The histone mark H3K36me3 regulates human DNA mismatch repair through its interaction with MutSα.

DNA mismatch repair (MMR) ensures replication fidelity by correcting mismatches generated during DNA replication. Although human MMR has been reconstituted in vitro, how MMR occurs in vivo is unknown. Here, we show that an epigenetic histone mark, H3K36me3, is required in vivo to recruit the mismatch recognition protein hMutSα (hMSH2-hMSH6) onto chromatin through direct interactions with the hMSH6 PWWP domain. The abundance of H3K36me3 in G1 and early S phases ensures that hMutSα is enriched on chromatin before mispairs are introduced during DNA replication. Cells lacking the H3K36 trimethyltransferase SETD2 display microsatellite instability (MSI) and an elevated spontaneous mutation frequency, characteristic of MMR-deficient cells. This work reveals that a histone mark regulates MMR in human cells and explains the long-standing puzzle of MSI-positive cancer cells that lack detectable mutations in known MMR genes.

Cancer-driving H3G34V/R/D mutations block H3K36 methylation and H3K36me3-MutSα interaction.

Somatic mutations on glycine 34 of histone H3 (H3G34) cause pediatric cancers, but the underlying oncogenic mechanism remains unknown. We demonstrate that substituting H3G34 with arginine, valine, or aspartate (H3G34R/V/D), which converts the non-side chain glycine to a large side chain-containing residue, blocks H3 lysine 36 (H3K36) dimethylation and trimethylation by histone methyltransferases, including SETD2, an H3K36-specific trimethyltransferase. Our structural analysis reveals that the H3 "G33-G34" motif is recognized by a narrow substrate channel, and that H3G34/R/V/D mutations impair the catalytic activity of SETD2 due to steric clashes that impede optimal SETD2-H3K36 interaction. H3G34R/V/D mutations also block H3K36me3 from interacting with mismatch repair (MMR) protein MutSα, preventing the recruitment of the MMR machinery to chromatin. Cells harboring H3G34R/V/D mutations display a mutator phenotype similar to that observed in MMR-defective cells. Therefore, H3G34R/V/D mutations promote genome instability and tumorigenesis by inhibiting MMR activity.

Regulation of PLPP3 gene expression by NF-κB family transcription factors.

Lipid phosphate phosphatase 3 (LPP3), encoded by the PLPP3 gene, is an integral membrane enzyme that dephosphorylates phosphate esters of glycero- and sphingophospholipids. Cell surface LPP3 can terminate the signaling actions of bioactive lysophosphatidic acid (LPA) and sphingosine 1 phosphate, which likely explains its role in developmental angiogenesis, vascular injury responses, and cell migration. Heritable variants in the final intron PLPP3 associate with interindividual variability in coronary artery disease risk that may result from disruption of enhancer sequences that normally act in cis to increase expression of the gene. However, the mechanisms regulating PLPP3 expression are not well understood. We show that the human PLPP3 promoter contains three functional NF-κB response elements. All of these are required for maximal induction of PLPP3 promoter activity in reporter assays. The identified sequences recruit RelA and RelB components of the NF-κB transcription complex to chromatin, and these transcription factors bind to the identified target sequences in two different cell types. LPA promotes binding of Rel family transcription factors to the PLPP3 promoter and increases PLPP3 gene expression through mechanisms that are attenuated by an NF-κB inhibitor, LPA receptor antagonists, and inhibitors of phosphoinositide 3 kinase. These findings indicate that up-regulation of PLPP3 during inflammation and atherosclerosis results from canonical activation of the NF-κB signaling cascade to increase PLPP3 expression through nuclear import and binding of RelA and RelB transcription factors to the PLPP3 promoter and suggest a mechanism by which the LPP3 substrate, LPA, can regulate PLPP3 expression.

Coronary Artery Disease Risk-Associated Plpp3 Gene and Its Product Lipid Phosphate Phosphatase 3 Regulate Experimental Atherosclerosis.

