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FASL –844C polymorphism is associated with increased activation-induced T cell death and risk of cervical cancer

The FAS receptor–ligand system plays a key role in regulating apoptotic cell death, and corruption of this signaling pathway has been shown to participate in tumor-immune escape and carcinogenesis. We have recently demonstrated (Sun, T., X. Miao, X. Zhang, W. Tan, P. Xiong, and D. Lin. 2004. J. Natl. Cancer Inst. 96:1030–1036; Zhang, X., X. Miao, T. Sun, W. Tan, S. Qu, P. Xiong, Y. Zhou, and D. Lin. 2005. J. Med. Genet. 42:479–484) that functional polymorphisms in FAS and FAS ligand (FASL) are associated with susceptibility to lung cancer and esophageal cancer; however, the mechanisms underlying this association have not been elucidated. We show that the FAS –1377G, FAS –670A, and FASL –844T variants are expressed more highly on ex vivo–stimulated T cells than the FAS –1377A, FAS –670G, and FASL –844C variants. Moreover, activation-induced cell death (AICD) of T cells carrying the FASL –844C allele was increased. We also found a threefold increased risk of cervical cancer among subjects with the FASL –844CC genotype compared with those with the –844TT genotype in a case-control study in Chinese women. Together, these observations suggest that genetic polymorphisms in the FAS–FASL pathway confer host susceptibility to cervical cancers, which might be caused by immune escape of tumor cells because of enhanced AICD of tumor-specific T cells.

Activation-induced cell death (AICD) refers to the induction of apoptosis of previously activated T cells on subsequent encounter with antigen (1). This mechanism plays a crucial role in both central and peripheral deletion events involved in tolerance and homeostasis of the immune system (2,3). Accumulating evidence also demonstrates that AICD is one of the important mechanisms responsible for the increased apoptosis rate among tumor infiltration lymphocytes (TILs), which may protect transformed cells from elimination by antitumor immune responses and, therefore, contribute to carcinogenesis and cancer progression (4,5).
AICD is a FAS ligand (FASL)-dependent process (6). AICD in peripheral T cells is often caused by the induction of expression of the death ligand, FASL. Cytotoxic T cells increase expression of both FAS and FASL on activation by antigen or other stimulation and subsequently undergo "suicide" or "fratricide" by FASL liganding to FAS (7,8). Antigenic stimulation within the tumor microenvironment might also be involved in the enhanced expression and function of FASL on T cells, resulting in activation of autocrine or paracrine mechanisms of apoptosis (9). FASL-mediated AICD is directly regulated by the level of this death ligand (1), whereas FASL transcriptional regulation happens in both T cells and nonlymphoid cells, such as a variety of tumor cells. In a tumor microenvironment, tumor cells can also counterattack by expressing FASL and inducing the apoptosis of T cells (10,11).
Cervical cancer is strongly associated with infection by oncogenic types of human papilloma virus (HPV) (12). However, only a portion of infected individuals develop cervical cancer during their life, indicating that genetic factors may also contribute to the formation of this cancer. A large body of epidemiological T. Sun, Y. Zhou, H. Li, and X. Han contributed equally to this work. evidence shows that genetic predisposition to cervical cancer may exist (13,14). It has been shown that cervical tumor cells have the ability to induce apoptosis of activated tumor cell-specific cytotoxic T lymphocytes, and this process can be blocked by an inhibitory anti-FAS antibody, suggesting that it is mediated by FAS-FASL interaction (15). Because human cervical tumors (HeLas) indeed express FASL (15)(16)(17), these tumor cells are expected to induce AICD of TILs in vivo. Thus, the expression levels of FASL and FAS might be of importance in determining individual susceptibility to cervical cancer development.
Single nucleotide polymorphisms (SNPs) in the promoter regions of FASL and FAS have been linked to the differential expression of these two genes. It has been shown that the T → C transition at position Ϫ 844 in the promoter region of FASL is located in a binding motif for transcription factor CAAT/enhancer-binding protein ␤ , and a considerably higher basal expression of FASL is associated with the FASL -844C allele compared with the -844T allele (18). In regards to the FAS gene, G → A transition at position -1377 and A → G transition at position Ϫ 670 in the promoter region destroy stimulatory protein 1 and signal transducer and activator of transcription 1 protein binding element, respectively, and thus diminish promoter activity and decrease FAS expression (19,20). We have recently shown that these functional polymorphisms in FASL and FAS have great impact on susceptibility to cancers of the esophagus and lung (21,22). The effect of the FAS polymorphism on the risk of cervical cancer has been reported, but the studies were limited to the Ϫ 670A → G polymorphism, and the results were controversial (23)(24)(25). To date, no study has been reported to examine the association between functional polymorphisms in FASL and the risk for the development of cervical cancer.
We wanted to determine whether the polymorphisms in FAS-FASL have effects on the expression of FAS and FASL and AICD of T cells induced by PHA and HeLa cells in vitro. We also performed a case-control study to examine the association between FAS-FASL polymorphism and the risk of cervical cancer.

