Omics research project on prospective cohort studies from the Tohoku Medical Megabank Project.

Population-based prospective cohort studies are indispensable for modern medical research as they provide important knowledge on the influences of many kinds of genetic and environmental factors on the cause of disease. Although traditional cohort studies are mainly conducted using questionnaires and physical examinations, modern cohort studies incorporate omics and genomic approaches to obtain comprehensive physical information, including genetic information. Here, we report the design and midterm results of multi-omics analysis on population-based prospective cohort studies from the Tohoku Medical Megabank (TMM) Project. We have incorporated genomic and metabolomic studies in the TMM cohort study as both metabolome and genome analyses are suitable for high-throughput analysis of large-scale cohort samples. Moreover, an association study between the metabolome and genome show that metabolites are an important intermediate phenotype connecting genetic and lifestyle factors to physical and pathologic phenotypes. We apply our metabolome and genome analyses to large-scale cohort samples in the following studies.

Methionine adenosyltransferase II serves as a transcriptional corepressor of Maf oncoprotein.

Protein methylation pathways comprise methionine adenosyltransferase (MAT), which produces S-adenosylmethionine (SAM) and SAM-dependent substrate-specific methyltransferases. However, the function of MAT in the nucleus is largely unknown. MafK represses or activates expression of heme oxygenase-1 (HO-1) gene, depending on its heterodimer partners. Proteomics analysis of MafK revealed its interaction with MATIIα, a MAT isozyme. MATIIα was localized in nuclei and found to form a dense network with chromatin-related proteins including Swi/Snf and NuRD complexes. MATIIα was recruited to Maf recognition element (MARE) at HO-1 gene. When MATIIα was knocked down in murine hepatoma cell line, expression of HO-1 was derepressed at both basal and induced levels. The catalytic activity of MATIIα, as well as its interacting factors such as MATIIß, BAF53a, CHD4, and PARP1, was required for HO-1 repression. MATII serves as a transcriptional corepressor of MafK by interacting with chromatin regulators and supplying SAM for methyltransferases.

Regulatory signatures of liver regeneration distilled by integrative analysis of mRNA, histone methylation, and proteomics.

The capacity of the liver to regenerate is likely to be encoded as a plasticity of molecular networks within the liver. By applying a combination of comprehensive analyses of the epigenome, transcriptome, and proteome, we herein depict the molecular landscape of liver regeneration. We demonstrated that histone H3 Lys-4 was trimethylated at the promoter regions of many loci, among which only a fraction, including cell-cycle-related genes, were transcriptionally up-regulated. A cistrome analysis guided by the histone methylation patterns and the transcriptome identified FOXM1 as the key transcription factor promoting liver regeneration, which was confirmed in vitro using a hepatocarcinoma cell line. The promoter regions of cell-cycle-related genes and Foxm1 acquired higher levels of trimethylated histone H3 Lys-4, suggesting that epigenetic regulations of these key regulatory genes define quiescence and regeneration of the liver cells. A quantitative proteome analysis of the regenerating liver revealed that conditional protein degradation also mediated regeneration-specific protein expression. These sets of informational resources should be useful for further investigations of liver regeneration.

Microproteomics with microfluidic-based cell sorting: Application to 1000 and 100 immune cells.

Ultimately, cell biology seeks to define molecular mechanisms underlying cellular functions. However, heterogeneity within cell populations must be considered for optimal assay design and data interpretation. Although single-cell analyses are desirable for addressing this issue, practical considerations, including assay sensitivity, limit their broad application. Therefore, omics studies on small numbers of cells in defined subpopulations represent a viable alternative for elucidating cell functions at the molecular level. MS-based proteomics allows in-depth proteome exploration, although analyses of small numbers of cells have not been pursued due to loss during the multistep procedure involved. Thus, optimization of the proteomics workflow to facilitate the analysis of rare cells would be useful. Here, we report a microproteomics workflow for limited numbers of immune cells using non-damaging, microfluidic chip-based cell sorting and MS-based proteomics. Samples of 1000 or 100 THP-1 cells were sorted, and after enzymatic digestion, peptide mixtures were subjected to nano-LC-MS analysis. We achieved reasonable proteome coverage from as few as 100-sorted cells, and the data obtained from 1000-sorted cells were as comprehensive as those obtained using 1 µg of whole cell lysate. With further refinement, our approach could be useful for studying cell subpopulations or limited samples, such as clinical specimens.