OBJECTIVE: Genome-wide association studies identified novel loci in PLPP3(phospholipid phosphatase 3) that associate with coronary artery disease risk independently of traditional risk factors. PLPP3 encodes LPP3 (lipid phosphate phosphatase 3), a cell-surface enzyme that can regulate the availability of bioactive lysophopsholipids including lysophosphatidic acid (LPA). The protective allele of PLPP3 increases LPP3 expression during cell exposure to oxidized lipids, however, the role of LPP3 in atherosclerosis remains unclear. Approach and Results: In this study, we sought to validate LPP3 as a determinate of the development of atherosclerosis. In experimental models of atherosclerosis, LPP3 is upregulated and co-localizes with endothelial, smooth muscle cell, and CD68-positive cell markers. Global post-natal reductions in Plpp3 expression in mice substantially increase atherosclerosis, plaque-associated LPA, and inflammation. Although LPP3 expression increases during ox-LDL (oxidized low-density lipoprotein)-induced phenotypic modulation of bone marrow-derived macrophages, myeloid Plpp3 does not appear to regulate lesion formation. Rather, smooth muscle cell LPP3 expression is a critical regulator of atherosclerosis and LPA content in lesions. Moreover, mice with inherited deficiency in LPA receptor signaling are protected from experimental atherosclerosis. CONCLUSIONS: Our results identify a novel lipid signaling pathway that regulates inflammation in the context of atherosclerosis and is not related to traditional risk factors. Pharmacological targeting of bioactive LPP3 substrates, including LPA, may offer an orthogonal approach to lipid-lowering drugs for mitigation of coronary artery disease risk.

Effects of diet and hyperlipidemia on levels and distribution of circulating lysophosphatidic acid.

Lysophosphatidic acids (LPAs) are bioactive radyl hydrocarbon-substituted derivatives of glycerol 3-phosphate. LPA metabolism and signaling are implicated in heritable risk of coronary artery disease. Genetic and pharmacological inhibition of these processes attenuate experimental atherosclerosis. LPA accumulates in atheromas, which may be a consequence of association with LDLs. The source, regulation, and biological activity of LDL-associated LPA are unknown. We examined the effects of experimental hyperlipidemia on the levels and distribution of circulating LPA in mice. The majority of plasma LPA was associated with albumin in plasma from wild-type mice fed normal chow. LDL-associated LPA was increased in plasma from high-fat Western diet-fed mice that are genetically prone to hyperlipidemia (LDL receptor knockout or activated proprotein convertase subtilisin/kexin type 9-overexpressing C57Bl6). Adipose-specific deficiency of the ENPP2 gene encoding the LPA-generating secreted lysophospholipase D, autotaxin (ATX), attenuated these Western diet-dependent increases in LPA. ATX-dependent increases in LDL-associated LPA were observed in isolated incubated plasma. ATX acted directly on LDL-associated lysophospholipid substrates in vitro. LDL from all human subjects examined contained LPA and was decreased by lipid-lowering drug therapies. Human and mouse plasma therefore contains a diet-sensitive LDL-associated LPA pool that might contribute to the cardiovascular disease-promoting effects of LPA.

Coordinated processing of 3' slipped (CAG)n/(CTG)n hairpins by DNA polymerases ß and δ preferentially induces repeat expansions.

Expansion of CAG/CTG trinucleotide repeats causes certain familial neurological disorders. Hairpin formation in the nascent strand during DNA synthesis is considered a major path for CAG/CTG repeat expansion. However, the underlying mechanism is unclear. We show here that removal or retention of a nascent strand hairpin during DNA synthesis depends on hairpin structures and types of DNA polymerases. Polymerase (pol) δ alone removes the 3'-slipped hairpin using its 3'-5' proofreading activity when the hairpin contains no immediate 3' complementary sequences. However, in the presence of pol ß, pol δ preferentially facilitates hairpin retention regardless of hairpin structures. In this reaction, pol ß incorporates several nucleotides to the hairpin 3'-end, which serves as an effective primer for the continuous DNA synthesis by pol δ, thereby leading to hairpin retention and repeat expansion. These findings strongly suggest that coordinated processing of 3'-slipped (CAG)n/(CTG)n hairpins by polymerases δ and ß on during DNA synthesis induces CAG/CTG repeat expansions.

Specific sequence determinants of miR-15/107 microRNA gene group targets.

