Identification Of New Target Genes Research Articles

Abstract 5221: Identifying acute lymphoblastic leukemia risk loci in latino children via admixture mapping

Abstract Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and accounts for 25% of malignancies before the age of 20. Despite decades of inquiry into ALL causes, Latino children have up to 1.4 times the rate of ALL compared to their non-Latino White counterparts. The etiology of ALL is complex and this disparity in risk has not been fully explained by social factors alone, suggesting the role of genetic variants and ancestry. Previous literature has implicated variants in IKZF1, ARID5B, GATA3, PIP42KA, and ERG with greater risk allele frequencies or population-specific risk effects in Latinos. Indigenous American ancestry has also been previously associated with ALL risk, relapse, and poor prognosis. To elucidate the genetic and ancestral etiology of ALL, we conducted the first admixture mapping analysis of 1930 childhood ALL cases and 8520 controls of self-identified Latino ethnicity. We uncovered putative admixture associations on chromosomes 2, 7, 10, and 15 (P = 3.21 x 10−5 to 7.32 x 10−8 among top associations across the four loci). Two of the four loci, on chromosomes 2 and 15, were previously not known to be associated with ALL. Following imputation with the TOPMed reference panel, we fine-mapped each putative locus by testing the ALL risk association of each variant with a minor allele frequency greater than 1%. The top associated variants at each locus showed substantial frequency differences between ancestries from the gnomAD or 1000Genomes project. For example, the top associated variant on chromosome 2 has a risk allele frequency of 43% in Native Americans from the Human Genome Diversity Panel, but a frequency of 0.10% in Non-Finnish Europeans in gnomAD. Including these variants in the admixture mapping model greatly attenuated the admixture signals at each locus, suggesting that they are likely the causal SNPs or close proxies of the causal SNPs. The top associated variants from three out of the four loci passed regional multiple testing burden and mapped to genes MGAT5, IKFZ1 and ARID5B on chromosomes 2, 7 and 10, respectively. The top two SNPs on chromosome 15, which narrowly missed the regional threshold, mapped to genes RAB11A and MEGF11. We are pursuing replication of putative novel loci on chromosomes 2 and 15 in additional Latino GWAS datasets. Our results suggest that ALL risk variants with higher frequencies in individuals with Native American ancestry may contribute to the observed increased risk of ALL in Latino children. Investigation of such risk loci can contribute to the identification of new target genes for ALL prediction and therapeutics and new insights for precision medicine, which will reduce the burden of ALL. Citation Format: Jalen Langie, Soyoung Jeon, Xiaomei Ma, Catherine Metayer, Adam J. de Smith, Joseph L. Wiemels, Charleston W. Chiang. Identifying acute lymphoblastic leukemia risk loci in latino children via admixture mapping. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5221.

ScRNA-sequencing reveals subtype-specific transcriptomic perturbations in DRG neurons of PirtEGFPf mice in neuropathic pain condition.

Functionally distinct subtypes/clusters of dorsal root ganglion (DRG) neurons may play different roles in nerve regeneration and pain. However, details about their transcriptomic changes under neuropathic pain conditions remain unclear. Chronic constriction injury (CCI) of the sciatic nerve represents a well-established model of neuropathic pain, and we conducted single-cell RNA-sequencing (scRNA-seq) to characterize subtype-specific perturbations of transcriptomes in lumbar DRG neurons on day 7 post-CCI. By using PirtEGFPf mice that selectively express an enhanced green fluorescent protein in DRG neurons, we established a highly efficient purification process to enrich neurons for scRNA-seq. We observed the emergence of four prominent CCI-induced clusters and a loss of marker genes in injured neurons. Importantly, a portion of injured neurons from several clusters were spared from injury-induced identity loss, suggesting subtype-specific transcriptomic changes in injured neurons. Moreover, uninjured neurons, which are necessary for mediating the evoked pain, also demonstrated cell-type-specific transcriptomic perturbations in these clusters, but not in others. Notably, male and female mice showed differential transcriptomic changes in multiple neuronal clusters after CCI, suggesting transcriptomic sexual dimorphism in DRG neurons after nerve injury. Using Fgf3 as a proof-of-principle, RNAscope study provided further evidence of increased Fgf3 in injured neurons after CCI, supporting scRNA-seq analysis, and calcium imaging study unraveled a functional role of Fgf3 in neuronal excitability. These findings may contribute to the identification of new target genes and the development of DRG neuron cell-type-specific therapies for optimizing neuropathic pain treatment and nerve regeneration.

