• NF-Y Recruits Both Transcription Activator and Repressor to Modulate Tissue- and Developmental Stage-Specific Expression of Human γ-Globin Gene

      Zhu, Xingguo; Wang, Yongchao; Pi, Wenhu; Liu, Hui; Wickrema, Amittha; Tuan, Dorothy; Department of Biochemistry and Molecular Biology (2012-10-10)
      The human embryonic, fetal and adult β-like globin genes provide a paradigm for tissue- and developmental stage-specific gene regulation. The fetal γ-globin gene is expressed in fetal erythroid cells but is repressed in adult erythroid cells. The molecular mechanism underlying this transcriptional switch during erythroid development is not completely understood. Here, we used a combination of in vitro and in vivo assays to dissect the molecular assemblies of the active and the repressed proximal γ-globin promoter complexes in K562 human erythroleukemia cell line and primary human fetal and adult erythroid cells. We found that the proximal γ-globin promoter complex is assembled by a developmentally regulated, general transcription activator NF-Y bound strongly at the tandem CCAAT motifs near the TATA box. NF-Y recruits to neighboring DNA motifs the developmentally regulated, erythroid transcription activator GATA-2 and general repressor BCL11A, which in turn recruit erythroid repressor GATA-1 and general repressor COUP-TFII to form respectively the NF-Y/GATA-2 transcription activator hub and the BCL11A/COUP-TFII/GATA-1 transcription repressor hub. Both the activator and the repressor hubs are present in both the active and the repressed γ-globin promoter complexes in fetal and adult erythroid cells. Through changes in their levels and respective interactions with the co-activators and co-repressors during erythroid development, the activator and the repressor hubs modulate erythroid- and developmental stage-specific transcription of γ-globin gene.
    • Modulation of Dnmt3b function in vitro by interactions with Dnmt3L, Dnmt3a and Dnmt3b splice variants

      Van Emburgh, Beth O.; Robertson, Keith D.; Department of Biochemistry and Molecular Biology; GHSU Cancer Center (2011-07-4)
      DNA methylation, an essential regulator of transcription and chromatin structure, is established and maintained by the coordinated action of three DNA methyltransferases: DNMT1, DNMT3A and DNMT3B, and the inactive accessory factor DNMT3L. Disruptions in DNMT3B function are linked to carcinogenesis and genetic disease. DNMT3B is also highly alternatively spliced in a tissue- and disease-specific manner. The impact of intra-DNMT3 interactions and alternative splicing on the function of DNMT3 family members remains unclear. In the present work, we focused on DNMT3B. Using a panel of in vitro assays, we examined the consequences of DNMT3B splicing and mutations on its ability to bind DNA, interact with itself and other DNMT3's, and methylate DNA. Our results show that, while the C-terminal catalytic domain is critical for most DNMT3B functions, parts of the N-terminal region, including the PWWP domain, are also important. Alternative splicing and domain deletions also influence DNMT3Bâ s cellular localization. Furthermore, our data reveal the existence of extensive DNMT3B self-interactions that differentially impact on its activity. Finally, we show that catalytically inactive isoforms of DNMT3B are capable of modulating the activity of DNMT3Aâ DNMT3L complexes. Our studies therefore suggest that seemingly â inactiveâ DNMT3B isoforms may influence genomic methylation patterns in vivo.
    • A Monte Carlo test of linkage disequilibrium for single nucleotide polymorphisms

      Xu, Hongyan; George, Varghese; Department of Biostatistics and Epidemiology (2011-04-14)
      Background: Genetic association studies, especially genome-wide studies, make use of linkage disequilibrium(LD) information between single nucleotide polymorphisms (SNPs). LD is also used for studying genome structure and has been valuable for evolutionary studies. The strength of LD is commonly measured by r2, a statistic closely related to the Pearson's x2 statistic. However, the computation and testing of linkage disequilibrium using r2 requires known haplotype counts of the SNP pair, which can be a problem for most population-based studies where the haplotype phase is unknown. Most statistical genetic packages use likelihood-based methods to infer haplotypes. However, the variability of haplotype estimation needs to be accounted for in the test for linkage disequilibrium.
    • Metabolites of Purine Nucleoside Phosphorylase (NP) in Serum Have the Potential to Delineate Pancreatic Adenocarcinoma