MicroRNAs (miRNAs) target mRNAs in human cells via complex mechanisms that are still incompletely understood. Using anti-Argonaute (anti-AGO) antibody co-immunoprecipitation, followed by microarray analyses and downstream bioinformatics, 'RIP-Chip' experiments enable direct analyses of miRNA targets. RIP-Chip studies (and parallel assessments of total input mRNA) were performed in cultured H4 cells after transfection with miRNAs corresponding to the miR-15/107 gene group (miR-103, miR-107, miR-16 and miR-195), and five control miRNAs. Three biological replicates were run for each condition with a total of 54 separate human Affymetrix Human Gene 1.0 ST array replicates. Computational analyses queried for determinants of miRNA:mRNA binding. The analyses support four major findings: (i) RIP-Chip studies correlated with total input mRNA profiling provides more comprehensive information than using either RIP-Chip or total mRNA profiling alone after miRNA transfections; (ii) new data confirm that miR-107 paralogs target coding sequence (CDS) of mRNA; (iii) biochemical and computational studies indicate that the 3' portion of miRNAs plays a role in guiding miR-103/7 to the CDS of targets; and (iv) there are major sequence-specific targeting differences between miRNAs in terms of CDS versus 3'-untranslated region targeting, and stable AGO association versus mRNA knockdown. Future studies should take this important miRNA-to-miRNA variability into account.

Rad5 and Its Human Homologs, HLTF and SHPRH, Are Novel Interactors of Mismatch Repair.

DNA mismatch repair (MMR) repairs replication errors, and MMR defects play a role in both inherited cancer predisposition syndromes and in sporadic cancers. MMR also recognizes mispairs caused by environmental and chemotherapeutic agents; however, in these cases mispair recognition leads to apoptosis and not repair. Although mutation avoidance by MMR is fairly well understood, MMR-associated proteins are still being identified. We performed a bioinformatic analysis that implicated Saccharomyces cerevisiae Rad5 as a candidate for interacting with the MMR proteins Msh2 and Mlh1. Rad5 is a DNA helicase and E3 ubiquitin ligase involved in post-replicative repair and damage tolerance. We confirmed both interactions and found that the Mlh1 interaction is mediated by a conserved Mlh1-interacting motif (MIP box). Despite this, we did not find a clear role for Rad5 in the canonical MMR mutation avoidance pathway. The interaction of Rad5 with Msh2 and Mlh1 is conserved in humans, although each of the Rad5 human homologs, HLTF and SHPRH, shared only one of the interactions: HLTF interacts with MSH2, and SHPRH interacts with MLH1. Moreover, depletion of SHPRH, but not HLTF, results in a mild increase in resistance to alkylating agents although not as strong as loss of MMR, suggesting gene duplication led to specialization of the MMR-protein associated roles of the human Rad5 homologs. These results provide insights into how MMR accessory factors involved in the MMR-dependent apoptotic response interact with the core MMR machinery and have important health implications into how human cells respond to environmental toxins, tumor development, and treatment choices of tumors with defects in Rad5 homologs.

Erratum: Author Correction: Identification of novel genetic variants predisposing to familial oral squamous cell carcinomas.

[This corrects the article DOI: 10.1038/s41421-019-0126-6.].

Identification and characterization of OGG1 mutations in patients with Alzheimer's disease.

Patients with Alzheimer's disease (AD) exhibit higher levels of 8-oxo-guanine (8-oxoG) DNA lesions in their brain, suggesting a reduced or defective 8-oxoG repair. To test this hypothesis, this study investigated 14 AD patients and 10 age-matched controls for mutations of the major 8-oxoG removal gene OGG1. Whereas no alterations were detected in any control samples, four AD patients exhibited mutations in OGG1, two carried a common single base (C796) deletion that alters the carboxyl terminal sequence of OGG1, and the other two had nucleotide alterations leading to single amino acid substitutions. In vitro biochemical assays revealed that the protein encoded by the C796-deleted OGG1 completely lost its 8-oxoG glycosylase activity, and that the two single residue-substituted OGG1 proteins showed a significant reduction in the glycosylase activity. These results were consistent with the fact that nuclear extracts derived from a limited number of AD patients with OGG1 mutations exhibited greatly reduced 8-oxoG glycosylase activity compared with age-matched controls and AD patients without OGG1 alterations. Our findings suggest that defects in OGG1 may be important in the pathogenesis of AD in a significant fraction of AD patients and provide new insight into the molecular basis for the disease.

Identification of novel genetic variants predisposing to familial oral squamous cell carcinomas.

