Liver Nuclear Proteins Research Articles

Age-related expression analysis of mouse liver nuclear protein binding to 3'-untranslated region of Period2 gene.

In mammals, both circadian rhythm and aging play important roles in regulating time-dependent homeostasis. We previously discovered an age-related increase element binding protein, hnRNP A3, which binds to the 3'-untranslated region (UTR) of blood coagulation factor IX (FIX). Here, we describe other members of this protein family, hnRNP C and hnRNP H, which bind to the 3'-UTR of the mouse circadian clock gene Period 2 (mPer2). RNA electrophoretic mobility shift assays using a (32)P-labeled Per2 RNA probe coupled with two-dimensional gel electrophoresis followed by MALDI-TOF/MS peptide mass fingerprint analysis was used to analyze these proteins. Western blotting suggested that the total expression of these proteins in mouse liver cell nuclei does not increase with age. Two-dimensional gel electrophoresis analysis of age-related protein expression showed that many isoforms of these proteins exist in the liver and that each protein exhibits a complex age-related expression pattern. These results suggest that many isoforms of proteins are regulated by different aging systems and that many age regulation systems function in the liver.

Functional analysis of four LDLR 5'UTR and promoter variants in patients with familial hypercholesterolaemia.

Familial hypercholesterolaemia (FH) is an autosomal dominant inherited disease characterised by increased low-density lipoprotein cholesterol (LDL-C) levels. The functionality of four novel variants within the LDLR 5′UTR and promoter located at c.-13A>G, c.-101T>C, c.-121T>C and c.-215A>G was investigated using in silico and in vitro assays, and a systemic bioinformatics analysis of all 36 reported promoter variants are presented. Bioinformatic tools predicted that all four variants occurred in sites likely to bind transcription factors and that binding was altered by the variant allele. Luciferase assay was performed for all the variants. Compared with wild type, the c.-101T>C and c.-121T>C variants showed significantly lower mean (±SD) luciferase activity (64±8 and 72±8%, all P<0.001), suggesting that these variants are causal of the FH phenotype. No significant effect on gene expression was seen for the c.-13A>G or c.-215A>G variants (96±15 and 100±12%), suggesting these variants are not FH causing. Similar results were seen for the c.-101T>C and c.-121T>C variants in lipid-depleted serum. However, a significant reduction in luciferase activity was seen in the c.-215A>G variant in lipid-depleted serum. Electrophoretic-mobility shift assays identified allele-specific binding of liver (hepatoma) nuclear proteins to c.-121T>C and suggestive differential binding to c.-101T>C but no binding to c.-215A>G. These data highlight the importance of in vitro testing of reported LDLR promoter variants to establish their role in FH. The functional assays performed suggest that the c.-101T>C and c.-121T>C variants are pathogenic, whereas c.-13A>G variant is benign, and the status of c.-215A>G remains unclear.

Differential localization of transferrin receptors and lipocalin-2 in rat hepatic and extra-hepatic organs during acute phase response

The transferrin receptors (TfR1, TfR2) and lipocalin-2 (LCN-2) are mainly involved in iron transport. We previously reported increasing amounts of liver cytoplasmic and nuclear iron content during acute phase response (ARP). Aim of the present study is to investigate intracellular localization of transferrin receptors and lipocalin-2 in liver compared to extra-hepatic organs under physiological and acute phase conditions. Immunohistochemistry was performed on 5 µm thick sections of hepatic and extra-hepatic tissues. Liver, spleen and heart cytoplasmic and nuclear proteins were used for Western blot analysis. By means of immunohistology TfR1 was detected in membranes and cytoplasm of liver and spleen cells while TfR2 showed nuclear expression mostly. In heart TfR1 and TfR2 both showed nuclear expression along with membranous and cytoplasmic localization, colocalization of TfR1 and TfR2 was also observed in spleen and heart. LCN-2 immuno-reactivity was detected in neutrophils and hepatocytes both. However, LCN-2 was detected in macrophages mostly, in spleen. Colocalization of LCN-2 with both transferrin receptors (TfR1, TfR2) was detected in hepatic and extra-hepatic tissues. The data presented demonstrate the differential and colocalization of transferrin receptors and LCN-2 in liver, spleen and heart. Western blot analysis of cytoplasmic and nuclear proteins from hepatic and extra-hepatic organs confirmed the immunihistology findings. Differential localization of iron transport proteins in different organs suggest their diverse role in these organs under physiological and pathological conditions. Colocalization of LCN2 with transferrin receptors clearly demonstrates its role in iron transport during acute phase conditions and presence of TfR1, TfR2 in cell nuclei demonstrate transport of iron to the cell nuclei needed for the integrity of life i.e. DNA repair, DNA replication and RNA metabolism.