Identification of c-di-GMP/FleQ-Regulated New Target Genes, Including cyaA, Encoding Adenylate Cyclase, in Pseudomonas putida.

The bacterial second messenger cyclic diguanylate (c-di-GMP) modulates plankton-to-biofilm lifestyle transition of Pseudomonas species through its transcriptional regulatory effector FleQ. FleQ regulates transcription of biofilm- and flagellum-related genes in response to c-di-GMP. Through transcriptomic analysis and FleQ-DNA binding assay, this study identified five new target genes of c-di-GMP/FleQ in P. putida, including PP_0681, PP_0788, PP_4519 (lapE), PP_5222 (cyaA), and PP_5586 Except lapE encoding an outer membrane pore protein and cyaA encoding an adenylate cyclase, the functions of the other three genes encoding hypothetical proteins remain unknown. FleQ and c-di-GMP coordinately inhibit transcription of PP_0788 and cyaA and promote transcription of PP_0681, lapE, and PP_5586 Both in vitro and in vivo assays show that FleQ binds directly to promoters of the five genes. Further analyses confirm that LapE plays a central role of in the secretion of adhesin LapA and that c-di-GMP/FleQ increases lapE transcription, thereby promoting adhesin secretion and biofilm formation. The adenylate cyclase CyaA is responsible for synthesis of another second messenger, cyclic AMP (cAMP). FleQ and c-di-GMP coordinate to decrease the content of cAMP, suggesting that c-di-GMP and FleQ coregulate cAMP by modulating cyaA expression. Overall, this study adds five new members to the c-di-GMP/FleQ-regulated gene family and reveals the role of c-di-GMP/FleQ in LapA secretion and cAMP synthesis regulation in P. putida IMPORTANCE c-di-GMP/FleQ promotes the plankton-to-biofilm lifestyle transition at the transcriptional level via FleQ in Pseudomonas species. Identification of new target genes directly regulated by c-di-GMP/FleQ helps to broaden the knowledge of c-di-GMP/FleQ-mediated transcriptional regulation. Regulation of lapE by c-di-GMP/FleQ guarantees highly efficient LapA secretion and biofilm formation. The mechanism of negative correlation between c-di-GMP and cAMP in both P. putida and P. aeruginosa remains unknown. Our result concerning transcriptional inhibition of cyaA by c-di-GMP/FleQ reveals the mechanism underlying the decrease of cAMP content by c-di-GMP in P. putida.

The CRISP(Y) Future of Pediatric Soft Tissue Sarcomas.

The RNA-guided clustered regularly interspaced palindromic repeats (CRISPR)/associated nuclease 9 (Cas9)-based genome editing technology has increasingly become a recognized method for translational research. In oncology, the ease and versatility of CRISPR/Cas9 has made it possible to obtain many results in the identification of new target genes and in unravel mechanisms of resistance to therapy. The majority of the studies have been made on adult tumors so far. In this mini review we present an overview on the major aspects of CRISPR/Cas9 technology with a focus on a group of rare pediatric malignancies, soft tissue sarcomas, on which this approach is having promising results.

New Insights into Molecular Oncogenesis and Therapy of Uveal Melanoma.

Uveal melanoma (UM), which is the most common cancer of the eye, was investigated in recent years by many teams in the field of biomedical sciences and eye clinicians. New knowledge was acquired on molecular pathways found to be dysregulated during the multistep process of oncogenesis, whereas novel therapeutic approaches gave significant results in the clinical applications. Uveal melanoma-affected patients greatly benefited from recent advances of the research in this eye cancer. Tumour biology, genetics, epigenetics and immunology contributed significantly in elucidating the role of different genes and related pathways during uveal melanoma onset/progression and UM treatments. Indeed, these investigations allowed identification of new target genes and to develop new therapeutic strategies/compounds to cure this aggressive melanoma of the eye. Unfortunately, the advances reported in the treatment of cutaneous melanoma have not produced analogous benefits in metastatic uveal melanoma. Nowadays, no systemic adjuvant therapy has been shown to improve overall survival or reduce the risk of metastasis. However, the increasing knowledge of this disease, and the encouraging results seen in clinical trials, offer promise for future effective therapies. Herein, different pathways/genes involved in uveal melanoma onset/progression were taken into consideration, together with novel therapeutic approaches.

