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Luquan Wang

Publications and source records attributed to Luquan Wang.

17 recordsLinked to original sources
PubMed2003

Global transcriptional program of p53 target genes during the process of apoptosis and cell cycle progression.

The temporal gene expression profile during the entire process of apoptosis and cell cycle progression in response to p53 in human ovarian cancer cells was explored with cDNA microarrays representing 33 615 individual human genes. A total of 1501 genes (4.4%) were found to respond to p53 (approximately 80% of these were repressed by p53) using 2.5-fold change as a cutoff. It was anticipated that most of p53 responsive genes resulted from the secondary effect of p53 expression at late stage of apoptosis. To delineate potential p53 direct and indirect target genes during the process of apoptosis and cell cycle progression, microarray data were combined with global p53 DNA-binding site analysis. Here we showed that 361 out of 1501 p53 responsive genes contained p53 consensus DNA-binding sequence(s) in their regulatory region, approximately 80% of which were repressed by p53. This is the first time that a large number of p53-repressed genes have been identified to contain p53 consensus DNA-binding sequence(s) in their regulatory region. Hierarchical cluster analysis of these genes revealed distinct temporal expression patterns of transcriptional activation and repression by p53. More genes were activated at early time points, while more repressed genes were found after the onset of apoptosis. A small-scale quantitative chromatin immunoprecipitation analysis indicated that in vivo p53-DNA interaction was detected in eight out of 10 genes, most of which were repressed by p53 at the early onset of apoptosis, suggesting that a portion of p53 target genes in the human genome could be negatively regulated by p53 via sequence-specific DNA binding. The approaches and genes described here should aid the understanding of global gene regulatory network of p53.

Adenoviridae↗
PubMed2003

Identification and characterization of a novel RF-amide peptide ligand for orphan G-protein-coupled receptor SP9155.

Orphan G-protein-coupled receptors are a large class of receptors whose cognate ligands are unknown. SP9155 (also referred to as AQ27 and GPR103) is an orphan G-protein-coupled receptor originally cloned from a human brain cDNA library. SP9155 was found to be predominantly expressed in brain, heart, kidney, retina, and testis. Phylogenetic analysis shows that SP9155 shares high homology with Orexin, NPFF, and cholecystokinin (CCK) receptors, but identification of the endogenous ligand for SP9155 has not been reported. In this study, we have used a novel method to predict peptides from genome data bases. From these predicted peptides, a novel RF-amide peptide, P52 was shown to selectively activate SP9155-transfected cells. We subsequently cloned the precursor gene of the P52 ligand and characterized the activity of other possible peptides encoded by the precursor. This revealed an extended peptide, P518, which exhibited high affinity for SP9155 (EC50 = 7 nm). mRNA expression analysis revealed that the peptide P518 precursor gene is predominantly expressed in various brain regions, coronary arteries, thyroid and parathyroid glands, large intestine, colon, bladder, testes, and prostate. These results indicate the existence of a novel RF-amide neuroendocrine peptide system, and suggest that SP9155 is likely the relevant G-protein-coupled receptor for this peptide.

Algorithms↗
PubMed2003

Genomic targets of the human c-Myc protein.

The transcription factor Myc is induced by mitogenic signals and regulates downstream cellular responses. If overexpressed, Myc promotes malignant transformation. Myc modulates expression of diverse genes in experimental systems, but few are proven direct targets. Here, we present a large-scale screen for genomic Myc-binding sites in live human cells. We used bioinformatics to select consensus DNA elements (CACGTG or E-boxes) situated in the 5' regulatory region of genes and measured Myc binding to those sequences in vivo by quantitative chromatin immunoprecipitation. Strikingly, most promoter-associated E-boxes showed selective recovery with Myc, unlike non-E-box promoters or E-boxes in bulk genomic DNA. Promoter E-boxes were distributed in two groups bound by Myc at distinct frequencies. The high-affinity group included an estimated 11% of all cellular loci, was highly conserved among different cells, and was bound independently of Myc expression levels. Overexpressed Myc associated at increased frequency with low-affinity targets and, at extreme levels, also with other sequences, suggesting that some binding was not sequence-specific. The strongest DNA-sequence parameter defining high-affinity targets was the location of E-boxes within CpG islands, correlating with an open, preacetylated state of chromatin. Myc further enhanced histone acetylation, with or without accompanying induction of mRNA expression. Our findings point to a high regulatory and biological diversity among Myc-target genes.