Heterogeneous Nuclear Ribonucleoprotein A3 Is the Liver Nuclear Protein Binding to Age Related Increase Element RNA of the Factor IX Gene

BackgroundIn the ASE/AIE-mediated genetic mechanism for age-related gene regulation, a recently identified age-related homeostasis mechanism, two genetic elements, ASE (age-related stability element) and AIE (age-related increase element as a stem-loop forming RNA), play critical roles in producing specific age-related expression patterns of genes.Principal FindingWe successfully identified heterogeneous nuclear ribonucleoprotein A3 (hnRNP A3) as a major mouse liver nuclear protein binding to the AIE-derived RNAs of human factor IX (hFIX) as well as mouse factor IX (mFIX) genes. HnRNP A3 bound to the AIE RNA was not phosphorylated at its Ser359, while hnRNP A3 in the mouse liver nuclear extracts was a mixture of phosphorylated and unphosphorylated Ser359. HepG2 cells engineered to express recombinant hFIX transduced with adenoviral vectors harboring an effective siRNA against hnRNP A3 resulted in a substantial reduction in hFIX expression only in the cells carrying a hFIX expression vector with AIE, but not in the cells carrying a hFIX expression vector without AIE. The nuclear hnRNP A3 protein level in the mouse liver gradually increased with age, while its mRNA level stayed age-stable.ConclusionsWe identified hnRNP A3 as a major liver nuclear protein binding to FIX-AIE RNA. This protein plays a critical role in age-related gene expression, likely through an as yet unidentified epigenetic mechanism. The present study assigned a novel functional role to hnRNP A3 in age-related regulation of gene expression, opening up a new avenue for studying age-related homeostasis and underlying molecular mechanisms.

An age-related homeostasis mechanism is essential for spontaneous amelioration of hemophilia B Leyden

Regulation of age-related changes in gene expression underlies many diseases. We previously discovered the first puberty-onset gene switch, the age-related stability element (ASE)/age-related increase element (AIE)-mediated genetic mechanism for age-related gene regulation. Here, we report that this mechanism underlies the mysterious puberty-onset amelioration of abnormal bleeding seen in hemophilia B Leyden. Transgenic mice robustly mimicking the Leyden phenotype were constructed. Analysis of these animals indicated that ASE plays a central role in the puberty-onset amelioration of the disease. Human factor IX expression in these animals was reproducibly nullified by hypophysectomy, but nearly fully restored by administration of growth hormone, being consistent with the observed sex-independent recovery of factor IX expression. Ets1 was identified as the specific liver nuclear protein binding only to the functional ASE, G/CAGGAAG, and not to other Ets consensus elements. This study demonstrates the clinical relevance of the first discovered puberty-onset gene switch, the ASE/AIE-mediated regulatory mechanism.

Solution Structure of the DNA Binding Domain of Rice Telomere Binding Protein RTBP1,