FAS and FASL genotypes and cervical cancer risk
Baseline clinical characteristics of cases and controls are summarized in Table I. The age distribution and smoking status in patients were not significantly different from those in controls (P ϭ 0.583 and 0.537, respectively). An absolute majority of cases (97.8%) was infected with HPV. Of 314 patients, 95.2% were classified as squamous cell carcinoma, whereas 3.5 and 1.3% were adenocarcinoma and adenosquamous carcinoma, respectively. According to the International Federation of Gynecology and Obstetrics classification, 21.3% of patients had carcinoma in situ (stage 0), whereas 21.3, 37.6, and 18.5% of patients had stage I, II, and III disease, respectively. Only 1.3% of patients had stage IV cervical cancer.
Genotype analyses of FAS and FASL polymorphisms were successful for all patients, but 13 controls failed to be The heterozygous CT genotype also presented a higher risk for the cancer (OR ϭ 1.68; 95% CI ϭ 0.78-3.66), although the association was not statistically significant (P ϭ 0.187). In the stratification analysis, age and smoking had no effect on the risk of cervical cancer related to FASL genotypes (unpublished data). Because of tight linkage, the haplotype of FAS Ϫ 1377G → A and FAS Ϫ 670A → G did not further increase the risk of the cancer (unpublished data). In view of the fact that FAS and FASL work together in apoptotic cell death, we examined whether there was a statistical interaction between the SNP in FAS and FASL that was associated with the risk of cervical cancer. However, no such interaction was evident (unpublished data).

FAS and FASL genotypes and their expression on T cells
To determine the effects of the SNPs in FAS and FASL on expression of the genes, PBMCs isolated from 40 healthy donors carrying different genotypes were incubated with PHA or mitomycin C (MMC)-treated HeLa cells. The proportion of CD25 ϩ /CD4 ϩ cells to CD3 ϩ cells was determined as an index of T cell activation. In addition, the levels of IL-2 in the supernatants of culture media of PBMCs were also determined as an indicator of T cell activation. It was found that the proportion of CD25 ϩ /CD4 ϩ cells (mean Ϯ SD, n ϭ 40) in the PBMC cultures with PHA was 50.83 Ϯ 14.08%, which was significantly higher than that in the cultures without PHA (2.27 Ϯ 0.88%; P Ͻ 0.001). In parallel, the IL-2 level (mean Ϯ SD, n ϭ 40) in the PBMC culture media with PHA was significantly higher than that in the PBMC culture media without PHA (191. 25  conditions. Moreover, neither the elevated population of CD25 ϩ /CD4 ϩ cells nor IL-2 concentration in the media was correlated to FAS -1377, FAS -670, or FASL -844 genotypes (unpublished data). Next, we examined whether the FAS -1377 or FAS -670 and FASL -844 SNPs had effects on the expression of FAS and FASL on T cells in PHA-or HeLa cell-stimulated PBMCs. The representative flow cytometry determination of FAS and FASL expression is shown in Fig. 1, A and B. We observed a significant difference (P Ͻ 0.05) in the FAS expression on T cells after incubation with PHA as a function of FAS genotype ( Fig. 1 C). The FAS -1377GG or GA genotype had a significantly heightened FAS expression levels (mean Ϯ SD) compared with the AA genotype (66.63 Ϯ 3.53% [ n ϭ 12] and 57.92 Ϯ 2.61% [n ϭ 22] vs. 46.31 Ϯ 5.02% [n ϭ 6]; P ϭ 0.002 and 0.048, respectively), whereas the levels were not significantly different (P ϭ 0.355) between the GG and GA genotype. Similar results were obtained for the FAS -670 polymorphism, which is in almost complete linkage disequilibrium with the FAS -1377 polymorphism. However, the expression of FAS in PBMCs incubated with HeLa cells among three genotypes was not statistically significant (P Ͼ 0.05; Fig. 1 C). For FASL expression after stimulation by PHA, the FASL -844CC genotype had significantly higher levels than the CT or TT genotype (1.72 Ϯ 0.10% [n ϭ 18] vs. 1.22 Ϯ 0.10% [n ϭ 18] and 0.32 Ϯ 0.25% [n ϭ 4]; P ϭ 0.002 and P Ͻ 0.0001, respectively), and the difference between the heterozygous CT genotype and the variant homozygous TT genotype was also statistically significant (P ϭ 0.002). After stimulation by HeLa cells, the FASL expression in subjects with the CC genotype was 0.48 Ϯ 0.04%, which was significantly higher than that in subjects with the CT (0.36 Ϯ 0.03%; P ϭ 0.018) or TT (0.19 Ϯ 0.09%; P ϭ 0.004) genotypes, although the levels between the CT and TT genotypes were not statistically different, possibly because of limited statistical power ( Fig. 1 D). These findings clearly demonstrated that the investigated polymorphisms in the promoter region of FAS and FASL have a substantial impact on the activationinduced expression of FAS and FASL on T cells.