Novel Mechanisms for Heme-dependent Degradation of ALAS1 Protein as a Component of Negative Feedback Regulation of Heme Biosynthesis.

In eukaryotic cells, heme production is tightly controlled by heme itself through negative feedback-mediated regulation of nonspecific 5-aminolevulinate synthase (ALAS1), which is a rate-limiting enzyme for heme biosynthesis. However, the mechanism driving the heme-dependent degradation of the ALAS1 protein in mitochondria is largely unknown. In the current study, we provide evidence that the mitochondrial ATP-dependent protease ClpXP, which is a heteromultimer of CLPX and CLPP, is involved in the heme-dependent degradation of ALAS1 in mitochondria. We found that ALAS1 forms a complex with ClpXP in a heme-dependent manner and that siRNA-mediated suppression of either CLPX or CLPP expression induced ALAS1 accumulation in the HepG2 human hepatic cell line. We also found that a specific heme-binding motif on ALAS1, located at the N-terminal end of the mature protein, is required for the heme-dependent formation of this protein complex. Moreover, hemin-mediated oxidative modification of ALAS1 resulted in the recruitment of LONP1, another ATP-dependent protease in the mitochondrial matrix, into the ALAS1 protein complex. Notably, the heme-binding site in the N-terminal region of the mature ALAS1 protein is also necessary for the heme-dependent oxidation of ALAS1. These results suggest that ALAS1 undergoes a conformational change following the association of heme to the heme-binding motif on this protein. This change in the structure of ALAS1 may enhance the formation of complexes between ALAS1 and ATP-dependent proteases in the mitochondria, thereby accelerating the degradation of ALAS1 protein to maintain appropriate intracellular heme levels.

Epigenetic Regulation of the Blimp-1 Gene (Prdm1) in B Cells Involves Bach2 and Histone Deacetylase 3.

B lymphocyte-induced maturation protein 1 (Blimp-1) encoded by Prdm1 is a master regulator of plasma cell differentiation. The transcription factor Bach2 represses Blimp-1 expression in B cells to stall terminal differentiation, by which it supports reactions such as class switch recombination of the antibody genes. We found that histones H3 and H4 around the Prdm1 intron 5 Maf recognition element were acetylated at higher levels in X63/0 plasma cells expressing Blimp-1 than in BAL17 mature B cells lacking its expression. Conversely, methylation of H3-K9 was lower in X63/0 cells than BAL17 cells. Purification of the Bach2 complex in BAL17 cells revealed its interaction with histone deacetylase 3 (HDAC3), nuclear co-repressors NCoR1 and NCoR2, transducin ß-like 1X-linked (Tbl1x), and RAP1-interacting factor homolog (Rif1). Chromatin immunoprecipitation confirmed the binding of HDAC3 and Rif1 to the Prdm1 locus. Reduction of HDAC3 or NCoR1 expression by RNA interference in B cells resulted in an increased Prdm1 mRNA expression. Bach2 is suggested to cooperate with HDAC3-containing co-repressor complexes in B cells to regulate the stage-specific expression of Prdm1 by writing epigenetic modifications at the Prdm1 locus.

Methionine adenosyltransferase II-dependent histone H3K9 methylation at the COX-2 gene locus.

BACKGROUND: MATII biosynthesizes AdoMet, which supplies methyl group for methylation of molecules, including histone. RESULTS: MATII interacts with histone methyltransferase SETDB1 and inhibits COX-2 gene expression. CONCLUSION: AdoMet synthesis and histone methylation are coupled on chromatin by a physical interaction of MATII and SETDB1 at the MafK target genes. SIGNIFICANCE: MATII may be important for both gene-specific and epigenome-wide regulation of histone methylation. Methionine adenosyltransferase (MAT) synthesizes S-adenosylmethionine (AdoMet), which is utilized as a methyl donor in transmethylation reactions involving histones. MATIIα, a MAT isozyme, serves as a transcriptional corepressor in the oxidative stress response and forms the AdoMet-integrating transcription regulation module, affecting histone methyltransferase activities. However, the identities of genes regulated by MATIIα or its associated methyltransferases are unclear. We show that MATIIα represses the expression of cyclooxygenase 2 (COX-2), encoded by Ptgs2, by specifically interacting with histone H3K9 methyltransferase SETDB1, thereby promoting the trimethylation of H3K9 at the COX-2 locus. We discuss both gene-specific and epigenome-wide functions of MATIIα.