      Vareed, Shaiju K.; Bhat, Vadiraja B.; Thompson, Christopher; Vasu, Vihas T.; Fermin, Damian; Choi, Hyungwon; Creighton, Chad J.; Gayatri, Sitaram; Lan, Ling; Putluri, Nagireddy; et al. (2011-03-23)
      Pancreatic Adenocarcinoma (PDAC), the fourth highest cause of cancer related deaths in the United States, has the most aggressive presentation resulting in a very short median survival time for the affected patients. Early detection of PDAC is confounded by lack of specific markers that has motivated the use of high throughput molecular approaches to delineate potential biomarkers. To pursue identification of a distinct marker, this study profiled the secretory proteome in 16 PDAC, 2 carcinoma in situ (CIS) and 7 benign patients using label-free mass spectrometry coupled to 1D-SDS-PAGE and Strong Cation-Exchange Chromatography (SCX). A total of 431 proteins were detected of which 56 were found to be significantly elevated in PDAC. Included in this differential set were Parkinson disease autosomal recessive, early onset 7 (PARK 7) and Alpha Synuclein (aSyn), both of which are known to be pathognomonic to Parkinson's disease as well as metabolic enzymes like Purine Nucleoside Phosphorylase (NP) which has been exploited as therapeutic target in cancers. Tissue Microarray analysis confirmed higher expression of aSyn and NP in ductal epithelia of pancreatic tumors compared to benign ducts. Furthermore, extent of both aSyn and NP staining positively correlated with tumor stage and perineural invasion while their intensity of staining correlated with the existence of metastatic lesions in the PDAC tissues. From the biomarker perspective, NP protein levels were higher in PDAC sera and furthermore serum levels of its downstream metabolites guanosine and adenosine were able to distinguish PDAC from benign in an unsupervised hierarchical classification model. Overall, this study for the first time describes elevated levels of aSyn in PDAC as well as highlights the potential of evaluating NP protein expression and levels of its downstream metabolites to develop a multiplex panel for non-invasive detection of PDAC.
    • Uracils at nucleotide position 9â 11 are required for the rapid turnover of miR-29 family

      Zhang, Zhuo; Zou, Jun; Wang, Guo-Kun; Zhang, Jun-Tao; Huang, Shuang; Qin, Yong-Wen; Jing, Qing; Department of Biochemistry and Molecular Biology (2011-02-1)
      MicroRNAs are endogenous small RNA molecules that regulate gene expression. Although the biogenesis of microRNAs and their regulation have been thoroughly elucidated, the degradation of microRNAs has not been fully understood. Here by using the pulseâ chase approach, we performed the direct measurement of microRNA lifespan. Five representative microRNAs demonstrated a general feature of relatively long lifespan. However, the decay dynamic varies considerably between these individual microRNAs. Mutation analysis of miR-29b sequence revealed that uracils at nucleotide position 9â 11 are required for its rapid decay, in that both specific nucleotides and their position are critical. The effect of uracil-rich element on miR-29b decay dynamic occurs in duplex but not in single strand RNA. Moreover, analysis of published data on microRNA expression profile during development reveals that a substantial subset of microRNAs with the uracil-rich sequence tends to be down-regulated compared to those without the sequence. Among them, Northern blotting shows that miR-29c and fruit fly bantam possess a relatively rapid turnover rate. The effect of uracil-rich sequence on microRNA turnover depends on the sequence context. The present work indicates that microRNAs contain sequence information in the middle region besides the sequence element at both ends.
    • TNFα Cooperates with IFN-γ to Repress Bcl-xL Expression to Sensitize Metastatic Colon Carcinoma Cells to TRAIL-mediated Apoptosis

      Liu, Feiyan; Hu, Xiaolin; Zimmerman, Mary; Waller, Jennifer L.; Wu, Ping; Hayes-Jordan, Andrea; Lev, Dina; Liu, Kebin; Department of Biochemistry and Molecular Biology; Department of Biostatistics and Epidemiology (2011-01-17)
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    • Amino acid derivatives are substrates or non-transported inhibitors of the amino acid transporter PAT2 (slc36a2)

