predicted to be deleterious (pD). Patients with EOD carrying pD variants had higher fasting C-peptide, postprandial C-peptide, and HOMA2-IR levels than those without variants. The frequency of ALMS1 pD variants in the subgroup with more insulin-resistant EOD was higher than that in other EOD subgroups. Two patients with EOD, obesity, and IR who carried one heterozygous pathogenic/likely pathogenic rare variant of ALMS1 according to ACMG were identified. Moreover, rare heterozygous pD variants of ALMS1 were found in participants from cohorts of postprandial hyperinsulinemia as well as in pre-diabetes with fasting IR. CONCLUSIONS: ALMS1 rare pD variants are enriched in the populations with significant IR, which is a major hallmark of diabetes pathogenesis. Accordingly, our exploratory study provides insights and hypotheses for further studies of gene function.

Depletion of CNOT4 modulates the DNA damage responses following ionizing radiation (IR).

BACKGROUND: The Ccr4-Not complex (CNOT complex in mammals) is a unique and highly conserved complex with numerous cellular functions. Until now, there has been relatively little known about the importance of the CNOT complex subunits in the DNA damage response (DDR) in mammalian cells. CNOT4 is a subunit of the complex with E3 ubiquitin ligase activity that interacts transiently with the CNOT1 subunit. Here, we attempt to investigate the role of human CNOT4 subunit in the DDR in human cells. MATERIAL AND METHODS: In this study, cell viability in the absence of CNOT4 was assessed using a Cell Titer-Glo Luminescence assay up to 4 days post siRNA transfection. In a further experiment, CNOT4-depleted HeLa cells were exposed to 3Gy ionizing radiation (IR). Ataxia telangiectasia-mutated (ATM) and ATM Rad3-related (ATR) signaling pathways were then investigated by western blotting for phosphorylated substrates. In addition, foci formation of histone 2A family member X (γH2AX), replication protein A (RPA), TP53 binding protein 1 (53BP1), and DNA repair protein RAD51 homolog 1 was also determined by immunofluorescence microscopy comparing control and CNOT4-depleted HeLa cells 0, 8, and 24 h post IR treatment. RESULTS: Our results from cell viability assays showed a significant reduction of cell growth activity at 24 (P value 0.02) and 48 h (P value 0.002) post siRNA. Western blot analysis showed slightly reduced or slightly delayed DDR signaling in CNOT4-depleted HeLa cells after IR. More significantly, we observed increased formation of γH2AX, RPA, 53BP1, and RAD51 foci after IR in CNOT4-depleted cells compared with the control cells. CONCLUSION: We conclude that depletion of CNOT4 affects various aspects of the cellular response to DNA damage.