Oral squamous cell carcinoma (OSCC) is a common subtype of head and neck squamous cell carcinoma (HNSCC), but the pathogenesis underlying familial OSCCs is unknown. Here, we analyzed whole-genome sequences of a family with autosomal dominant expression of oral tongue cancer and identified proto-oncogenes VAV2 and IQGAP1 as the primary factors responsible for oral cancer in the family. These two genes are also frequently mutated in sporadic OSCCs and HNSCCs. Functional analysis revealed that the detrimental variants target tumorigenesis-associated pathways, thus confirming that these novel genetic variants help to establish a predisposition to familial OSCC.

Altered 8-oxoguanine glycosylase in mild cognitive impairment and late-stage Alzheimer's disease brain.

Eight-hydroxy-2'-deoxyguanosine (8-OHdG) is increased in the brain in late-stage Alzheimer's disease (LAD) and mild cognitive impairment (MCI). To determine if decreased base-excision repair contributes to these elevations, we measured oxoguanine glycosylase 1 (OGG1) protein and incision activities in nuclear and mitochondrial fractions from frontal (FL), temporal (TL), and parietal (PL) lobes from 8 MCI and 7 LAD patients, and 6 age-matched normal control (NC) subjects. OGG1 activity was significantly (P<0.05) decreased in nuclear specimens of FL, TL, and PL in MCI and LAD and in mitochondria from LAD FL and TL and MCI TL. Nuclear OGG1 protein was significantly decreased in LAD FL and MCI and LAD PL. No differences in mitochondrial OGG1 protein levels were found. Overall, our results suggest that decreased OGG1 activity occurs early in the progression of AD, possibly mediated by 4-hydroxynonenal inactivation and may contribute to elevated 8-OHdG in the brain in MCI and LAD.

Purification and relationship with gastric disease of a 130 kDa (CagA) protein of Helicobacter pylori.

OBJECTIVE: The aims of this research were to purify and identify the 130 kDa (CagA) protein of H. pylori clinical isolate HP97002 and evaluate the relationships between the purified 130 kDa (CagA) protein and gastric diseases. METHODS: The procedure for isolating the protein included 6 mol/L guanidine extract, size exclusion chromatography and elusion from gel. Sera of 68 patients with gastric diseases (44 with chronic gastritis, 15 with atrophic gastritis, 7 with peptic ulcer disease, 2 with gastric cancer) were obtained, and the serological response to CagA was studied by Western-blot using the purified protein. RESULTS: The purified protein was 130 kDa and preserved good antigenicity and revealed basic isoelectric point about of 8.1. Among 68 sera, 43 sera could recognize the purified protein associated with chronic gastritis 47.7% (21/44), atrophic gastritis 86.7% (13/15), peptic ulcer disease 100% (77), gastric cancer 100% (2/2). Compared with each other, the difference was significant (chi2 = 13.327, P = 0.004), and 130 kDa (CagA) protein was associated with severe gastric diseases ( r(S) = 0.442, P = 0.001). CONCLUSION: The 130 kDa (CagA) protein was associated with severe gastric diseases.

[Effect of eucalyptus globulus oil on activation of nuclear factor-kappaB in THP-1 cells].

OBJECTIVE: To study the effect of eucalyptus globulus oil on the activity of nuclear factor-kappaB(NF-kappaB) in THP-1 cell line. METHODS: THP-1 cells were cultured with or without eucalyptus globulus oil at different concentrations (1, 10, 100 mg x L(-1), 30 min) before being stimulated with lipopolysaccharide (LPS, 1 mg x L(-1), 30 min). The location of NF-kappaB p65 subunit (NF-kappaB/p65) in THP-1 cells was detected by indirect immunofluorescence and laser scanning confocal microscope. The expression of NF-kappaB/p65 in nuclei was measured by Western-blot analysis. RESULT: The FITC-label NF-kappaB/p65 was mainly located in the nuclei after THP-1 cells were stimulated with LPS. Whereas, no fluorescence were seen in the nuclei of cells pretreated with eucalyptus globulus oil. This effect on NF-kappaB/p65 nuclear translocation was in a concentration dependent manner. CONCLUSION: Eucalyptus globulus oil inhibits the nuclear translocation of NF-kappaB induced by LPS in THP-1 cells.

[Changes of phosphorylation states of Cx43 protein in CCL-64 cells induced by SiO2].