Metabolic aspects of epigenome: coupling of S-adenosylmethionine synthesis and gene regulation on chromatin by SAMIT module.

Histone and DNA methyltransferases utilize S-adenosyl-L-methionine (SAM), a key intermediate of sulfur amino acid metabolism, as a donor of methyl group. SAM is biosynthesized by methionine adenosyltransferase (MAT) using two substrates, methionine and ATP. Three distinct forms of MAT (MATI, MATII and MATIII), encoded by two distinct genes (MAT1A and MAT2A), have been identified in mammals. MATII consists of α2 catalytic subunit encoded by MAT2A and ß regulatory subunit encoded by MAT2B, but the physiological function of the ß subunit is not clear. MafK is a member of Maf oncoproteins and functions as both transcription activator and repressor by forming diverse heterodimers to bind to DNA elements termed Maf recognition elements. Proteomics analysis of MafK-interactome revealed its interaction with both MATIIα and MATIIß. They are recruited specifically to MafK target genes and are required for their repression by MafK and its partner Bach1. Because the catalytic activity of MATIIα is required for the MafK target gene repression, MATIIα is suggested to provide SAM locally on chromatin where it is recruited. One of the unexpected features of MATII is that MATIIα interacts with many chromatin-related proteins of diverse functions such as histone modification, chromatin remodeling, transcription regulation, and nucleo-cytoplasmic transport. MATIIα appears to generate multiple, heterogenous regulatory complexes where it provides SAM. Considering their function, the heterooligomer of MATIIα and ß is named SAMIT (SAM-integrating transcription) module within their interactome where it serves SAM for nuclear methyltransferases.

Solution structure of clostridial collagenase H and its calcium-dependent global conformation change.

Collagenase H (ColH) from Clostridium histolyticum is a multimodular protein composed of a collagenase module (activator and peptidase domains), two polycystic kidney disease-like domains, and a collagen-binding domain. The interdomain conformation and its changes are very important for understanding the functions of ColH. In this study, small angle x-ray scattering and limited proteolysis were employed to reveal the interdomain arrangement of ColH in solution. The ab initio beads model indicated that ColH adopted a tapered shape with a swollen head. Under calcium-chelated conditions (with EGTA), the overall structure was further elongated. The rigid body model indicated that the closed form of the collagenase module was preferred in solution. The limited proteolysis demonstrated that the protease sensitivity of ColH was significantly increased under the calcium-chelated conditions, and that the digestion mainly occurred in the domain linker regions. Fluorescence measurements with a fluorescent dye were performed with the limited proteolysis products after separation. The results indicated that the limited proteolysis products exhibited fluorescence similar to that of the full-length ColH. These findings suggested that the conformation of full-length ColH in solution is the elongated form, and this form is calcium-dependently maintained at the domain linker regions.

Bach1-dependent and -independent regulation of heme oxygenase-1 in keratinocytes.

Bach1 is a member of the basic leucine zipper transcription factor family, and the Bach1/small Maf heterodimer specifically represses transcriptional activity directed by the Maf recognition element (MARE). Because Bach1 is a repressor of the oxidative stress response, we examined the function(s) of Bach1 in keratinocytes subjected to oxidative stress. Oxidative stress induced by H(2)O(2) led to an increase in MARE activity and expression of heme oxygenase-1 (HO-1), an inducible antioxidant defense enzyme. Bach1 depletion by small interfering RNAs or by deletion of Bach1 enhanced HO-1 expression in the absence of H(2)O(2), indicating that Bach1 is a critical repressor of HO-1 in keratinocytes. Although Bach1-deficient or -reduced keratinocytes expressed higher levels of HO-1 than control cells in response to H(2)O(2), Bach1 down-regulation did not attenuate the production of reactive oxygen species by H(2)O(2). In contrast, Bach1 overexpression abolished HO-1 induction by H(2)O(2), which led to increased reactive oxygen species accumulation. HO-1 was induced during keratinocyte differentiation, but MARE activity did not change during differentiation. Furthermore, Bach1 overexpression did not inhibit differentiation-associated induction of HO-1 expression, suggesting that HO-1 induction in differentiation is independent of Bach1. Thus, in response to oxidative stress, Bach1 regulates the oxidation state through the negative control of HO-1 expression prior to terminal keratinocyte differentiation. However, Bach1-mediated repression is negated during keratinocyte differentiation.