Identification and function of FAR protein family genes from a transcriptome analysis of Aphelenchoides besseyi.

The rice white tip nematode (RWTN) Aphelenchoides besseyi is a migratory plant parasitic nematode that infects the aboveground parts of plants. Fatty acid- and retinoid-binding (FAR) proteins are nematode-specific proteins that are involved in many important biological processes. Genes encoding FAR proteins have been identified in many species of nematodes, which indicated that nematodes may produce more than one type of FAR protein. The main goal of this study is to find new molecular targets including new far genes that will help control RWTN, and reduce the economic damage caused by RWTN. Two RWTN populations with different levels of pathogenicity and reproduction were sequenced and analyzed with next-generation sequencing. 17 087 transcripts were annotated using six databases and 1696 differentially expressed genes (DEGs) were identified between the two RWTN populations. Seven new Ab-far genes were identified from the transcriptome data of the two RWTN populations which is the first to identify multiple far genes in plant parasitic nematodes. This study is the first to identify far genes in the nervous system of nematodes and the first to report a transcriptome sequencing analysis of different RWTN populations. The results help elucidate the genes related to parasitism and pathogenicity and also contribute to the identification of new target genes and development of new methods to control RWTN. Our data are publicly available at Sequence Read Archive (SRA) database and GenBank database. Supplementary data are available at Bioinformatics online.

Abstract 2401: Identification of new target genes in microsatellite unstable colorectal cancer by exome sequencing

Abstract Colorectal cancer (CRC) is the third most common cancer in Western countries. Approximately 15% of CRCs display microsatellite instability (MSI) caused by defective cellular mismatch repair. Most of the MSI CRCs are sporadic and result from biallelic inactivation of the MLH1 gene, which is most often due to hypermethylation of its promoter. Cells displaying MSI accumulate a high number of mutations throughout the genome, especially in short repeat areas called microsatellites. These mutations typically lead to a premature stop codon resulting in a truncated protein product that may inactivate the gene, a mechanism known typical of tumor suppressor genes. The aim of this study is to identify new target genes in MSI CRC. To date, several genes have been proposed as MSI target genes based on high mutation frequency in targeted searches of microsatellites. The availability of new sequencing and bioinformatic technologies has enabled genome-wide investigation of mutations in human cancers. This has led to identification of a vast amount of mutations in cancer, most of which are passenger mutations that do not confer selective growth advantage. The challenge is therefore to distinguish true driver genes from passengers. The high number of mutations in MSI tumors augments this challenge. In this study, 25 sporadic MSI CRCs and their corresponding normal samples have been exome sequenced to identify changes of somatic origin. An analysis and visualization program developed in our group, “RikuRator” (unpublished), will be utilized along with a script to estimate the accurate somatic mutation frequency of each coding mononucleotide repeat. A statistical model based on exome sequencing data will be developed that takes into account the background mutation frequency of microsatellites depending on the repeat length and nucleotide context. The most mutated repeats will be validated by Sanger sequencing in a set of additional MSI CRCs. Functional studies will be carried out to further investigate the pathogenic effect of the new target genes identified. The systematic screening of mutations in microsatellites will improve our understanding on the mutation profile typical of these tumors. With our approach that takes into account the background mutation frequency of microsatellites we aim to identify true driver genes in MSI CRC. A detailed understanding of the molecular background of this tumor type is important for the development of more efficient screening methods and personalized treatments to improve the prognosis of patients diagnosed with MSI type CRC. Citation Format: Johanna Kondelin, Esa Pitkänen, Alexandra E. Gylfe, Kimmo Palin, Heikki Ristolainen, Riku Katainen, Eevi Kaasinen, Minna Taipale, Jussi Taipale, Laura Renkonen-Sinisalo, Heikki Järvinen, Jan Böhm, Jukka-Pekka Mecklin, Pia Vahteristo, Sari Tuupanen, Lauri A. Aaltonen. Identification of new target genes in microsatellite unstable colorectal cancer by exome sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2401. doi:10.1158/1538-7445.AM2014-2401

Novel Molecular Pathways Elicited by Mutant FGFR2 May Account for Brain Abnormalities in Apert Syndrome