Cell Line↗
PubMed2003

Gene expression profiles and transcription factors involved in parathyroid hormone signaling in osteoblasts revealed by microarray and bioinformatics.

Parathyroid hormone (PTH) binds to its receptor PTH1R (parathyroid hormone 1 receptor) in osteoblastic cells to regulate bone remodeling and calcium homeostasis. While prolonged exposure to PTH causes increased bone resorption, intermittent injections of PTH have an anabolic effect on bone. The molecular mechanisms regulating these processes are still largely unknown. Here, we present our results on gene expression profile changes in the PTH-treated osteoblastic cell line, UMR 106-01, using DNA microarray analysis. A total of 125 known genes and 30 unknown expressed sequence tags (ESTs) were found to have at least 2-fold expression changes after PTH treatment at 4, 12, and 24 h. 14 genes were previously known to be PTH-regulated but many were unknown to be regulated by PTH prior to our experiments. Real-time reverse transcriptase-PCR confirmed that 90 and 50% of the genes are regulated more than 2-fold by PTH in UMR 106-01 and rat primary osteoblastic cells, respectively. Most genes belong to the following protein families: hormones, growth factors, and receptors; signal transduction pathway proteins; transcription factors; proteases; metabolic enzymes; structural and matrix proteins; transporters; etc. These results provide a comprehensive and deeper knowledge about PTH regulation of osteoblastic gene expression. Next, we designed a computational method to extract information about transcription factors likely involved in regulating these genes. These factors include those previously known to be involved in PTH signaling (AP-1 and the cAMP response element-binding protein), those that were identified by microarray data (C/EBP), and some novel transcription factors (AP-2, AP-4, SP1, FoxD3, etc.). Our results suggest that a reliable bioinformatics approach can be easily applied for other systems.

Animals↗
PubMed2003

Comparative promoter analysis and its application in analysis of PTH-regulated gene expression.

Taking advantage of the "working draft" of the human genome and the MIT shotgun assembly of the mouse genome, we performed a comparative promoter analysis of human RefSeq mRNA (sequences from GenBank's RefSeq database). By combining this analysis with a transcription factor (TF) binding site analysis using a TRANSFAC position weight matrix (PWM) search, 86% of non-specific TF sites were removed. Using a set of genes that are regulated by parathyroid hormone (PTH), a statistical analysis was performed on the conserved TF binding sites among a set of eight human and mouse genes. From among the eight genes tested, we obtained a set of 31 TFs, suggesting possible roles for associated genes in PTH-mediated pathways. All three known PTH-responsive TFs (AP1, RUNX2, CREB) were correctly predicted by this analysis as well as two other potential TFs (VDR and CEBP Delta). Additionally, a model was made to describe the TF site characteristic module of PTH-regulated genes. This model was then used to search all human RefSeq gene promoters with established human-mouse ortholog relationships to identify other PTH-regulated genes. This comparative approach combined with statistical analysis proved to be sufficiently specific to decipher critical TFs involved in PTH-regulated pathways.

Algorithms↗
PubMed2002

Domain-oriented functional analysis based on expression profiling.

BACKGROUND: Co-regulation of genes may imply involvement in similar biological processes or related function. Many clusters of co-regulated genes have been identified using microarray experiments. In this study, we examined co-regulated gene families using large-scale cDNA microarray experiments on the human transcriptome. RESULTS: We present a simple model, which, for each probe pair, distills expression changes into binary digits and summarizes the expression of multiple members of a gene family as the Family Regulation Ratio. The set of Family Regulation Ratios for each protein family across multiple experiments is called a Family Regulation Profile. We analyzed these Family Regulation Profiles using Pearson Correlation Coefficients and derived a network diagram portraying relationships between the Family Regulation Profiles of gene families that are well represented on the microarrays. Our strategy was cross-validated with two randomly chosen data subsets and was proven to be a reliable approach. CONCLUSION: This work will help us to understand and identify the functional relationships between gene families and the regulatory pathways in which each family is involved. Concepts presented here may be useful for objective clustering of protein functions and deriving a comprehensive protein interaction map. Functional genomic approaches such as this may also be applicable to the elucidation of complex genetic regulatory networks.