FAS and FASL genotypes and AICD of T cells
To examine the effects of FAS and FASL genotypes on AICD of T cells, PBMCs incubated with PHA or HeLa cells were labeled with FITC-Annexin V, propidium iodide (PI), and PerCP-Cy5.5-CD3, and the proportion of Annexin V ϩ cells in the CD3 ϩ gate were determined by flow cytometry (Fig. 2  A). We found that neither FAS nor FASL genotypes had an effect on the spontaneous apoptosis of T cells during culture. However, when T cells were incubated with either PHA or HeLa cells, the differences in AICD among three different FASL -844 genotypes were striking. As shown in Fig. 2 B, after incubation with HeLa cells, the mean proportion (ϮSD) of Annexin V ϩ cells was significantly higher in T cells carrying the FASL -844CC genotype (11.59 Ϯ 5.39) compared with that in T cells carrying the -844CT (7.36 Ϯ 3.45; P ϭ 0.006) or -844TT (6.76 Ϯ 0.34; P ϭ 0.068) genotype. Similarly, after incubation with PHA, the differences in AICD of T cells among three FASL genotypes were also significant, with the proportion of Annexin V ϩ cells being higher in T

ARTICLE
cells carrying the -844CC genotype than that in T cells carrying the -844TC or -844TT genotype (P Ͻ 0.05 for both). However, the differences in AICD of T cells stimulated either with HeLa cells or with PHA were not significant (P Ͼ 0.05) among different FAS -1377 genotypes (Fig. 2 C).