RTBP1 is a rice telomeric protein that binds to the duplex array of TTTAGGG repeats at chromosome ends. The DNA binding domain of RTBP1 contains a Myb-type DNA binding motif and a highly conserved C-terminal Myb extension that is unique to plant telomeric proteins. Using an electrophoretic mobility shift assay, we identified the C-terminal 110-amino acid region (RTBP1(506-615)) as the minimal telomeric DNA binding domain, suggesting that the Myb extension is required for binding plant telomeric DNA. Like other telomeric proteins such as human TRF1 and yeast Rap1, RTBP1 induced a DNA bending in the telomeric repeat sequence, suggesting that RTBP1 may play a role in establishing and/or maintaining an active telomere configuration in vivo. To elucidate the DNA binding mode of RTBP1, we determined the three-dimensional structure of RTBP1(506-615) in solution by NMR spectroscopy. The overall structure of RTBP1(506-615) is composed of four alpha-helices and stabilized by three hydrophobic patches. The second and third helices in RTBP1 form a helix-turn-helix motif that interacts directly with DNA. The fourth helix located in the Myb extension is essential for binding to telomeric DNA via stabilization of the overall structure of the RTBP1 DNA binding domain. When DNA bound to RTBP1(506-615), large chemical shift perturbations were induced in the N-terminal arm, helix 3, and the loop between helices 3 and 4. These results suggest that helix 3 functions as a sequence-specific recognition helix while the N-terminal arm stabilizes the DNA binding.

Global Analyses of Age-Related Expression Profiles of Mouse Liver Proteins and Database Construction

Toward making an integrated understanding of age-related homeostasis, we carried out global analyses of age-related expression profiles of mouse liver proteins. Liver protein samples prepared from nuclear, cytoplasm and mitochondria fractions of mice (C57BL/6xSJL, male) at 1,3, 6, 12, 18, 21, and 24 months of age (n=10-20/age point) were subjected to quantitative analyses by 2DE (pH range 4-11) and MALDI-TOF/MS. For nuclear protein fraction, approximately 8000 protein spots separated on 2DE were analyzed by MALDI-TOF/MS for, and 4547 protein spots were identified by MASCOT protein identifier program with reasonable scores. After removing duplicated spots, 3113 protein spots were found unique, composed of 2534 single protein spots and 579 mixture protein spots. Single protein spots were subjected to quantitative analyses with a PDQuest program, generating agerelated expression profiles. GeneSpring software was then used for clustering and filtering analyses of the profiles. In addition to many complex age-related expression profiles, about a dozen unique and fundamental age-related profiles were identified. Many isomers, likely generated mostly by posttranslational modifications and/or by alternative splicings were found for about 40% of single protein spots. These findings suggested that there exist multiple, but a relatively small number of basic age-related regulatory mechanisms for liver nuclear proteins. These fundamental mechanisms may independently and/or in various combinations function, generating many complex age-related regulatory patterns. Similar studies have also been completed for cytosolic proteins. Analyses on mitochondrial proteins and female liver proteins are under progress. Continued

Differential regulation of human hepatic flavin containing monooxygenase 3 ( FMO3) by CCAAT/enhancer-binding protein β (C/EBPβ) liver inhibitory and liver activating proteins

Flavin-containing monooxygenase 3 (FMO3) is important for oxidative xenobiotic metabolism, but regulation of the FMO3 gene remains poorly understood. FMO3 is not expressed in HepG2 cells, a commonly employed model for hepatic gene regulation studies. Transcription factor transient expression and treatment with histone deacetylase or DNA methylase inhibitors identified decreased hepatic nuclear factor (HNF) 4α levels and DNA hypermethylation as mechanisms suppressing HepG2 FMO3 expression. The absence of major deficiencies in transcriptional machinery suggested that within limits, the HepG2 model is suitable for the study of FMO3 regulation. DNA–protein binding studies with HepG2 cell and hepatic tissue nuclear protein extracts and reporter construct transient expression experiments were performed to characterize FMO3 sequences from position −494 to −439 (domain I), previously demonstrated to significantly impact promoter function. Although both HNF3β and CCAAT enhancer-binding protein (C/EBP) were observed to specifically interact with this element using HepG2 cell nuclear proteins, only C/EBP DNA–protein interactions were observed using adult liver nuclear proteins. No specific DNA/protein interactions were observed using fetal liver nuclear proteins. Mutation of a putative HNF3β element had no effect on FMO3 promoter activity, while mutagenesis of a distinct, but overlapping C/EBP element resulted in a 55% reduction in activity. Furthermore, promoter activity was regulated as a function of defined C/EBPβ liver activating protein:liver inhibitory protein ratios through this same element. Chromatin immunoprecipitation demonstrated C/EBPβ binding to the FMO3 domain I element in intact cells and adult liver tissue. These results are consistent with C/EBPβ being important for regulating hepatic FMO3 expression.