Heme induces ubiquitination and degradation of the transcription factor Bach1.

The transcription repressor Bach1 is a sensor and effector of heme that regulates the expression of heme oxygenase 1 and globin genes. Heme binds to Bach1, inhibiting its DNA binding activity and inducing its nuclear export. We found that hemin further induced the degradation of endogenous Bach1 in NIH 3T3 cells, murine embryonic fibroblasts, and murine erythroleukemia cells. In contrast, succinylacetone, an inhibitor of heme synthesis, caused accumulation of Bach1 in murine embryonic fibroblasts, indicating that physiological levels of heme regulated the Bach1 turnover. Polyubiquitination and rapid degradation of overexpressed Bach1 were induced by hemin treatment. HOIL-1, an ubiquitin-protein ligase which recognizes heme-bound, oxidized iron regulatory protein 2, was found to bind with Bach1 when both were overexpressed in NIH 3T3 cells. HOIL-1 stimulated the polyubiquitination of Bach1 in a purified in vitro ubiquitination system depending on the intact heme binding motifs of Bach1. Expression of dominant-negative HOIL-1 in murine erythroleukemia cells resulted in higher stability of endogenous Bach1, raising the possibility that the heme-regulated degradation involved HOIL-1 in murine erythroleukemia cells. These results suggest that heme within a cell regulates the polyubiquitination and degradation of Bach1.

Bach1 promotes muscle regeneration through repressing Smad-mediated inhibition of myoblast differentiation.

It has been reported that Bach1-deficient mice show reduced tissue injuries in diverse disease models due to increased expression of heme oxygenase-1 (HO-1)that possesses an antioxidant function. In contrast, we found that Bach1 deficiency in mice exacerbated skeletal muscle injury induced by cardiotoxin. Inhibition of Bach1 expression in C2C12 myoblast cells using RNA interference resulted in reduced proliferation, myotube formation, and myogenin expression compared with control cells. While the expression of HO-1 was increased by Bach1 silencing in C2C12 cells, the reduced myotube formation was not rescued by HO-1 inhibition. Up-regulations of Smad2, Smad3 and FoxO1, known inhibitors of muscle cell differentiation, were observed in Bach1-deficient mice and Bach1-silenced C2C12 cells. Therefore, Bach1 may promote regeneration of muscle by increasing proliferation and differentiation of myoblasts.

Keap1 recruits Neh2 through binding to ETGE and DLG motifs: characterization of the two-site molecular recognition model.

The expression of the phase 2 detoxification enzymes and antioxidant proteins is induced at the transcriptional level by Nrf2 and negatively regulated at the posttranslational level by Keap1 through protein-protein interactions with and subsequent proteolysis of Nrf2. We found that the Neh2 domain of Nrf2 is an intrinsically disordered but biologically active regulatory domain containing a 33-residue central alpha-helix followed by a mini antiparallel beta-sheet. Isothermal calorimetry analysis indicated that one Neh2 molecule interacts with two molecules of Keap1 via two binding sites, the stronger binding ETGE motif and the weaker binding DLG motif. Nuclear magnetic resonance titration study showed that these two motifs of the Neh2 domain bind to an overlapping site on the bottom surface of the beta-propeller structure of Keap1. In contrast, the central alpha-helix of the Neh2 domain does not have any observable affinity to Keap1, suggesting that this region may serve as a bridge connecting the two motifs for the association with the two beta-propeller structures of a dimer of Keap1. Based on these observations, we propose that Keap1 recruits Nrf2 by the ETGE motif and that the DLG motif of the Neh2 domain locks its lysine-rich central alpha-helix in a correct position to benefit ubiquitin signaling.

A novel liver metastasis-correlated protein of pancreatic neuroendocrine neoplasm (PanNEN) discovered by proteomic analysis.

