Tissue-specific Gene Expression Analysis Research Articles

A large-scale analysis of tissue-specific pathology and gene expression of human disease genes and complexes

Heritable diseases are caused by germ-line mutations that, despite tissuewide presence, often lead to tissue-specific pathology. Here, we make a systematic analysis of the link between tissue-specific gene expression and pathological manifestations in many human diseases and cancers. Diseases were systematically mapped to tissues they affect from disease-relevant literature in PubMed to create a disease-tissue covariation matrix of high-confidence associations of >1,000 diseases to 73 tissues. By retrieving >2,000 known disease genes, and generating 1,500 disease-associated protein complexes, we analyzed the differential expression of a gene or complex involved in a particular disease in the tissues affected by the disease, compared with nonaffected tissues. When this analysis is scaled to all diseases in our dataset, there is a significant tendency for disease genes and complexes to be overexpressed in the normal tissues where defects cause pathology. In contrast, cancer genes and complexes were not overexpressed in the tissues from which the tumors emanate. We specifically identified a complex involved in XY sex reversal that is testis-specific and down-regulated in ovaries. We also identified complexes in Parkinson disease, cardiomyopathies, and muscular dystrophy syndromes that are similarly tissue specific. Our method represents a conceptual scaffold for organism-spanning analyses and reveals an extensive list of tissue-specific draft molecular pathways, both known and unexpected, that might be disrupted in disease.

Genome-Wide Microarray Expression Analysis of CD4+ T Cells from Nonobese Diabetic Congenic Mice Identifies Cd55 (Daf1) and Acadl as Candidate Genes for Type 1 Diabetes

NOD.Idd3/5 congenic mice have insulin-dependent diabetes (Idd) regions on chromosomes 1 (Idd5) and 3 (Idd3) derived from the nondiabetic strains B10 and B6, respectively. NOD.Idd3/5 mice are almost completely protected from type 1 diabetes (T1D) but the genes within Idd3 and Idd5 responsible for the disease-altering phenotype have been only partially characterized. To test the hypothesis that candidate Idd genes can be identified by differential gene expression between activated CD4+ T cells from the diabetes-susceptible NOD strain and the diabetes-resistant NOD.Idd3/5 congenic strain, genome-wide microarray expression analysis was performed using an empirical Bayes method. Remarkably, 16 of the 20 most differentially expressed genes were located in the introgressed regions on chromosomes 1 and 3, validating our initial hypothesis. The two genes with the greatest differential RNA expression on chromosome 1 were those encoding decay-accelerating factor (DAF, also known as CD55) and acyl-coenzyme A dehydrogenase, long chain, which are located in the Idd5.4 and Idd5.3 regions, respectively. Neither gene has been implicated previously in the pathogenesis of T1D. In the case of DAF, differential expression of mRNA was extended to the protein level; NOD CD4+ T cells expressed higher levels of cell surface DAF compared with NOD.Idd3/5 CD4+ T cells following activation with anti-CD3 and -CD28. DAF up-regulation was IL-4 dependent and blocked under Th1 conditions. These results validate the approach of using congenic mice together with genome-wide analysis of tissue-specific gene expression to identify novel candidate genes in T1D.

Genomic organization and tissue-specific expression analysis of hepcidin-like genes from black porgy ( Acanthopagrus schlegelii B.)