Piperonyl butoxide activates c-Jun and ATF-2 in the hepatocytes of mice

In order to clarify the possible mechanism of hepatocarcinogenesis induced by piperonyl butoxide, we attempted to identify the transcription factor activated by piperonyl butoxide in the male ICR mouse liver. Administration of 0.6% piperonyl butoxide for 24 h elevated the level of liver nuclear proteins that bind to an AP-1 consensus oligonucleotide, and these proteins demonstrated a supershift with the anti-c-Jun antibody. Additionally, immunoblot analysis revealed that piperonyl butoxide induced c-Jun phosphorylation within 8 h of administration, and phosphorylated ATF-2 was detected after 24 h of piperonyl butoxide treatment. Immunohistochemical analysis also demonstrated the presence of phosphorylated ATF-2 in the hepatocyte nuclei of mice fed with 0.6% piperonyl butoxide for 24 h. Furthermore, piperonyl butoxide induced ATF-2 phosphorylation in TLR-3, a mouse immortalized hepatocyte cell line. These results indicated that piperonyl butoxide activated c-Jun and ATF-2 in mouse hepatocytes during the early stage of hepatocarcinogenesis.

Dexamethasone treatment affects nuclear glucocorticoid receptor and glucocorticoid response element binding activity in liver of rats ( Rattus norvegicus) during aging

Aging is associated with marked changes in the biochemical processes of many organs. Basal and glucocorticoid induced of liver nuclear glucocorticoid receptor (GR) on the level of protein expression and DNA-binding activity were investigated at different ages (3, 6, 12, 18 and 24 months old) in two groups of rats in: untreated and dexamethasone treated. The results showed a significant decline of GR protein immunopurified from untreated rats of advanced age. In dexamethasone-treated rats, the quantity of GR protein was lower than in controls at all ages. The interactions of liver nuclear proteins with radioactively labelled synthetic oligonucleotide analogue containing consensus GRE sequence were analysed during aging. The results showed that GRE binding activity demonstrated a decrease both in untreated and in dexamethasone treated rats. However, relative to untreated rats, dexamethasone treatment resulted in a significant increase in GRE binding at all ages, except that of three months old animals. In conclusion, the observed alterations in GR protein expression and its DNA binding activity may play a role in the changes of the cell response to glucocorticoid during aging.

Replication-deficient Adenovirus Induces Host Topoisomerase I Activity: Implications for Adenovirus-mediated Gene Expression

Replication-deficient adenoviruses are useful vectors for the transfer of therapeutic transgenes to malignant and non-malignant tissues. Yet their clinical application is limited by the potential toxicity of viral infection and the transient nature of transgene expression. Although transgene expression from adenovirus vectors is initially higher than expression of transgenes transduced by other viral or non-viral vectors, it is often insufficient to generate a significant therapeutic effect. We addressed this issue by searching for DNA-targeted viral-induced host responses potentially restricting transgene expression. Nuclear protein extracts from livers of rats systemically infected with replication-deficient adenovirus exhibited enhanced topoisomerase I activity compared with extracts from uninfected animals. Consequently, the inhibition of topoisomerase I by the anti-cancer drug topotecan greatly enhanced transgene expression in adenovirus-infected hepatic cells, colon cancer and prostate cancer cell cultures, mouse liver, human ex vivo tumor specimens, and mouse tumor in vivo. The enhancement could not be ascribed to non-specific genotoxic stress, cell death, or cell-cycle perturbation. These findings are significant for gene therapy as they reveal novel aspects of the host anti-adenovirus response and set the stage for the development of a rational molecular-pharmacological approach to increase the effectiveness, and safety, of adenovirus-mediated cancer therapeutics.