The aim of this study was to identify novel liver metastasis-correlated proteins of PanNEN by proteomics to compare pancreatic tumor (PT) with paired metastatic liver tumor (LT). Of 118 surgical cases with PanNEN, 7 cases with formalin-fixed paraffin-embedded (FFPE) tissues of both PT and paired LT were evaluated by proteomics. Tumor cells were selectively collected from FFPE tissues by laser capture microdissection. A total of 3,722 proteins were detected from extracted peptides by mass spectrometry-based shotgun analysis. Selection of the candidate proteins expressed differently between PT and LT were performed by semi-quantitative comparison in silico and confirmation with immunohistochemistry. We focused on ANXA6, CNPY2, RAB11B and TUBB3, all of which had higher expressions in LT. In all surgical cases with FFPE samples, liver recurrence-free survival (RFS) was evaluated in correlation to the expression of the candidate proteins in PT by immunohistochemistry. Liver RFS was significantly poorer in CNPY2 positive patients than in negative patients (10-year liver RFS; 39.8% vs. 92.3%, p = 0.012). Also, liver RFS tended to be poorer in ANXA6 positive patients than in those who were negative (10-year liver RFS; 51.4% vs. 95.0%, p = 0.099). In the multivariate analysis, the independent predictors of liver RFS were CNPY2 positivity (HR: 6.19, 95 % CI: 1.47-42.79, p = 0.011) and tumor size ≥ 42 mm (HR: 4.63, 95 % CI: 1.03-23.23, p = 0.045). In conclusion, CNPY2 is a novel liver metastasis-correlated protein of PanNEN.

Actin Family Proteins in the Human INO80 Chromatin Remodeling Complex Exhibit Functional Roles in the Induction of Heme Oxygenase-1 with Hemin.

Nuclear actin family proteins, comprising of actin and actin-related proteins (Arps), are essential functional components of the multiple chromatin remodeling complexes. The INO80 chromatin remodeling complex, which is evolutionarily conserved and has roles in transcription, DNA replication and repair, consists of actin and actin-related proteins Arp4, Arp5, and Arp8. We generated Arp5 knockout (KO) and Arp8 KO cells from the human Nalm-6 pre-B cell line and used these KO cells to examine the roles of Arp5 and Arp8 in the transcriptional regulation mediated by the INO80 complex. In both of Arp5 KO and Arp8 KO cells, the oxidative stress-induced expression of HMOX1 gene, encoding for heme oxygenase-1 (HO-1), was significantly impaired. Consistent with these observations, chromatin immunoprecipitation (ChIP) assay revealed that oxidative stress caused an increase in the binding of the INO80 complex to the regulatory sites of HMOX1 in wild-type cells. The binding of INO80 complex to chromatin was reduced in Arp8 KO cells compared to that in the wild-type cells. On the other hand, the binding of INO80 complex to chromatin in Arp5 KO cells was similar to that in the wild-type cells even under the oxidative stress condition. However, both remodeling of chromatin at the HMOX1 regulatory sites and binding of a transcriptional activator to these sites were impaired in Arp5 KO cells, indicating that Arp5 is required for the activation of the INO80 complex. Collectively, these results suggested that these nuclear Arps play indispensable roles in the function of the INO80 chromatin remodeling complex.

Ubiquitylation of Ku80 by RNF126 Promotes Completion of Nonhomologous End Joining-Mediated DNA Repair.

Repair of damaged DNA is critical for maintenance of genetic information. In eukaryotes, DNA double-strand breaks (DSBs) are recognized by the Ku70-Ku80 heterodimer, which then recruits proteins that mediate repair by nonhomologous end joining (NHEJ). Prolonged retention of Ku70/80 at DSBs prevents completion of repair, however, with ubiquitylation of Ku80 having been implicated in Ku70/80 dissociation from DNA. Here, we identify RNF126 as a ubiquitin ligase that is recruited to DSBs and ubiquitylates Ku80, with UBE2D3 serving as an E2 enzyme. Knockdown of RNF126 prevented Ku70/80 dissociation from DSBs and inhibited break repair. Attenuation of Ku80 ubiquitylation by replacement of ubiquitylation site lysines with arginine residues delayed Ku70/80 release from chromatin after DSB induction by genotoxic insults. Together, our data indicate that RNF126 is a novel regulator of NHEJ that promotes completion of DNA repair by ubiquitylating Ku80 and releasing Ku70/80 from damaged DNA.