DISCUSSION
Nearly every cell in the body has the potential to threaten the life of an individual if it transforms into cancer. Therefore, the immune surveillance of T cells plays an essential role in maintaining homeostasis (1-3). The FAS-FASL system regulates the AICD process of T cells, and genetic variations in these cell death pathway genes may thus influence susceptibility to cancers. Functional germline and somatic mutations in the FAS gene, and perhaps also in the FASL gene, that impair apoptotic signal transduction are associated with susceptibility to cancer (26)(27)(28)(29)(30). We have recently reported that functional SNPs in FAS and FASL increase the risk of esophageal cancer and lung cancer in a Chinese population (21,22). Cervical cancer is a unique tumor that tightly links to infection of oncogenic types of HPV. So far, no specific oncogenes or tumor suppressor genes have been defined in this cancer (12). Therefore, we performed a case-control study to investigate whether genetic polymorphisms in FAS and FASL confer susceptibility to cervical cancer, a typical viral infection-associated cancer, in a Chinese population. Based on the analysis of 314 patients and 615 frequencymatched controls, we found that subjects carrying the FASL -844CC genotype had a threefold increased risk for cervical cancer compared with those carrying the FASL -844TT genotype, whereas the FAS polymorphisms seemed not to be associated with the risk of this cancer. In addition, we observed no associations between genotypes and histological types or clinical stages of cervical cancer. However, this might be the result of a limited sample size in each stratum, and more studies with stronger statistical power would be helpful to clarify the associations.
FASL -844T→C polymorphism has been shown to have a substantial impact on promoter activity of the FASL genes in an in vitro assay system because it is located in a binding motif for transcription factor CAAT/enhancer-binding protein ␤ (18). However, little or nothing is known so far about genotype and phenotype association; i.e., the effect of this polymorphism on the expression of FASL in vivo in cells. Our data in the present study provide evidence for the first time that the -844T→C polymorphism in FASL strongly in- fluences the expression of FASL ex vivo on T cells stimulated by either cervical cancer cells or PHA. More importantly, our study demonstrates that the FASL -844C allele, which had higher expression of FASL on T cells, was associated with an enhanced rate of AICD of T cells. This genotype and phenotype correlation between FASL -844T→C polymorphism and AICD of T cells was consistent with molecular epidemiological findings, demonstrating the FASL -844C allele as an at-risk allele for cervical cancer. Because FASL-FAS-mediated AICD is considered to be one of the important mechanisms responsible for the TIL apoptosis occurring in the tumor microenvironment (4-7), individuals carrying the FASL -844C allele and, thus, with higher FASL expression on T cells on tumor antigenic stimulation would be anticipated to be susceptible to cancer development. On the other hand, it has been shown that various types of tumors express FASL, and aberrant FASL expression has been linked to cervical carcinogenesis and tumor progression (15)(16)(17). Because of the functional consequence of the FASL -844T→C polymorphism, it would be expected that individuals carrying the FASL -844C allele may also have higher FASL expression on tumor cells compared with those carrying the FASL -844T allele. Heightened expression of FASL may facilitate transformed cells to counterattack the FASexpressing TILs (4,10,11,15,31), resulting in immune evasion of these malignant cells. This mechanism may also contribute to an increased risk for developing cervical cancer among subjects carrying the risk FASL allele.
We did not observe any substantial association between the investigated functional polymorphisms in FAS and the rate of AICD of T cells stimulated by PHA or HeLa cells, although the polymorphisms did somewhat influence the expression of the FAS gene ex vivo on T cells. This finding was consistent with the population data in the present study but was inconsistent with our previous results from large molecular epidemiological studies on esophageal and lung cancer showing a positive association between the FAS polymorphisms and risk of these cancers (21,22). This discrepancy may reflect the essential difference in carcinogenesis between cervical cancer and lung or esophageal cancer. However, these results may also suggest that, unlike FASL, a subtle change of widely expressed FAS might not be sensitive enough to detect a small change of AICD of T cells and the risk of the cancer. More studies with a larger sample size and stronger statistical power are needed to get conclusive results.