Mechanisms Regulating Human FMO3 Transcription

Flavin-containing monooxygenases (FMOs) are important oxidative drug metabolizing enzymes. FMO3 is the primary human adult liver FMO enzyme, but is developmentally regulated. FMO3 promoter characterization using in vitro DNA binding assays with HepG2 cell and fetal and adult liver nuclear protein, as well as FMO3/reporter construct transient expression in HepG2 cells, provided evidence for specific mechanisms contributing to both developmental and constitutive adult regulation. NFY, USF1, an unidentified GC box binding protein, and YY1 appear to play major roles regulating constitutive FMO3 transcription, while Pbx2 as a heterodimer with an unidentified Hox isoform also may contribute to FMO3 developmental expression.

Age-Related Homeostasis: Molecular Mechanism, Disease and Predictions from Global Analyses of Mouse Liver Proteins.

We previously reported the first molecular mechanism underlying the age-related homeostasis, ASE/AIE-mediated genetic mechanism for age-related regulation of gene expression, which turned out to be a mechanism of puberty-onset gene switching, specifically controlling a group of genes for their expression from puberty into old age (Kurachi & Kurachi J Thromb Haemost 2005; Zahng et al J Biol Chem 277:4532, 2002; Kurachi et al Science 285:739, 1999). This led us to successful construction of a transgenic mouse model of hemophilia B Leyden, a unique subset of hemophilia B with its mechanism being remained mysterious, robustly mimicking its unusual pattern of puberty-onset spontaneous amelioration. With this background, we hypothesized that besides the ASE/AIE-mediated mechanism, there exist more unidentified fundamental regulatory mechanisms for age-related homeostasis, which individually or in various combinations generate age-related complex and dynamic regulatory patterns of liver proteins. We launched a series of global and quantitative analysis of age-related changes in expression of liver nuclear proteins of mice (C57BL/6xSJL, [male], 1 through-24 month of age) by taking a procedure composed of two-dimensional gel electrophoresis (2DE) for separation and quantification of liver nuclear protein spots and of MALDI-TOF/MS PMF analyses to identify proteins in the spots. Out of over 6000 spots recognized and quantified in 2DE, 4547 protein spots were subjected to MALDI-TOF/MS analysis for protein identification. Finally, 2765 protein spots including many isomers were found unique. Systematic analyses of their age-related expression identified several major phases in protein expression throughout the lifespan. These findings supported our hypothesis that there exist multiple novel molecular mechanisms responsible for maintaining age-related homeostasis. The comprehensive liver nuclear protein data set was then used to construct a comprehensive database, which allows rapid and reliable searches for expression of specific proteins, their age-related dynamic profiles, isomers, protein identification from 2DE image, and other related information. This will serve as a valuable platform resource for studying epigenetic challenges, evaluation of drugs as well as gaining further insights into the molecular mechanisms underlying age-related homeostasis.

Analysis of nuclear proteome in C57 mouse liver tissue by a nano‐flow 2‐D‐LC–ESI‐MS/MS approach

The analysis of whole cell or tissue extracts is too complex for current protein identification technology and not suitable for the study of proteins with low copy levels. To concentrate and enrich low abundance proteins, organelle proteomics is a promising strategy. This approach can not only reduce the protein sample complexity but also provide information about protein location in cells, organs, or tissues under analysis. Nano-flow two-dimensional strong-cation exchange chromatography (SCX)-RPLC-ESI-MS/MS is an ideal platform for analyzing organelle extracts because of its advantages of sample non-bias, low amounts of sample required, powerful separation capability, and high detection sensitivity. In this study, we apply nano-scale multidimensional protein identification technology to the analysis of C57 mouse liver nuclear proteins. Organelle isolation has been optimized to obtain highly pure nuclei. Evaluation of nucleus integrity and purity has been performed to demonstrate the effectiveness of the optimized isolation procedure. The extracted nuclear proteins were identified by five independent nano-flow on-line SCX-RPLC-ESI-MS/MS analyses to improve the proteome coverage. Finally, a total of 462 proteins were identified. Corresponding analyses of protein molecular mass and pI distribution and biological function categorization have been undertaken to further validate our identification strategy.