Pi class glutathione S-transferase genes are regulated by Nrf 2 through an evolutionarily conserved regulatory element in zebrafish.

Pi class GSTs (glutathione S-transferases) are a member of the vertebrate GST family of proteins that catalyse the conjugation of GSH to electrophilic compounds. The expression of Pi class GST genes can be induced by exposure to electrophiles. We demonstrated previously that the transcription factor Nrf 2 (NF-E2 p45-related factor 2) mediates this induction, not only in mammals, but also in fish. In the present study, we have isolated the genomic region of zebrafish containing the genes gstp1 and gstp2. The regulatory regions of zebrafish gstp1 and gstp2 have been examined by GFP (green fluorescent protein)-reporter gene analyses using microinjection into zebrafish embryos. Deletion and point-mutation analyses of the gstp1 promoter showed that an ARE (antioxidant-responsive element)-like sequence is located 50 bp upstream of the transcription initiation site which is essential for Nrf 2 transactivation. Using EMSA (electrophoretic mobility-shift assay) analysis we showed that zebrafish Nrf 2-MafK heterodimer specifically bound to this sequence. All the vertebrate Pi class GST genes harbour a similar ARE-like sequence in their promoter regions. We propose that this sequence is a conserved target site for Nrf 2 in the Pi class GST genes.

The Mediator Subunit MED16 Transduces NRF2-Activating Signals into Antioxidant Gene Expression.

The KEAP1-NRF2 system plays a central role in cytoprotection. NRF2 is stabilized in response to electrophiles and activates transcription of antioxidant genes. Although robust induction of NRF2 target genes confers resistance to oxidative insults, how NRF2 triggers transcriptional activation after binding to DNA has not been elucidated. To decipher the molecular mechanisms underlying NRF2-dependent transcriptional activation, we purified the NRF2 nuclear protein complex and identified the Mediator subunits as NRF2 cofactors. Among them, MED16 directly associated with NRF2. Disruption of Med16 significantly attenuated the electrophile-induced expression of NRF2 target genes but did not affect hypoxia-induced gene expression, suggesting a specific requirement for MED16 in NRF2-dependent transcription. Importantly, we found that 75% of NRF2-activated genes exhibited blunted inductions by electrophiles in Med16-deficient cells compared to wild-type cells, which strongly argues that MED16 is a major contributor supporting NRF2-dependent transcriptional activation. NRF2-dependent phosphorylation of the RNA polymerase II C-terminal domain was absent in Med16-deficient cells, suggesting that MED16 serves as a conduit to transmit NRF2-activating signals to RNA polymerase II. MED16 indeed turned out to be essential for cytoprotection against oxidative insults. Thus, the KEAP1-NRF2-MED16 axis has emerged as a new regulatory pathway mediating the antioxidant response through the robust activation of NRF2 target genes.

Establishment of Protocols for Global Metabolomics by LC-MS for Biomarker Discovery.

Metabolomics is a promising avenue for biomarker discovery. Although the quality of metabolomic analyses, especially global metabolomics (G-Met) using mass spectrometry (MS), largely depends on the instrumentation, potential bottlenecks still exist at several basic levels in the metabolomics workflow. Therefore, we established a precise protocol initially for the G-Met analyses of human blood plasma to overcome some these difficulties. In our protocol, samples are deproteinized in a 96-well plate using an automated liquid-handling system, and conducted either using a UHPLC-QTOF/MS system equipped with a reverse phase column or a LC-FTMS system equipped with a normal phase column. A normalization protocol of G-Met data was also developed to compensate for intra- and inter-batch differences, and the variations were significantly reduced along with our normalization, especially for the UHPLC-QTOF/MS data with a C18 reverse-phase column for positive ions. Secondly, we examined the changes in metabolomic profiles caused by the storage of EDTA-blood specimens to identify quality markers for the evaluation of the specimens' pre-analytical conditions. Forty quality markers, including lysophospholipids, dipeptides, fatty acids, succinic acid, amino acids, glucose, and uric acid were identified by G-Met for the evaluation of plasma sample quality and established the equation of calculating the quality score. We applied our quality markers to a small-scale study to evaluate the quality of clinical samples. The G-Met protocols and quality markers established here should prove useful for the discovery and development of biomarkers for a wider range of diseases.