The data from the genotype-phenotype analysis in the present study are reliable because our experimental model using ex vivo PBMCs treated with HeLa cells mimicked the situation in vivo. HeLa cells are integrated with HPV-18 (32), and the antigen extracted from these cancer cells contains HPV oncoproteins, which are suitable for testing the response of cytotoxic T lymphocytes to both HPV infection and cervical tumor cells. In addition, it has been shown that HeLa cells persistently express FASL (15), which allows tumor cells in our experimental model to counterattack the Fas-expressing T lymphocytes using the T cell's own principal mechanism of cytotoxicity. Finally, all PBMC samples we selected for testing were of the HLA-A2 genotype, which are matched to HeLa cells (33). Therefore, the T cells sensitized by HeLa cell antigen could be activated by the following treatment with HeLa cells. Our results showing increased AICD caused by interaction of T cells with HeLa cells or PHA clearly demonstrate that our experimental system is valid.
Although little is known about polymorphisms in FASL and susceptibility to cervical cancer, at least three studies have been published regarding the FAS -670 polymorphism and the risk of cervical cancer. Lai et al. (23) conducted a study in a Chinese population in Taiwan and reported that the FAS -670A allele was associated with an increased risk of squamous intraepithelial lesions and squamous cell carcinoma. However, Dybikowska et al. (24) and Engelmark et al. (25) have recently shown that the FAS -670 genotype is not associated with a risk of the cancer in Caucasian populations. In this study, we analyzed both FAS -670 and -1377 polymorphisms and found that these two tightly linked polymorphisms were not associated with an increased risk of cervical cancer. The reasons for the inconsistent results among these studies are not clear, but geographic or ethnic differences in genotype frequencies should be considered, as have been extensively discussed in our previous reports (21,34). In this study with 615 healthy controls, we observed a frequency of 12.2% for the variant FAS -670GG genotype, which is not significantly different from that reported by Dybikowska et al. (18.5%; P ϭ 0.06) but significantly lower than that reported by Lai et al. (20.5%; P Ͻ 0.001). The genotype frequencies are not available in the report of Engelmark et al. because of the sib-pair design in their study (25). These geographic or ethnic differences in genotype frequencies might partially explain the dissimilar results.
Both FAS Ϫ670A→G and FAS Ϫ1377G→A SNPs were shown to have a biological impact on the transcriptional activity of the FAS gene, although the effect of Ϫ670A→G is still ambiguous (20,35). We found a heightened expression of FAS on T cells stimulated by PHA that was associated with either the FAS Ϫ1377AA or FAS Ϫ670GG genotype. However, the role which SNP actually plays in influencing the gene expression remains unclear, because these two SNPs are in almost completely linkage disequilibrium in our donor population. It would be interesting and important to distinguish between an effect at Ϫ1377 and the effect at Ϫ670, and studies are underway to explore this issue. Another limitation of our study is that the HPV status of controls is unknown, and this did not allow us to explore whether the findings reflect the association of the genotypes with control of HPV infection (i.e., the ability to prevent persistent HPV infection) or the ability to eliminate infected cells before they undergo the transformation process.
In conclusion, our study demonstrates an association between the genotype and phenotype of FASL polymorphism ARTICLE and suggests that heightened AICD of T lymphocytes and/ or counterattack of tumor cells against TILs resulting from functional polymorphisms in the FAS-FASL pathway may be involved in the mechanism underlying an individual's susceptibility to the development of cervical cancer.