Apert syndrome (AS), the most severe form craniosynostosis, is characterized by premature fusion of coronal sutures. Approximately 70% of AS patients carry S252W gain-of-function mutation in FGFR2. Besides the cranial phenotype, brain dysmorphologies are present and are not seen in other FGFR2-asociated craniosynostosis, such as Crouzon syndrome (CS). Here, we hypothesized that S252W mutation leads not only to overstimulation of FGFR2 downstream pathway, but likewise induces novel pathological signaling. First, we profiled global gene expression of wild-type and S252W periosteal fibroblasts stimulated with FGF2 to activate FGFR2. The great majority (92%) of the differentially expressed genes (DEGs) were divergent between each group of cell populations and they were regulated by different transcription factors. We than compared gene expression profiles between AS and CS cell populations and did not observe correlations. Therefore, we show for the first time that S252W mutation in FGFR2 causes a unique cell response to FGF2 stimulation. Since our gene expression results suggested that novel signaling elicited by mutant FGFR2 might be associated with central nervous system (CNS) development and maintenance, we next investigated if DEGs found in AS cells were also altered in the CNS of an AS mouse model. Strikingly, we validated Strc (stereocilin) in newborn Fgfr2S252W/+ mouse brain. Moreover, immunostaining experiments suggest a role for endothelial cells and cerebral vasculature in the establishment of characteristic CNS dysmorphologies in AS that has not been proposed by previous literature. Our approach thus led to the identification of new target genes directly or indirectly associated with FGFR2 which are contributing to the pathophysiology of AS.

Global gene expression reveals a set of new genes involved in the modification of cells during erythroid differentiation

Erythroid differentiation is a dynamic process in which a pluripotent stem cell undergoes a series of developmental changes that commit it to a specific lineage. These alterations involve changes in gene expression profiles. In this study, gene expression profiles during differentiation of human erythroid cells of a normal blood donor were evaluated using SAGE. Global gene expression was evaluated in cells collected immediately before addition of erythropoietin (0 h) and 192 and 336 h after addition of this hormone. Real-time PCR was used to evaluate activation of differentially expressed genes. The data indicate that global aspects of the transcriptome were similar during differentiation of the majority of the genes and that a relatively small set of genes is probably involved in modification of erythroid cells during differentiation. We have identified 93 differentially expressed genes during erythroid development, and expression of some of these was confirmed by qPCR. Various genes including EYA3, ERH, HES6, TIMELESS and TRIB3 were found to be homologous to those of Drosophila melanogaster and here are described for the first time during erythroid development. An important and unique carboxypeptidase inhibitor described in mammalians, LXN, was also identified. The results of this study amplify previously published data and may contribute to comprehension of erythroid differentiation and identification of new target genes involved in some erythroid concerning diseases.

Anopheles gambiaemales produce and transfer the vitellogenic steroid hormone 20-hydroxyecdysone to females during mating

In female insects, the steroid hormone 20-hydroxyecdysone (20E) plays a major role in activating vitellogenesis, a process required for egg development. By contrast with vertebrates, production of large amounts of hormonal steroids has not been reported in adult male insects. In the present study, we analyzed steroidogenesis in both male and female adult of the malaria mosquito Anopheles gambiae and we found that A. gambiae male mosquitoes produce high amounts of the steroid hormone 20E. Importantly, we found that male accessory glands, but not testes, are the source of 20E. Moreover, this steroid hormone is stored in male accessory glands and delivered to females during mating. These findings suggest that male 20E may not act as a true male sex steroid, but more likely as an allohormone. Our results give new insights into species-specific physiological processes that govern the reproductive success of the malaria mosquito. This could thus lead to the identification of new target genes for manipulating male and/or female reproductive success, a promising way to reduce or eliminate mosquito population and therefore to control malaria transmission.