Functional Analysis of a Metal Response Element in the Regulatory Region of Flounder Cytochrome P450 1A and Implications for Environmental Monitoring of Pollutants

Cytochrome P450 1A (CYP 1A) is a member of a multigene family of xenobiotic metabolizing enzymes. CYP 1A is highly inducible by numerous environmental contaminants including polycyclic aromatic hydrocarbons (PAHs) and is widely used in biomonitoring studies. Therefore, understanding the regulation of this gene is important for accurate interpretation of biomarker data. We describe here the functional role of a metal response element (MRE) in the European flounder CYP 1A promoter region. To help elucidate the potential role of this MRE, reporter gene constructs, with or without site-directed mutagenesis, were used in conjunction with a dual-luciferase assay. The electrophoretic mobility shift assay (EMSA) was also used to investigate potential protein binding at this MRE site. Treatment with the prototypical PAH 3-methylcholanthrene (3MC) (1.0 microM) produced a dose-dependent response at the CYP 1A promoter, whereas treatment with cadmium (0-1.0 microM) produced little transcriptional activity at either the wild-type or mutated promoter. Cotreatment with cadmium (1.0 microM) and 3MC (1.0 microM) reduced induction at this promoter to 1.83-fold compared to 3MC treatment alone (4.0-fold induction). Mutation of the MRE site resulted in abolishment of this cadmium-related loss of 3MC-dependent activity. Furthermore, a retarded band was observed in the EMSA when the MRE was used as a probe and incubated with liver nuclear protein from flounder treated with cadmium. The results not only add to knowledge of the diversity in vertebrate CYP 1A regulation but also raise the complexity of interpretation of CYP 1A induction in monitoring studies that involve mixtures of PAHs and metals.

Molecular properties and intracellular localization of rat liver nuclear scaffold protein P130

We examined the molecular basis of rat P130, a nuclear scaffold protein, and its functions. P130 comprising 845 amino acid residues possesses several functional domains and yields an electrophoretically distinctive isoform, P123, by altering its phosphorylation status in association with translocation across the nuclear membrane and from the digitonin-extractable fraction of the nucleus to the nuclear scaffold. The functional domains, NLS, NES, and zinc-finger bearing DNA-binding domains, ZF1 and ZF2, aid these translocations. P130 binds RNA through two RNA-binding domains (RB1 and RB2) similar to those of hnRNPs I and L. Microsome- and polysome-localized P130 and P123 were found in rat liver and Ac2F hepatoma cells. This localization required prior entry of P130 to the nucleus, but did not require RB1 and RB2. Thus, P130 initially purified from rat liver nuclear scaffold has the potential to play a variety of roles in biological events not only in the nuclear scaffold but also in various subcellular compartments. P130 (AB205483) is identical to matrin 3 (M63485 and BC062231), although the primary structure of rat matrin 3 has been revised, since it was first published.

Identification of zinc finger binding protein 89 (ZBP-89) as a transcriptional activator for a major bovine growth hormone receptor promoter

The objective of this study was to identify the transcription factors that regulate the expression of growth hormone receptor (GHR) 1A mRNA, a major GHR mRNA variant in the bovine liver. A deoxyribonuclease I footprint analysis revealed that the GHR1A promoter region −69 to −30 (relative to the transcription start site for GHR1A mRNA) contained binding sites for bovine liver nuclear proteins. Using a yeast one-hybrid analysis, zinc finger binding protein 89 (ZBP-89) was identified as a binding protein to this promoter region. Binding of ZBP-89 to the GHR1A promoter region −69 to −30 was further confirmed by an electrophoretic mobility shift assay. In cotransfection analyses, overexpression of ZBP-89 enhanced ( P < 0.01) the activity of the GHR1A promoter and this enhancement was dependent on the putative ZBP-89 binding site in the promoter. These results together indicate that ZBP-89 is a transcription factor that regulates the expression of GHR1A mRNA.

Age Related Expression Patterns of Mouse Liver Nuclear Proteins.