MATERIALS AND METHODS
Study subjects. The case-control study consisted of 314 patients with cervical cancer and 628 controls. All subjects were ethnic Han Chinese and residents of Beijing and its surrounding regions. Patients were recruited between June 2001 and March 2002 at the Cancer Hospital, Chinese Academy of Medical Sciences (Beijing, China). All eligible patients diagnosed with cervical carcinoma at the hospital during the study period were recruited, with a response rate of 94%. Healthy controls were from a nutritional survey conducted in the same region during the period of case collection. The control subjects were randomly selected from a database consisting of 2,500 individuals based on a physical examination, including chest radiography and abdominal ultrasonography. Gynecological and cervical cytological examinations were also performed. The selection criteria included a history of no cancer or previous operations on the uterine cervix and free of cervical intraepithelial neoplasia. Controls were frequency matched to cervical cancer patients by age (Ϯ5 yr). Most control subjects (n ϭ 520) were characterized in a breast case control study as reported previously (36). We added 108 more controls selected from the same database to make a 2:1 matching to the cases and to increase the statistical power. For FAS/FASL expression and AICD assays, blood samples were obtained from a group of 40 healthy volunteers (22 males and 18 females) working in our institution aged 19-49 yr (mean Ϯ SD ϭ 31.8 Ϯ 8.6). All these volunteers were HLA-A2 positive with no history of malignant disease and clinically evident HPV infection. At recruitment, informed consent was obtained from each subject, and this study was approved by the Institutional Review Board of the Chinese Academy of Medical Sciences Cancer Institute.
Genotype analysis. Genotypes of FAS -1377G→A, FAS -670A→G, and FASL Ϫ844T→C polymorphisms were determined by PCR-based restriction fragment length polymorphism assays as described previously (21). All subjects were successfully genotyped except for 13 controls whose DNA samples failed to be amplified. To ensure quality control, genotyping was performed without knowledge of case/control status of the subjects, and a 15% random sample of cases and controls was genotyped twice by different persons; the reproducibility was 100%.
HeLa cell culture, antigen preparation, and MMC treatment. HeLa cells (HLA-A2.1) were cultured in RPMI 1640 medium with 10% FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin at 37ЊC in a humidified 5% CO 2 atmosphere. For production of HeLa cell antigen, portions of logarithmic growth HeLa cells were collected and washed with cold PBS. These cells were then shattered by freezing in liquid nitrogen and melted in a 37ЊC water bath alternately five times, followed by centrifugation at 2,200 g for 15 min at 4ЊC. The supernatants were collected and the total amount of protein was determined. Another portion of 10 6 HeLa cells/ml in RPMI 1640 medium was treated with 100 mg/ml MMC for 1 h at 37ЊC with occasional shaking to inhibit proliferation. These cells were then washed four times with the medium before use in the activation of PBMCs as described below.
PBMC isolation and culture. 5 ϫ 10 6 PBMC/ml were isolated by Ficoll-Paque (Sigma-Aldrich) density-gradient centrifugation and suspended in RPMI 1640 medium, as used for HeLa cells. PBMCs were distributed into 24-well tissue culture plates at 1 ml/well and cultured in the presence of 25 g/ml PHA for 6 h or cultured with 50 g/ml HeLa cell antigen for the first 4 h, followed by the addition of MMC-treated HeLa cells (1:10 PBMCs) and cultured for the next 68 h. PBMCs cultured without PHA or HeLa antigen/cells served as respective controls. Cultured PBMCs were used to assay expression of FAS, FASL, and CD25 and the apoptotic index among CD3 ϩ or CD4 ϩ cells, and the cell culture supernatants were collected to determine IL-2 production as described below.
Flow cytometry analysis. A flow cytometer (FACSCalibur; BD Biosciences) was used to determine the cell surface expression of FAS, FASL, CD25, CD3, and CD4 and cell apoptosis index indicated by Annexin V and PI staining. Cultured PBMCs, as described above, were washed twice in cold PBS and incubated in PBS plus 3% FCS with 5 l PE-labeled anti-FASL, 5 l FITC-labeled anti-FAS, and 5 l PerCP-Cy5.5-labeled anti-CD3 (tube 1); 5 l FITC-labeled anti-CD25, 5 l PE-labeled anti-CD3, and 5 l PerCP-Cy5.5-labeled mouse anti-CD4 (RPA-T4, IgG1; tube 2); or 5 l FITC-labeled Annexin V, 5 l PI staining solution, and 5 l PerCP-Cy5.5-labeled anti-CD3 (tube 3) in a 100-l volume for 15 min in the dark at room temperature (25ЊC). The samples were washed twice with PBS-FCS, followed by the addition of 500 l PBS with 1% paraformaldehyde to tubes 1 and 2 or 400 l 1ϫ BD Annexin V binding buffer to tube 3. The Annexin V-PI was analyzed by flow cytometry within 1 h, and other samples were stored at 4ЊC in the dark and analyzed within 24 h. A total of 50,000 events were counted within the lymphocyte gate for each samples.
IL-2 assay. PBMC culture media were collected, and IL-2 concentrations were determined by a human IL-2 ELISA kit (R&D Systems), according to the manufacturer's protocol. ODs were measured at 450 nm with a multilabel counter (Wallac Victor 2 1420; PerkinElmer Life and Analytical Sciences).