Hepcidin is an antimicrobial peptide and putative iron regulatory hormone previously described in mice and humans. Dozens of fish hepcidins have been isolated and characterized so far. Here we present seven hepcidin-like cDNA sequences named AS-hepc1–7, amplified from the normal commercially cultured fish (black porgy) by RACE–PCR. Sequence analysis reveals that these seven potential hepcidin peptides have highly conserved sequences with other known hepcidins, but they are different from each other in constitution and characteristics of predicted mature amino acids. Based on the study, it is deduced that AS-hepc1–7 represent different variants of a family of hepcidin genes in black porgy. To understand the organization of these hepcidin-like genes, we sequenced AS-hepc2 DNA, AS-hepc3 DNA, AS-hepc4 DNA, AS-hepc7 DNA and AS-hepc2 upstream region; and all of the four genomic DNAs consisted of two introns and three exons, the same organization as other reported hepcidins. The tissue-specific gene expression of hepcidins in normal black porgy was evaluated using RT–PCR and dot blot approaches. RT–PCR showed that transcripts of hepcidin-like mRNAs were present in each tested tissue of normal juvenile black porgy, including liver, spleen, kidney, heart, brain, stomach, intestine, gill, skin and blood, but abundant hepcidin-like mRNA transcripts were only detected in the liver, kidney, spleen, intestine and stomach by dot blot assay. In addition, using dot blot and Northern blot approach, a significant increase of hepcidin mRNA transcription was observed in the liver within 48 h after immersion in a suspension of live bacteria, which suggested that the expression pattern of hepcidin-like genes in black porgy might be different in the liver from the other tissues as previously reported in several hepcidin studies.

Evaluation of Global RNA Amplification and Its Use for High-Throughput Transcript Analysis of Laser-Microdissected Endosperm

Laser microdissection (LM) provides a useful method for isolating specific cells or tissues from biological samples. Here, we adapted microdissection protocols to allow high-resolution transcript analysis of different tissues from developing Arabidopsis seed. Sufficient RNA (∼50 ng) was extracted from endosperm tissue for RT-PCR. However, to obtain enough RNA for microarray analyses, it was necessary to amplify the RNA. PCR- and IVT-based amplification methods were investigated and several important technical aspects of amplification were identified (such as target truncation and alterations in signal intensity). We found that when starting from only 50 ng of RNA, amplification methods based on PCR and IVT produced sufficient product for reliable microarray hybridizations, with two-round IVT giving the best results. Microarray analyses, using endosperm-derived RNA amplified by two-round IVT, reproducibly identified endosperm enriched marker genes. Thus, when combined with RNA-amplification protocols, LM is a robust and reliable technique for high-throughput tissue-specific gene expression analysis.

Analysis of Local Tissue-Specific Gene Expression in Cellular Micropatterns

While cellular micropatterning approaches are employed extensively in cell biology and tissue engineering, only a limited number of methods for analysis of local function in the context of a complex, microfabricated environment are currently available. The present study develops a novel strategy for analysis of local tissue-specific function in cellular micropatterns. Model hepatocytes (HepG2 cells) were seeded onto silane-modified glass slides containing robotically printed arrays of collagen type I. These model hepatocytes formed cell arrays with individual cell cluster dimensions (150 or 500 microm) corresponding in size to the printed collagen spots. Non-parenchymal cells (3T3 fibroblasts) were added to hepatocellular micropatterns to create heterotypic cocultures. Expression of hepatic phenotype in HepG2 cells was first verified by traditional techniques including intracellular staining and ELISA for albumin. In order to evaluate local liver function in the cellular microarray, individual array members composed of approximately 400 hepatocytes were retrieved using laser capture microdissection and analyzed with real-time reverse transcriptase (RT)-polymerase chain reaction (PCR). Hepatic function was assessed based on expression of four genes associated with differentiated liver phenotype: albumin, transferrin, alpha-fetoprotein, and alpha1-antitrypsin. "Titration" experiments, carried out to identify the smallest population of HepG2 cells yielding detectable mRNA levels and RT-PCR signals, showed that extraction area of 12,500 microm2 (corresponding to approximately 70 cells) provided detectable gene expression signals. All four liver-specific genes were routinely evaluated after extraction of approximately 400 HepG2 from the micropatterned surfaces. Significantly, selective retrieval and subsequent analysis of tissue-specific function was demonstrated for hepatic cells micropatterned alone and in coculture with non-parenchymal cells. In the future, methods described in this study will offer the possibility to investigate dynamic and reciprocal interactions between two or more cell types positioned on a microfabricated cell culture surface. We also envision the proposed approaches to be ideally suited for cell analysis in the context of combinatorial microenvironment.