Amino Acid Sequence↗
PubMed2002

A reference database for tumor-related genes co-expressed with interleukin-8 using genome-scale in silico analysis.

BACKGROUND: The EST database provides a rich resource for gene discovery and in silico expression analysis. We report a novel computational approach to identify co-expressed genes using EST database, and its application to IL-8. RESULTS: IL-8 is represented in 53 dbEST cDNA libraries. We calculated the frequency of occurrence of all the genes represented in these cDNA libraries, and ranked the candidates based on a Z-score. Additional analysis suggests that most IL-8 related genes are differentially expressed between non-tumor and tumor tissues. To focus on IL-8's function in tumor tissues, we further analyzed and ranked the genes in 16 IL-8 related tumor libraries. CONCLUSIONS: This method generated a reference database for genes co-expressed with IL-8 and could facilitate further characterization of functional association among genes.

Journal Article↗
PubMed2002

Hepatitis C virus whole genome position weight matrix and robust primer design.

BACKGROUND: The high degree of sequence heterogeneity found in Hepatitis C virus (HCV) isolates, makes robust nucleic acid-based assays difficult to generate. Polymerase chain reaction based techniques, require efficient and specific sequence recognition. Generation of robust primers capable of recognizing a wide range of isolates is a difficult task. RESULTS: A position weight matrix (PWM) and a consensus sequence were built for each region of HCV and subsequently assembled into a whole genome consensus sequence and PWM. For each of the 10 regions, the number of occurrences of each base at a given position was compiled. These counts were converted to frequencies that were used to calculate log odds scores. Using over 100 complete and 14,000 partial HCV genomes from GenBank, a consensus HCV genome sequence was generated along with a PWM reflecting heterogeneity at each position. The PWM was used to identify the most conserved regions for primer design. CONCLUSIONS: This approach allows rapid identification of conserved regions for robust primer design and is broadly applicable to sets of genomes with all levels of genetic heterogeneity.

Conserved Sequence↗
PubMed2002

Human members of the eukaryotic protein kinase family.

BACKGROUND: Eukaryotic protein kinases (EPKs) constitute one of the largest recognized protein families represented in the human genome. EPKs, which are similar to each other in sequence, structure and biochemical properties, are important players in virtually every signaling pathway involved in normal development and disease. Near completion of projects to sequence the human genome and transcriptome provide an opportunity to identify and perform sequence analysis on a nearly complete set of human EPKs. RESULTS: Publicly available genetic sequence data were searched for human sequences that potentially represent EPK family members. After removal of duplicates, splice variants and pseudogenes, this search yielded 510 sequences with recognizable similarity to the EPK family. Protein sequences of putative EPK catalytic domains identified in the search were aligned, and a phonogram was constructed based on the alignment. Representative sequence records in GenBank were identified, and derived information about gene mapping and nomenclature was summarized. CONCLUSIONS: This work represents a nearly comprehensive census and early bioinformatics overview of the EPKs encoded in the human genome. Evaluation of the sequence relationships between these proteins contributes contextual information that enhances understanding of individual family members. This curation of human EPK sequences provides tools and a framework for the further characterization of this important class of enzymes.

Binding Sites↗
PubMed2002

Transcriptional regulation during p21WAF1/CIP1-induced apoptosis in human ovarian cancer cells.