OBJECTIVE: To investigate whether the cellular gap junctional communication(GJIC) down-regulation in alveolar epithelial cells (CCL-64 cells) induced by silica-stimulated pulmonary alveolar macrophages (PAM) supernatant is related with the phosphorylation states of connexin 43(Cx43) protein. METHOD: Western-blot analysis was used to identify phosphorylated Cx43 species. RESULTS: Western-blot analyses of SiO2- and phorbol 12-myristate 13-acetate(TPA)-treated CCL-64 cells showed the same phosphorylation states of Cx43 as the control group. There were no Cx43 protein in nucleus of CCL-64 cells. CONCLUSION: The inhibition on GJIC induced by SiO2 and TPA in CCL-64 cells may not be brought about by altering the phosphorylation states of Cx43.

[Localization of the connexin 43 gap-junction protein in silica-exposed alveolar epithelial cells].

OBJECTIVE: To study the effect of supernatants from silicon dioxide(SiO2) stimulated pulmonary alveolar macrophages(PAM) on the localization of connexin 43(Cx43) so as to explore the inhibition level of SiO2 on alveolar epithelial cellular gap-junctional communication(GJIC). METHODS: The supermatants from the primary cultured PAM were prepared, and then added 5% (v/v) SiO2 into 2% (v/v) NBS RPMI 1640 to stimulate the normal mink lung epithelial cell line CCL-64 for 24 hours. The localizations of Cx43 in CCL-64 were analyzed by indirect immunofluorescence histochemistry and laser confocal scanning microscopy(LCSM). RESULTS: The normal cultured CCL-64 cells displayed bright membrane-associated Cx43 plaques labeling and formed dashes at regions of intercellular junction. Being exposed to supernatants from SiO2-stimulated PAM, the CCL-64 cells retained a relative low degree of Cx43 labeling at the cell periphery, localized in cytoplasm, and the individual spot, rather than plaques, were smaller compared to normal cultured cells. Along with the increase of the concentrations of SiO2, the cells displayed a different staining pattern, with clear cluster labeling aggregating towards the nucleus. CONCLUSION: The altered localization of the gap-junctional protein Cx43 in alveolar epithelial cells, mediated by SiO2, indicated that the internalization of Cx43 may contribute to the inhibition on GJIC in silica-induced lung epithelium injury.

[Changes of phosphorylation states of Cx43 protein in Chinese hamster lung cells induced by SiO2].

OBJECTIVE: To investigate whether cellular gap-junctional communication(GJIC) down-regulation and the internalization of connexin 43(Cx43) in Chinese hamster lung fibroblasts (CHL) induced by silica-stimulated pulmonary alveolar macrophages (PAM) supermatant is related with the phosphorylation states of Cx43 protein. METHODS: Western-blot analysis was used to identify phosphorylated Cx43 species. RESULTS: Samples from membrane protein, total protein and nucleoprotein in CHL cells with 50-500 micrograms/ml doses of silica-stimulated PAM supernatants showed NP, P1, P2, P3 four immunoreactive bands of Cx43 protein by contrast with the control group and 0 microgram/ml SiO2 group. And with the dose of SiO2 increased, the increment of the levels of P2 and P3 was observed. Moreover, the groups treated with SiO2 and protein kinase C inhibitor, Palmitoyl-DL-Camitine chloride (PMC), simutaneously showed reduced level of P2 and P3, as compared with the groups treated with SiO2 only. CONCLUSION: The inhibition of GJIC and the internalization of Cx43 by SiO2 in CHL cells may relate to the changes of phosphorylation states of Cx43, and its mechanism may be similar to that of phorbol 12-myristate 13-acetate (TPA), i.e. via PKC activation pathway.

[Downregulation of gap junctional intercellular communication induced by silicon dioxide in the pulmonary alveolar epithelial cell].