Previously we reported the discovery of the ASE/AIE-type age related regulatory mechanism of gene expression, the first molecular mechanism for age dimension homeostasis (Kurachi et al, Science 1999; Zhang et al, J Biol Chem 2002; Kurachi & Kurachi J Thromb Haemost 2005). Here we report a comprehensive analysis of the age associated changes of mouse liver nuclear proteins, an experimental model for studying the dynamic regulatory mechanisms of the age-dimension homeostasis. Liver nuclei obtained from mice (C57BL/6xSJL, male) at 1, 3, 6, 12, 18 and 21 months of age (n=10–20/age) were used for preparing protein extracts. The protein extracts were then subjected to 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) (pH range of 4–11) and protein identification by MALDI-TOF/MS. As analyzed by the PDQuest program combined with Mascot Protein Identifier, out of over 5000 protein spots identified by 2D-PAGE, approximately 2800 protein spots were found unique including isomers. Intensity changes of these protein spots along the age axis were determined and analyzed in details with the PDQuest software. Twelve proteins were found to show no significant changes in their intensity throughout the life-span (smaller than 20% fluctuation). Besides some complex patterns, at least 6 unique age-related patterns were found, including those of puberty-onset increase, puberty-onset decrease, puberty-onset increase up to 6 months of age followed by decrease, unique increase only at around 12 months of age, ageing associated increase or decrease. Age related pattern changes were observed for some isomers among many, which were likely generated by differential posttranslational modifications or conformational changes. These findings support that in addition to the ASE/AIE type mechanism, there exist multiple novel age-dimension regulatory mechanisms, and their various combinations are responsible for producing complex and subtle age related regulatory patterns for a large number of genes. This is the first comprehensive analysis of the liver nuclear proteins in relation to age, and would provide a valuable basis for studying many age related phenomena, diseases as well as epigenetic effects.

Proteome analysis of a rat liver nuclear insoluble protein fraction and localization of a novel protein, ISP36, to compartments in the interchromatin space

A rat liver nuclear insoluble protein fraction was analyzed to investigate candidate proteins participating in nuclear architecture formation. Proteins were subjected to two-dimensional separation by reversed-phase HPLC in 60% formic acid and SDS/PAGE. The method produced good resolution of insoluble proteins. One hundred and thirty-eight proteins were separated, and 28 of these were identified. The identified proteins included one novel protein, seven known nuclear proteins and 12 known nuclear matrix proteins. The novel 36 kDa protein was further investigated for its subnuclear localization. The human ortholog of the protein was expressed in Escherichia coli and antibodies were raised against the recombinant protein. Exclusive localization of the protein to the nuclear insoluble protein fraction was confirmed by cell fractionation followed by immunoblotting. Immunostaining of mouse C3H cells suggested that the 36 kDa protein was a constituent of an insoluble macromolecular complex spread throughout the interchromatin space of the nucleus. The protein was designated 'interchromatin space protein of 36 kDa', ISP36.

Silencing of human methionine adenosyltransferase 1A expression by methylation of the coding region

Two genes (MAT1A and MAT2A) encode for the essential enzyme methionine adenosyltransferase (MAT). MAT1A is silenced in hepatocellular carcinoma (HCC), and absence of MAT1A leads to spontaneous development of HCC in mice. Here we investigated the role of methylation in regulating MAT1A expression. There are three MspI/HpaII sites from -1913 to +160 of the human MAT1A gene (numbered relative to the translational start site) at position -977, +10, and +88. Bisulfite treatment and DNA sequencing, and Southern blot analysis showed that methylation at +10 and +88, but not -977, correlated with lack of MAT1A expression. MAT1A promoter construct methylated at -977, +10 or +88 position has 0.7-fold, 3-fold, and 1.6-fold lower promoter activity, respectively. Methylation at -977 and +10 did not inhibit the promoter more than methylation at +10 alone; while methylation at +10 and +88 resulted in a 6-fold reduction of promoter activity. The promoter activity is not affected if these sites are mutated and cannot be methylated. Reactivation of MAT1A correlated with demethylation of +10 and +88. DNase I footprinting analysis using the probe containing nucleotides -346 to +160 and human liver nuclear extract or recombinant TATA binding protein (TBP) showed that HpaII methylation at +10 and +88 prevented TBP binding to the TATA box, but not if the sites were mutated. ChIP analysis confirmed TBP binding to MAT1A only in MAT1A expressing cells. Collectively our data support the novel finding that methylation of the MAT1A coding region can influence TBP binding to the TATA box and shut down gene transcription.