      Ganapathy, Vadivel; Department of Biochemistry and Molecular Biology (2011-01)
      The H+-coupled amino acid transporter PAT2 (SLC36A2) transports the amino acids proline, glycine, alanine and hydroxyproline. A physiological role played by PAT2 in amino acid reabsorption in the renal proximal tubule is demonstrated by mutations in SLC36A2 that lead to an iminoglycinuric phenotype (imino acid and glycine uria) in humans. A number of proline, GABA and tryptophan derivatives were examined to determine if they function either as transported substrates or non-transported inhibitors of PAT2. The compounds were investigated following heterologous expression of rat PAT2 in Xenopus laevis oocytes. PAT2 function was characterised by: radiotracer uptake and competition (cis-inhibition) studies; radiotracer efflux and trans-stimulation; and measurement of substrate-induced positive inward current by two-electrode voltage-clamp. In general, the proline derivatives appeared to be transported substrates and the relative ability to induce current flow was closely related to the inhibitory effects on PAT2-mediated
    • Epigenetic mechanisms and genome stability

      Putiri, Emily L.; Robertson, Keith D.; Department of Biochemistry and Molecular Biology; GHSU Cancer Center (2010-12-29)
      Keywords: DNA methylation
    • IFN-c Upregulates Survivin and Ifi202 Expression to Induce Survival and Proliferation of Tumor-Specific T Cells

      Zimmerman, Mary; Yang, Dafeng; Hu, Xiaolin; Liu, Feiyan; Singh, Nagendra; Browning, Darren; Ganapathy, Vadivel; Chandler, Phillip; Choubey, Divaker; Abrams, Scott I.; et al. (2010-11-22)
      Background: A common procedure in human cytotoxic T lymphocyte (CTL) adoptive transfer immunotherapy is to expand tumor-specific CTLs ex vivo using CD3 mAb prior to transfer. One of the major obstacles of CTL adoptive immunotherapy is a lack of CTL persistence in the tumor-bearing host after transfer. The aim of this study is to elucidate the molecular mechanisms underlying the effects of stimulation conditions on proliferation and survival of tumor-specific CTLs.
    • Cj0596 is a periplasmic peptidyl prolyl cis-trans isomerase involved in Campylobacter jejuni motility, invasion, and colonization.

      Rathbun, Kimberly M; Hall, Johanna E; Thompson, Stuart A; Department of Biochemistry and Molecular Biology (2009-08-31)
      BACKGROUND: Campylobacter jejuni is a gastrointestinal pathogen of humans, but part of the normal flora of poultry, and therefore grows well at the respective body temperatures of 37 degrees C and 42 degrees C. Proteomic studies on temperature regulation in C. jejuni strain 81-176 revealed the upregulation at 37 degrees C of Cj0596, a predicted periplasmic chaperone that is similar to proteins involved in outer membrane protein folding and virulence in other bacteria. RESULTS: The cj0596 gene was highly conserved in 24 strains and species of Campylobacter, implying the importance of this gene. To study the role that Cj0596 plays in C. jejuni pathogenesis, a mutant derivative of strain 81-176 was constructed in which the cj0596 gene was precisely deleted. A revertant of this mutant was isolated by restoring the gene to its original chromosomal location using streptomycin counterselection. The cj0596 mutant strain demonstrated a slightly decreased growth rate and lower final growth yield, yet was more motile and more invasive of human intestinal epithelial cells than wild-type. In either single or mixed infections, the mutant was less able to colonize mice than 81-176. The cj0596 mutant also expressed altered levels of several proteins. CONCLUSION: Mutation of cj0596 has an effect on phenotypes related to C. jejuni pathogenesis, probably due to its role in the proper folding of critical outer membrane proteins.
    • Expression and localization of GPR109A (PUMA-G/HM74A) mRNA and protein in mammalian retinal pigment epithelium.