Activation of Wingless Targets Requires Bipartite Recognition of DNA by TCF

Specific recognition of DNA by transcription factors is essential for precise gene regulation. In Wingless (Wg) signaling in Drosophila, target gene regulation is controlled by T cell factor (TCF), which binds to specific DNA sequences through a high mobility group (HMG) domain. However, there is considerable variability in TCF binding sites, raising the possibility that they are not sufficient for target location. Some isoforms of human TCF contain a domain, termed the C-clamp, that mediates binding to an extended sequence in vitro. However, the significance of this extended sequence for the function of Wnt response elements (WREs) is unclear. In this report, we identify a cis-regulatory element that, to our knowledge, was previously unpublished. The element, named the TCF Helper site (Helper site), is essential for the activation of several WREs. This motif greatly augments the ability of TCF binding sites to respond to Wg signaling. Drosophila TCF contains a C-clamp that enhances in vitro binding to TCF-Helper site pairs and is required for transcriptional activation of WREs containing Helper sites. A genome-wide search for clusters of TCF and Helper sites identified two new WREs. Our data suggest that DNA recognition by fly TCF occurs through a bipartite mechanism, involving both the HMG domain and the C-clamp, which enables TCF to locate and activate WREs in the nucleus.

Endoglin (CD105) Expression Is Regulated by the Liver X Receptor Alpha (NR1H3) in Human Trophoblast Cell Line JAR1

Human implantation involves invasion of the uterine wall and remodeling of uterine arteries by extravillous cytotrophoblasts. Defects in these early steps of placental development lead to poor placentation and are often associated with preeclampsia, a frequent complication of human pregnancy. One of the complex mechanisms controlling trophoblast invasion involves the activation of the liver X receptor beta (or NR1H2, more commonly known as LXRbeta) by oxysterols known as potent LXR activators. This activation of LXRbeta leads to a decrease of trophoblast invasion. The identification of new target genes of LXR in the placenta could aid in the understanding of their physiological roles in trophoblast invasion. In the present study, we show that the endoglin (ENG) gene is a direct target of the liver X receptor alpha (NR1H3, also known as LXRalpha). ENG, whose gene is highly expressed in syncytiotrophoblasts, is part of the transforming growth factor (TGF) receptor complex that binds several members of the TGFbeta superfamily. In the human placenta, ENG has been shown to be involved in the inhibition of trophoblast invasion. Treatment of human choriocarcinoma JAR cells with T0901317, a synthetic LXR-selective agonist, leads to a significant increase in ENG mRNA and protein levels. Using transfection and electrophoretic mobility shift assays, we demonstrate that LXR (as a heterodimer with the retinoid X receptor) is able to bind the ENG promoter on an LXR response element and mediates the activation of ENG gene expression by LXRalpha in JAR cells. This study suggests a novel mechanism by which LXR may regulate trophoblast invasion in pathological pregnancy such as preeclampsia.

Cooperative genetic defects in TLX3 rearranged pediatric T-ALL

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder, in which multiple genetic abnormalities cooperate in the malignant transformation of thymocytes. About 20% of pediatric T-ALL cases are characterized by TLX3 expression due to a cryptic translocation t(5;14)(q35;q32). Although a number of collaborating genetic events have been identified in TLX3 rearranged T-ALL patients (NOTCH1 mutations, p15/p16 deletions, NUP214-ABL1 amplifications), further elucidation of additional genetic lesions could provide a better understanding of the pathogenesis of this specific T-ALL subtype. In this study, we used array-CGH to screen TLX3 rearranged T-ALL patients for new chromosomal imbalances. Array-CGH analysis revealed five recurrent genomic deletions in TLX3 rearranged T-ALL, including del(1)(p36.31), del(5)(q35), del(13)(q14.3), del(16)(q22.1) and del(19)(p13.2). From these, the cryptic deletion, del(5)(q35), was exclusively identified in about 25% of TLX3 rearranged T-ALL cases. In addition, 19 other genetic lesions were detected once in TLX3 rearranged T-ALL cases, including a cryptic WT1 deletion and a deletion covering the FBXW7 gene, an U3-ubiquitin ligase that mediates the degradation of NOTCH1, MYC, JUN and CyclinE. This study provides a genome-wide overview of copy number changes in TLX3 rearranged T-ALL and offers great new challenges for the identification of new target genes that may play a role in the pathogenesis of T-ALL.

Global Gene Expression Revealed a Set of Genes Involved in the Modification of Cells during Erythropoiesis.