Comparison of reference genes for quantitative real-time polymerase chain reaction analysis of gene expression in sugarcane

A protocol for reverse transcription followed by real-time quantitative PCR (RT-qPCR) analysis of tissue-specific and genotype-variable gene expression in sugarcane (Saccharum sp.) was developed. A key requirement for this analysis was the identification of a housekeeping gene with transcript levels that were relatively stable across tissues and genotypes, suitable for use as a reference. Primers for β-actin, β-tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes and 25S ribosomal RNA were designed and tested by RT-qPCR, and formation of product in the reactions was measured with the SYBR green I dye system. Ribosomal RNA was the most sensitive and consistent as a reference gene. Determination of the expression levels of β-actin, β-tubulin, and GAPDH transcripts relative to that of 25S rRNA showed that GAPDH had the most consistent mRNA expression of protein-coding genes across different tissues. GAPDH also showed low variation in expression in maturing stem internodes when compared across 2 cultivars and 3 otherSaccharum species. GAPDH therefore appears to be a suitable “housekeeping gene” in addition to 25S rRNA as a reference for measuring the relative expression of other genes in sugarcane. With use of GAPDH as a reference, the relative expression of the sugarcane sugar transporter genePst2a was assessed in a range of tissues. The result obtained was similar to our previously published Northern blot analysis. The protocol described here, using GAPDH as a reference gene, is recommended for studying the expression of other genes of interest in diverse tissues and genotypes of sugarcane.

Polar auxin transport in the wood-forming tissues of hybrid aspen is under simultaneous control of developmental and environmental signals.

Recent research has highlighted the importance of auxin concentration gradients during plant development. Establishment of these gradients is believed to involve polar auxin transport through specialized carrier proteins. We have used an experimental system, the wood-forming tissue of hybrid aspen, which allows tissue-specific expression analysis of auxin carrier genes and quantification of endogenous concentrations of the hormone. As part of this study, we isolated the putative polar auxin transport genes, PttLAX1-PttLAX3 and PttPIN1-PttPIN3, belonging to the AUX1-like family of influx and PIN1-like efflux carriers, respectively. Analysis of PttLAX and PttPIN expression suggests that specific positions in a concentration gradient of the hormone are associated with different stages of vascular cambium development and expression of specific members of the auxin transport gene families. We were also able demonstrate positive feedback of auxin on polar auxin transport genes. Entry into dormancy at the end of a growing season leads to a loss of auxin transport capacity, paralleled by reduced expression of PttLAX and PttPIN genes. Furthermore, data from field experiments show that production of the molecular components of the auxin transport machinery is governed by environmental controls. Our findings collectively demonstrate that trees have developed mechanisms to modulate auxin transport in the vascular meristem in response to developmental and environmental cues.

Characterization of the human protein S gene promoter: a possible role of transcription factors Sp1 and HNF3 in liver.

Protein S is a vitamin-K-dependent plasma glycoprotein that serves as a cofactor for activated protein C in the protein C anticoagulation pathway, which regulates blood coagulation by inactivating factors Va and VIIIa. Mechanisms regulating the expression of the protein S gene remain unknown to date. The aim of this study was to characterize the cis-acting DNA elements of the human protein S gene in liver. We found that liver cell lines (HepG2 and PLC) transcribed the human protein S gene to mRNA, whereas non-liver cell lines (HEK293 and HeLa cells) either transcribed the gene weakly or not at all. Isolation and analysis of tissue-specific gene expression in HepG2 and HeLa cells of the 5'-flanking region from -6183 to +294 of the protein S gene indicated that the consensus binding motifs to HNF3 and Sp1 or MAZ transcription factors in the flanking region are essential for protein S gene expression. Exogenous expression of the Sp1 gene augmented the protein S-reporter gene expression in HepG2 or PLC cells but had no effect in HeLa cells. Taken together, we would conclude that transcription factors of HNF3, MAZ, and Sp1 are required for high-level expression of the protein S gene in hepatic cells, but in non-hepatic cells such as HeLa cells, an unknown factor(s) binds to the Sp1 region and disturbs the action of Sp1 and MAZ.