In this study we used adenovirus vector-mediated transduction of either the p53 gene (rAd-p53) or the p21(WAF1/CIP1) gene (rAd-p21) to mimic both p53-dependent and -independent up-regulation of p21(WAF1/CIP1) within a human ovarian cancer cell line, 2774, and the derivative cell lines, 2774qw1 and 2774qw2. We observed that rAd-p53 can induce apoptosis in both 2774 and 2774qw1 cells but not in 2774qw2 cells. Surprisingly, overexpression of p21(WAF1/CIP1) also triggered apoptosis within these two cell lines. Quantitative reverse transcription-PCR analysis revealed that the differential expression of BAX, BCL2, and caspase 3 genes, specific in rAd-p53-induced apoptotic cells, was not altered in rAd-p21-induced apoptotic cells, suggesting p21(WAF1/CIP1)-induced apoptosis through a pathway distinguishable from p53-induced apoptosis. Expression analysis of 2774qw1 cells infected with rAd-p21 on 60,000 cDNA microarrays identified 159 genes in response to p21(WAF1/CIP1) expression in at least one time point with 2.5-fold change as a cutoff. Integration of the data with the parallel microarray experiments with rAd-p53 infection allowed us to extract 66 genes downstream of both p53 and p21(WAF1/CIP1) and 93 genes in response to p21(WAF1/CIP1) expression in a p53-independent pathway. The genes in the former set may play a dual role in both p53-dependent and p53-independent pathways, and the genes in the latter set gave a mechanistic molecular explanation for p53-independent p21(WAF1/CIP1)-induced apoptosis. Furthermore, promoter sequence analysis suggested that transcription factor E2F family is partially responsible for the differential expression of genes following p21(WAF1/CIP1). This study has profound significance toward understanding the role of p21(WAF1/CIP1) in p53-independent apoptosis.

Adenoviridae↗
PubMed2002

Transforming growth factor-beta 2 is a transcriptional target for Akt/protein kinase B via forkhead transcription factor.

Tumors evade cell death by constitutively activating cell survival pathways and suppressing intrinsic death machinery. Activation of cell survival pathways leads to transcriptional repression of genes associated with cell death and activation of ones promoting anti-apoptosis. Akt/protein kinase B phosphorylates forkhead transcription factors and prevents their nuclear localization, leading to repression of genes involved in apoptosis, such as Fas ligand (FasL). Using bioinformatic approaches, we have identified three consensus sequences for forkhead transcription factor binding in transforming growth factor beta2 (TGF-beta2) promoter. TGF-beta inhibits cell proliferation and induces apoptosis in many cell types, and acquisition of TGF-beta resistance is linked to tumorigenesis. In this study, we show that activated Akt down-regulates TGF-beta2 promoter, and sequences within the promoter that are related to consensus forkhead binding sites are necessary for repression. Forkhead factor FKHRL1 binds in vitro to the three consensus sequences and can activate TGF-beta2 promoter in normal and Akt-transformed cell lines. In human breast and pancreatic tumors, activated Akt expression correlated with down-regulation of TGF-beta 2 mRNA levels. A number of tumor cells expressing activated Akt were responsive to TGF-beta addition, indicating the presence of an intact TGF-beta-signaling pathway. These results suggest that repression of TGF-beta 2 promoter activity in cells expressing activated Akt may play a role in promoting tumorigenesis and escape from the growth-inhibitory and/or apoptotic effects of TGF-beta.

Base Sequence↗
PubMed2002

Human survivin is negatively regulated by wild-type p53 and participates in p53-dependent apoptotic pathway.

Survivin is an inhibitor of apoptosis protein, which is over-expressed in most tumors. Aberrant expression of survivin and loss of wild-type p53 in many tumors prompted us to investigate a possible link between these two events. Here we show that wild-type p53 represses survivin expression at both mRNA and protein levels. Transient transfection analyses revealed that the expression of wild-type p53, but not mutant p53, was associated with strong repression of the survivin promoter in various cell types. The over-expression of exogenous survivin protein rescues cells from p53-induced apoptosis in a dose-dependent manner, suggesting that loss of survivin mediates, at least, in part the p53-dependent apoptotic pathway. In spite of the presence of two putative p53-binding sites in the survivin promoter, deletion and mutation analyses suggested that neither site is required for transcriptional repression of survivin expression. This was confirmed by chromatin immunoprecipitation assays. Further analyses suggested that the modification of chromatin within the survivin promoter could be a molecular explanation for silencing of survivin gene transcription by p53.

Adenoviridae↗
PubMed2002

Analysis of a human brain transcriptome map.