Erythroid differentiation is a dynamic and complex process in which a pluripotent stem cell undergoes a series of developmental changes that commit it to a specific lineage. These alterations involve changes in gene expression profiles. Extensive studies have led to a considerable understanding of the cellular and molecular control of hemoglobin production during red blood cell differentiation, however, a complete understanding of human erythropoiesis will require a robust description of the entire transcriptome of these cells during differentiation. From a global point of view of cell metabolic regulation, where genomic information could be complemented with gene expression, the use of methods that enable quantification of the entire transcriptome of the red blood cell during differentiation is of great importance. In this study, the gene expression profiles during differentiation of Human erythroid cells of a normal blood donor in a two-phase liquid culture (Fibach & Rachmilewitz, 1993) were evaluated using Serial analysis of gene expression (SAGE). Global gene expression was evaluated in cells collected immediately before the addition of erythropoeitin (0 hour) and 192 and 336 hours after the addition of this hormone. We generated a total of 30512 tags at 0h, 30117 tags at 192h and 30189 tags at 336h, representing 12026, 11709 and 11337 unique tags, respectively. In the 0h library, a high expression of ferritin genes and CD74 antigen gene was observed. As expected, the expression of globin genes started during intermediate stages of differentiation (predominantly basophilic erythroblasts) and were the most expressed genes at the end of the culture (predominantly orthocromatic erythroblasts). Ribosomal genes were the most expressed genes at 192 hours, indicating an increase in protein synthesis. To identify the genes that were differentially expressed between the libraries, a P value < 0.01 and fold ≥ 5 were considered as statistically significant. In the comparison of the 0h and 192h libraries, 179 differentially expressed transcripts were identified. From these genes, in addition to the globin genes, we found an up-regulation of several genes related to protein binding (LXN, GSTM3, and TRIP6), transcription factor (GATA-1), hydrolase activity (TPSAB1), ion transport (SLC12A9) and regulation of apoptosis (PRDX2). Comparing the 192h and 336h libraries, 103 differentially expressed transcripts were identified. The up-regulated genes were generally related to hemoglobin synthesis, such as ALAS2, involved in the biosynthesis of the heme group or related to intracellular transport such as MSCP and NUDT4 and cell differentiation such as GDF15. The transcription of some of these genes, such as SLC12A9, TRB3, EYA3 and TWIST2 are described for the first time during erythroid differentiation. The results indicated that the global aspects of the transcriptome were similar during differentiation for the majority of the genes and that probably a relative small set of genes is involved in the modification of erythroid cells during differentiation. The results of this study amplify the previous published data (Komor et al, 2005) and may contribute to the comprehension of erythroid differentiation and identification of new target genes involved in some erythroid diseases.

19 Identification of new target genes modulated by HDL3 in human endothelial cells

19 Identification of new target genes modulated by HDL3 in human endothelial cells

A genome-wide transcription analysis of a fungal riboflavin overproducer

The production of many fine chemicals such as vitamins and amino acids is carried out in bioreactors using microorganisms. Usually, these strains are developed from wild-type organisms by classical mutation and selection. After several generations of strain improvement, no further enhancement can be achieved. Therefore, metabolic engineering (ME) is a rational approach to optimise such producer organisms beyond this point, or for starting all over from the beginning. Metabolic Engineering involves detailed analysis of the organism’s metabolic and genetic properties, leading to the identification of new target genes. The fungal riboflavin overproducer Ashbya gossypii converts vegetable oil to vitamin B2 in a “one-step reaction”. The productivity and selectivity of this microorganism have been optimised significantly over the years, first following a classical approach and now a rational one. The improvement is based on our understanding of vitamin B2 metabolism. We have been able to selectively enhance the pathways that are necessary for the formation of riboflavin and to inhibit those leading to unwanted side products. New targets for further improvements of this process have been found using a genome-wide transcript expression analysis; namely massive parallel signature sequencing (MPSS). With this analysis even completely unknown genes can be used for strain improvement.

Use of DNA Microarrays to Monitor Host Response to Virus and Virus-Derived Gene Therapy Vectors

Given the biological complexity of viral infections, the variability of the host response, and the safety concerns related to viral-mediated gene transfer, recent studies have made use of DNA mircoarrays to integrate multi-layered experimental approaches aimed at completely clarifying virus-host interactions. Particular attention has been given to those viruses that are implicated in clinical use and/or in life-threatening diseases. Examples of such use can be divided into three main categories, including: (i) the use of microarrays to study viral expression; (ii) the use of microarrays to analyze the host response to viral infection; and (iii) the use of microarrays to characterize the host response to viral vector-mediated transduction. Significant information on virus- and viral vector-host interactions can be obtained with the microarray approach, including the recognition of master pathways of virally-induced responses, the identification of new target genes for specific viruses, and indications on the molecular toxicity of specific gene transfer vectors currently used for gene therapy trials (in particular, adeno-associated viruses and adenovirus-derived vectors). We predict that the development of accessible repositories containing most of the DNA microarray data on viral infections will certainly help to elucidate the puzzling pictures of different viral infections. This will be crucially important for the correct handling of viral diseases and the intelligent amelioration of viral vectors for gene therapy.