OBJECTIVE: To explore the effect of silica dioxide(SiO2) on proliferation and downregulation of gap junctional intercellular communication (GJIC) in pulmonary alveolar epithelial cells (CCL-64 cells). METHODS: The pulmonary alveolar macrophages(PAMs) were incubated in the serum-free RPMI 1640 containing the various concentration of SiO2 for 24 hours. The supernatants were prepared and added 5% (V/V) into 2% (V/V) NBS RPMI 1640 to stimulate the proliferation of CCL-64 cells for 24 hours. A set of "blank control", run in parallel, contained RPMI 1640 + 2% (V/V) NBS alone. The proliferation of CCL-64 cells was detected using MTT assay(to show as the absorbency, A570nm). GJIC function was measured using the fluorescence redistribution after photobleaching(FRAP) assay [to express as the transfer rate of the fluorescence, K (x 10(-3)/s)], with a laser scanning confocal microscope(LSCM, Leica TCS SP). RESULTS: The silica-exposed PAM supernatants could induce both the proliferation(F = 9.679, P < 0.01) and downregulation of GJIC(F = 20.587, P < 0.01) of CCL-64 cells. In the range of 50-500 micrograms/ml SiO2 concentrations, the proliferation (A570nm values) and GJIC(the transfer rate, K) were fitted well in a dose-dependent manner(proliferation: r = 0.891, P < 0.05; GJIC: r = -0.943, P < 0.05). CONCLUSION: By way of stimulating the PAM, SiO2 could inhibit GJIC function in lung alveolar epithelial cells, and induce epithelial cell proliferation. In the pathogenesis of silicosis, the downregulation of GJIC of the pulmonary epithelial cells may play an important role in silica-mediated alveolar epithelial cell injury.

[Abnormal shift of connexin 43 gap-junction protein induced by 50 Hz electromagnetic fields in Chinese hamster lung cells].

OBJECTIVE: To study the effects of extremely low frequency magnetic fields(ELF MF) on the amount and localization of connexin 43(Cx43) gap-junction protein in the Chinese hamster lung(CHL) cells, and to explore the mechanism of ELF MF suppression on gap-junctional intercellular communication(GJIC). METHODS: The cells were irradiated for 24 h with 50 Hz sinusoidal magnetic field at 0.8 mT without or with 12-O-tetrade-canoylphorbol-3-acetate(TPA), 5 ng/ml for 1 h. The localization of Cx43 proteins were performed by indirect immunofluorescence histochemical analysis and detected by confocal microscopy. The second experiment was conducted to examine the quantity of Cx43 proteins level in nuclei or cytoplasm and detected by Western blotting analysis. RESULTS: The cells exposed to TPA for 1 h displayed less bright labelled spots in the regions of intercellular junction than the normal cells. Most of Cx43 labelled spots occurred in the cytoplasm and aggregated near the nuclei. At the same time, the amount of Cx43 protein in cytoplasm were increased[(2.03 +/- 0.89) in ELF group, (2.43 +/- 0.82) in TPA group] as compared to normal control(1.04 +/- 0.17) (P < 0.01). CONCLUSION: Inhibition on GJIC function by ELF MF alone or combined with TPA may be related with the shift of Cx43 from the regions of intercellular junction to the cytoplasm.

[The effect of silicon dioxide on the activation of nuclear factor-kappaB in THP-1 cells].

OBJECTIVE: To study the effect of silicon dioxide (SiO(2)) on the activation of nuclear factor-kappaB (NF-kappaB) in THP-1 cell line. METHODS: THP-1 cells were incubated with a series of doses of SiO(2) (0, 100, 200 micro g/ml). The location of NF-kappaB p65 subunit (NF-kappaB/p65) in THP-1 cells was detected by immunofluorescence and laser scanning confocal microscope (LSCM). The expression of NF-kappaB/p65 in nuclei was measured by Western blot analysis. RESULTS: The majority of fluorescein isothiocyanate (FITC)-labelled NF-kappaB/p65 located in the nuclei 30 min after stimulation by 100 micro g/ml SiO(2), whereas the FITC-labelled NF-kappaB/p65 were mainly seen in the plasma of normal control cells. The expression of NF-kappaB/p65 in THP-1 nuclear protein was low in control group (0 micro g/ml SiO(2)) while it increased after stimulation by 100 micro g/ml SiO(2) and 200 micro g/ml SiO(2) for 15 min and 30 min. The level of NF-kappaB/p65 was comparatively increased with the increasing of doses and time. Lipopolysaccharides (LPS), an activator of NF-kappaB, had similar effect as SiO(2) on the activation of NF-kappaB/p65 in THP-1 cells. CONCLUSION: SiO(2) could activate and internalize NF-kappaB in the THP-1 cell line.