BACKGROUND: Genome wide transcriptome maps can provide tools to identify candidate genes that are over-expressed or silenced in certain disease tissue and increase our understanding of the structure and organization of the genome. Expressed Sequence Tags (ESTs) from the public dbEST and proprietary Incyte LifeSeq databases were used to derive a transcript map in conjunction with the working draft assembly of the human genome sequence. RESULTS: Examination of ESTs derived from brain tissues (excluding brain tumor tissues) suggests that these genes are distributed on chromosomes in a non-random fashion. Some regions on the genome are dense with brain-enriched genes while some regions lack brain-enriched genes, suggesting a significant correlation between distribution of genes along the chromosome and tissue type. ESTs from brain tumor tissues have also been mapped to the human genome working draft. We reveal that some regions enriched in brain genes show a significant decrease in gene expression in brain tumors, and, conversely that some regions lacking in brain genes show an increased level of gene expression in brain tumors. CONCLUSIONS: This report demonstrates a novel approach for tissue specific transcriptome mapping using EST-based quantitative assessment.

Journal Article↗
PubMed2002

Microarray profile of differentially expressed genes in a monkey model of allergic asthma.

BACKGROUND: Inhalation of Ascaris suum antigen by allergic monkeys causes an immediate bronchoconstriction and delayed allergic reaction, including a pulmonary inflammatory infiltrate. To identify genes involved in this process, the gene-expression pattern of allergic monkey lungs was profiled by microarrays. Monkeys were challenged by inhalation of A. suum antigen or given interleukin-4 (IL-4) treatment; lung tissue was collected at 4, 18 or 24 h after antigen challenge or 24 h after IL-4. Each challenged monkey lung was compared to a pool of normal, unchallenged monkey lungs. RESULTS: Of the approximately 40,000 cDNAs represented on the microarray, expression levels of 169 changed by more than 2.5-fold in at least one of the pairwise probe comparisons; these cDNAs encoded 149 genes, of which two thirds are known genes. The largest number of regulated genes was observed 4 h after challenge. Confirmation of differential expression in the original tissue was obtained for 95% of a set of these genes using real-time PCR. Cluster analysis revealed at least five groups of genes with unique expression patterns. One cluster contained genes for several chemokine mediators including eotaxin, PARC, MCP-1 and MCP-3. Genes involved in tissue remodeling and antioxidant responses were also identified as regulated by antigen and IL-4 or by antigen only. CONCLUSION: This study provides a large-scale profile of gene expression in the primate lung following allergen or IL-4 challenge. It shows that microarrays, with real-time PCR, are a powerful tool for identifying and validating differentially expressed genes in a disease model.

Animals↗
PubMed2002

Computational analysis of composite regulatory elements.

Combinatorial regulation is a powerful mechanism for generating specificity in gene expression, and it is thought to play a pivotal role in the formation of the complex gene regulatory networks found in higher eukaryotes. The term "Composite Element" (CE) refers to a minimal functional unit where protein-DNA and protein-protein interactions contribute to a highly specific pattern of gene transcriptional regulation. Identification of composite elements will help to better understand gene regulation networks. Experimentally identified CEs are limited in number, and the currently available CE database COMPEL is based on such published information. Here, based on the statistical analysis of over-represented adjacent transcription factor binding sites, we describe a computational method to predict composite regulatory elements in genomic sequences. The algorithm proved to be efficient for extracting composite elements that had been experimentally confirmed and documented in the COMPEL database. Furthermore, putative new composite elements are predicted based on this method, and we have been able to confirm some of our predictions which are not included in the COMPEL database by searching published information.

Binding Sites↗
PubMed2002

The use of direct cDNA selection to rapidly and effectively identify genes in the fungus Aspergillus fumigatus.

Aspergillus fumigatus is one of the causes of invasive lung disease in immunocompromised individuals. To rapidly identify genes in this fungus, including potential targets for chemotherapy, diagnostics, and vaccine development, we constructed cDNA libraries. We began with non-normalized libraries, then to improve this approach we constructed a normalized cDNA library using direct cDNA selection. Normalization resulted in a reduction of the frequency of clones with highly expressed genes and an enrichment of underrepresented cDNAs. Expressed sequence tags generated from both the original and the normalized libraries were compared with the genomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans, indicating that a large proportion of A. fumigatus genes do not have orthologs in these fungal species. This method allowed the expeditious identification of genes in a fungal pathogen. The same approach can be applied to other human or plant pathogens to rapidly identify genes without the need for genomic sequence information.

Aspergillus fumigatus↗