Target Explorer: An automated tool for the identification of new target genes for a specified set of transcription factors.

With the increasing number of eukaryotic genomes available, high-throughput automated tools for identification of regulatory DNA sequences are becoming increasingly feasible. Several computational approaches for the prediction of regulatory elements were recently developed. Here we combine the prediction of clusters of binding sites for transcription factors with context information taken from genome annotations. Target Explorer automates the entire process from the creation of a customized library of binding sites for known transcription factors through the prediction and annotation of putative target genes that are potentially regulated by these factors. It was specifically designed for the well-annotated Drosophila melanogaster genome, but most options can be used for sequences from other genomes as well. Target Explorer is available at http://trantor.bioc.columbia.edu/Target_Explorer/

Nuclear Receptor Target Gene Discovery Using High-Throughput Chromatin Immunoprecipitation

Publisher Summary Nuclear receptors are master transcription factors that regulate the development, physiology, and homeostasis of whole organisms through the direct control of gene expression in response to diverse ligands and hormonal stimuli. Nuclear receptors regulate the expression of their target genes through association with specific DNA regulatory elements. While a significant number of nuclear receptor target genes have been identified to date, it is believed that these genes represent only a small fraction of the regulatory units likely to be under the control of nuclear receptors. Most nuclear receptor target genes identified so far were characterized through ‘‘gene oriented’’ approaches that study the regulation of one candidate gene at a time, and these studies are usually limited to the promoter region. To understand the complex nuclear receptor-driven transcriptional networks that operate in a living organism, a whole genome approach is required. This chapter describes a powerful ‘‘nuclear receptor/whole genome-oriented’’ approach to identify and more accurately study nuclear receptor regulatory networks. Prior to performing the actual Chromatin Immunoprecipitation (ChIP), it is important to remove and freeze an aliquot of the diluted fragmented chromatin corresponding to 10% of the total amount used for one IP. The development of high-throughput ChIP technology now permits a whole-genome analysis of gene regulation by nuclear receptors. The efficient cloning of new regulatory elements harboring high-affinity-binding sites for specific nuclear receptors can now be efficiently achieved, and allows for the identification of new target genes within a particular cell context.

Modulation of endogenous GATA-4 activity reveals its dual contribution to Müllerian inhibiting substance gene transcription in Sertoli cells.

Secretion of Müllerian inhibiting substance by fetal Sertoli cells is essential for normal male sex differentiation since it induces regression of the Müllerian ducts in the developing male embryo. Proper spatiotemporal expression of the MIS gene requires a specific combination of transcription factors, including the zinc finger factor GATA-4 and the nuclear receptor steroidogenic factor-1, which both colocalize with Müllerian inhibiting substance in Sertoli cells. To establish the molecular mechanisms through which GATA-4 contributes to MIS transcription, we have generated and characterized novel GATA-4 dominant negative competitors. The first one, which consisted solely of the GATA-4 zinc finger DNA-binding domain, was an efficient competitor of GATA transcription mediated both by direct GATA binding to DNA and protein-protein interactions involving GATA factors. The second type of competitor consisted of the same GATA-4 zinc finger DNA-binding domain but harboring mutations that prevented DNA binding. This second class of competitors repressed GATA-dependent transactivation by specifically competing for GATA protein-protein interactions without affecting the DNA-binding activity of endogenous GATA factors. These competitors, along with the GATA-4 cofactor FOG-2 (friend of GATA-2), were used to specifically modulate endogenous GATA-4 activity in Sertoli cells. Our results indicate that GATA-4 contributes to MIS promoter activity through two distinct mechanisms. Moreover, the GATA competitors described here should provide invaluable in vitro and in vivo tools for the study of GATA- dependent transcription and the identification of new target genes.