Primary structure of rat ceruloplasmin and analysis of tissue-specific gene expression during development.

cDNA clones corresponding to rat ceruloplasmin were isolated from newborn rat lung and liver cDNA libraries and the nucleotide sequence was obtained. The derived amino acid sequence of rat ceruloplasmin is 93% homologous to the corresponding human sequence and contains a 19-amino acid leader peptide plus 1040 amino acids of mature protein. Southern blot analysis indicates that the ceruloplasmin gene exists as a single copy in the rat haploid genome. Using these cDNA clones in RNA blot analysis, a single 3.7-kilobase ceruloplasmin-specific transcript is detected in fetal rat liver and lung by day 15 of gestation. During fetal development the abundance of this transcript increases selectively in these two tissues and at birth is 60% of that found in the adult liver. Postnatally the temporal pattern of ceruloplasmin gene expression in lung and liver differs. Within the first 3 weeks postpartum ceruloplasmin mRNA content decreases in lung to undetectable levels, while that in the liver reaches adult levels. Primer extension reveals a single identical start site of ceruloplasmin gene transcription in lung and liver and biosynthetic studies indicate that each tissue synthesizes a ceruloplasmin protein which is qualitatively similar to that synthesized by adult liver. Ceruloplasmin mRNA is also detected in human fetal lung explant and a human lung adenocarcinoma cell line suggesting that a similar pattern of expression occurs in the developing human lung. These data indicate that lung is the predominant extrahepatic site of ceruloplasmin gene expression during fetal development and suggest that this protein may play a previously unappreciated role in lung development or pulmonary antioxidant defense.

26] Cloning of the rat parvalbumin gene

This chapter describes the techniques used to obtain parvalbumin (PV)-specific cDNA probes that could be utilized to isolate and analyze the natural PV gene. This should help to understand molecular mechanisms involved in the evolution of Ca 2+ -binding proteins. PV is a high-affinity Ca 2+ -binding protein. It is a member of a superfamily of low-molecular-weight acidic proteins, which includes troponin C (TNC), calmodulin (CAM), calbindin D 10K and D 28K , certain types of myosin light chains (MLC), S-100 proteins, and other related proteins. Through the binding of Ca 2+ , they are able to regulate numerous cellular processes. To study the tissue-specific and development-dependent PV gene expression, Northern blot and RNA dot-blot analyses were carried out. PV mRNA levels are compared to CaM mRNA concentrations in different tissues. A 32 P-labeled chicken CaM cDNA (350-bp RsaI fragment from pCB 12 containing only coding region) was used as a probe. In contrast to PV mRNA, CaM mRNA is present in all tissues with highest amounts in brain and testis. The time period with the maximal increase of PV mRNA coincides with the differentiation of the gastrocnemius muscle cells into fast contracting/ relaxing fibers. This supports the hypothesis that PV is involved in Ca 2+ transport mechanisms, which are important for the function of mainly type IIB muscle fibers.

Gene transfer by direct pronuclei microinjection.

The technique of micromanipulating and injecting a mouse egg was first described in 1966 by T.P. Lin (8,9). With the advent of recombinant DNA technology, it has become possible to transform cultured cells by modifying this technique to microinject directly into the cell nuclei-specific cloned gene sequences (1,4). This approach is amenable only to established cell lines in culture, and so its general usefulness in the analysis of tissue-specific gene expression has been found to be somewhat limited. Subse-quently, transgenic animals have been produced by introduction of foreign genes at the pronuclear stage of fertilized, one-cell zygotes. Most of the successes have been with mouse eggs (3,5,6,11,12), and only recently has the successful production of transgenic animals been extended to the rabbit, pig, and sheep (7). This technique has become a powerful tool for studying gene regulation and physiological functions of gene products in a normal host environment. This chapter summarizes the current methodology used for mouse eggs in gene transfer experiments by direct pronuclear microinjection.