      Martin, Pamela M; Ananth, Sudha; Cresci, Gail A.; Roon, Penny; Smith, Sylvia B; Ganapathy, Vadivel; Department of Biochemistry and Molecular Biology; Department of Cellular Biology and Anatomy; Department of Ophthalmology; Department of Surgery (2009-02-19)
      PURPOSE: GPR109A has been identified as a G-protein-coupled receptor for niacin. beta-hydroxybutyrate (beta-HB) is a physiologic ligand for the receptor. beta-HB, the predominate ketone body in circulation, is an important energy source for neurons, including retinal neurons, under various physiologic and pathologic conditions. The identification of GPR109A as the receptor for beta-HB suggests additional, hitherto unknown, functions for this metabolite. The circulating levels of beta-HB increase in diabetes. Since retinopathy is a serious complication associated with diabetes, we investigated GPR109A expression in retina and in different retinal cell types to determine if the receptor may have a role in the pathophysiology of diabetic retinopathy. METHODS: RT-PCR, fluorescent in situ hybridization, and immunofluorescent techniques were used to analyze GPR109A expression in mouse retina and in three transformed retinal cell lines: ARPE-19 (RPE), RGC-5 (ganglion), and rMC-1 (M?�ller). Activation of GPR109A by niacin and beta-HB was demonstrated in ARPE-19 cells by cAMP assay. RESULTS: Studies conducted using mouse retinal tissues demonstrated that GPR109A is expressed in retina with its expression restricted to RPE, where it differentially polarizes to the basolateral membrane. These results were confirmed with cell lines, which demonstrated GPR109A expression in ARPE-19, but not in rMC-1 and RGC-5 cells. Primary cultures of mouse RPE also showed robust expression of GPR109A. cAMP assay demonstrated that GPR109A expressed in RPE is functional. CONCLUSIONS: These data represent the first report on GPR109A expression in retina. The exclusive expression of GPR109A in RPE basolateral membrane, which has access to beta-HB in blood, may have biologic importance in diabetic retinopathy.
    • Sodium-coupled monocarboxylate transporters in normal tissues and in cancer.

      Ganapathy, Vadivel; Thangaraju, Muthusamy; Gopal, Elangovan; Martin, Pamela M; Itagaki, Shiro; Miyauchi, Seiji; Prasad, Puttur D; Department of Biochemistry and Molecular Biology (2008-04-30)
      SLC5A8 and SLC5A12 are sodium-coupled monocarboxylate transporters (SMCTs), the former being a high-affinity type and the latter a low-affinity type. Both transport a variety of monocarboxylates in a Na(+)-coupled manner. They are expressed in the gastrointestinal tract, kidney, thyroid, brain, and retina. SLC5A8 is localized to the apical membrane of epithelial cells lining the intestinal tract and proximal tubule. In the brain and retina, its expression is restricted to neurons and the retinal pigment epithelium. The physiologic functions of SLC5A8 include absorption of short-chain fatty acids in the colon and small intestine, reabsorption of lactate and pyruvate in the kidney, and cellular uptake of lactate and ketone bodies in neurons. It also transports the B-complex vitamin nicotinate. SLC5A12 is also localized to the apical membrane of epithelial cells lining the intestinal tract and proximal tubule. In the brain and retina, its expression is restricted to astrocytes and M?�ller cells. SLC5A8 also functions as a tumor suppressor; its expression is silenced in tumors of colon, thyroid, stomach, kidney, and brain. The tumor-suppressive function is related to its ability to mediate concentrative uptake of butyrate, propionate, and pyruvate, all of which are inhibitors of histone deacetylases. SLC5A8 can also transport a variety of pharmacologically relevant monocarboxylates, including salicylates, benzoate, and gamma-hydroxybutyrate. Non-steroidal anti-inflammatory drugs such as ibuprofen, ketoprofen, and fenoprofen, also interact with SLC5A8. These drugs are not transportable substrates for SLC5A8, but instead function as blockers of the transporter. Relatively less is known on the role of SLC5A12 in drug transport.
    • A facilitated tracking and transcription mechanism of long-range enhancer function.

      Zhu, Xingguo; Ling, Jianhua; Zhang, Ling; Pi, Wenhu; Wu, Min; Tuan, Dorothy; Department of Biochemistry and Molecular Biology (2007-09-14)
      In the human epsilon-globin gene locus, the HS2 enhancer in the Locus Control Region regulates transcription of the embryonic epsilon-globin gene located over 10 kb away. The mechanism of long-range HS2 enhancer function was not fully established. Here we show that the HS2 enhancer complex containing the enhancer DNA together with RNA polymerase II (pol II) and TBP tracks along the intervening DNA, synthesizing short, polyadenylated, intergenic RNAs to ultimately loop with the epsilon-globin promoter. Guided by this facilitated tracking and transcription mechanism, the HS2 enhancer delivers pol II and TBP to the cis-linked globin promoter to activate mRNA synthesis from the target gene. An insulator inserted in the intervening DNA between the enhancer and the promoter traps the enhancer DNA and the associated pol II and TBP at the insulator site, blocking mid-stream the facilitated tracking and transcription mechanism of the enhancer complex, thereby blocking long-range enhancer function.