Statistical analysis.
Results of cell surface molecule and expression, apoptosis index, and secreted IL-2 were expressed as means Ϯ SD or as median. Statistical analysis of these results was done by the one-way analysis of variance test. Because the expression levels of FAS and FASL on T cells might be influenced by the activation status of T cells (37), an analysis of covariance was performed when analyzing FAS/FASL expression by using the expression level of CD25 ϩ /CD4 ϩ cells and the concentration of IL-2 in cell culture media as covariates. A p-value of Ͻ0.05 was used as the criterion of statistical significance, and all statistical tests were two-sided tests. The association between FAS and FASL polymorphisms and the risk of cervical carcinoma was estimated using ORs and their 95% CIs, which were calculated by unconditional logistic regression. All the ORs were adjusted for age and smoking status. We tested the null hypotheses of additive and multiplicative gene-gene interactions between the FAS and FASL polymorphisms and evaluated departures from additive and multiplicative interaction models (38) by including main effect variables and their product terms in the logistic regression model. All analyses were performed with Statistical Analysis System software (version 6.12; SAS Institute). Haplotype frequencies and linkage disequilibrium coefficient were estimated using PHASE (39) and 2LD software (40).


extfieldsofstudy: Biology Medicine

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A novel approach for inhibition of HIV-1 by RNA interference: counteracting viral escape with a second generation of siRNAs

RNA interference (RNAi) is an evolutionary conserved gene silencing mechanism in which small interfering RNA (siRNA) mediates the sequence specific degradation of mRNA. The recent discovery that exogenously delivered siRNA can trigger RNAi in mammalian cells raises the possibility to use this technology as a therapeutic tool against pathogenic viruses. Indeed, it has been shown that siRNAs can be used effectively to inhibit virus replication. The focus of this review is on RNA interference strategies against HIV-1 and how this new technology may be developed into a new successful therapy. One of the hallmarks of RNAi, its sequence specificity, also presents a way out for the virus, as single nucleotide substitutions in the target region can abolish the suppression. Strategies to prevent the emergence of resistant viruses have been suggested and involve the targeting of conserved sequences and the simultaneous use of multiple siRNAs, similar to current highly active antiretroviral therapy. We present an additional strategy aimed at preventing viral escape by using a second generation of siRNAs that recognize the mutated target sites.

INTRODUCTION
Highly-active antiretroviral therapy (HAART), which involves the use of multiple antiviral agents, has been very successful in reducing the rates of progression to AIDS and death in people with HIV-1. However, adverse effects of the long-term use of these drug regimens, like toxicity and the emergence of drug resistant variants, obviate the need for new and improved methods to suppress HIV-1 in infected individuals. Recently, it was shown that delivery of siRNAs in mammalian cells can trigger a highly specific RNA interference response resulting in knockdown of the gene of interest. This finding raised the possibility that RNA interference can be used as a potent antiviral tool against HIV-1. siRNAs targeting HIV-1 sequences can inhibit virus production and virus replication in transient transfection experiments (Capodici et al, 2002;Coburn and Cullen, 2002;Hu et al, 2002;Jacque et al, 2002;Lee et al, 2003;Park et al, 2003;Dave and Pomerantz, 2004). The transient nature of these methods is not desirable in a therapeutic setting dealing with a persistent viral infection. Although patients may be treated with a stabilized form of siRNAs, the risk of acquiring resistant HIV-1 mutants is relatively high because the concentration of siRNAs will vary in different body compartments. Furthermore, delivery and toxicity remain major issues for a treatment with synthetic siRNAs. In contrast, if patients are treated with a stably integrated viral vector expressing a steady supply of interfering RNAs, HIV-1 replication will be inhibited constantly. siRNAs can be expressed from polymerase-III transcription units. The sense and anti-sense strands can be expressed from two different promoters, and RNAi is triggered upon annealing of the two strands (Lee et al, 2002;Banerjea et al, 2003;Tran et al, 2003). More potent inhibition was achieved when the sense and antisense strand were expressed as a single transcript with the ability to from a duplex hairpin structure (Brummelkamp et al, 2002). HIV-1 replication can be effectively inhibited with such short hairpin RNAs (shRNA) in stably transduced cell lines (Boden et al, 2003a;Unwalla et al, 2004;Das et al, 2004;Boden et al, 2004a;Lee et al, 2005).
Because RNAi is highly sequence specific, a single point mutation can potentially result in full resistance to RNAi. Indeed, resistant variants can emerge when a single antiviral shRNA is used (Boden et al, 2003a;Das et al, 2004). The emergence of RNAi-resistant mutants poses a challenge for the development of RNAi based treatments for HIV-1 infected individuals. It has been suggested to use multiple highly-effective shRNAs that target conserved regions of the viral genome (Berkhout, 2004). More potent suppression will further reduce the ability of the virus to evolve, and escape from multiple shRNA inhibitors will require several mutations. In this report, we will present an overview of current publications on RNAi against HIV-1. Furthermore, we have analyzed all published HIV-1 targets and present an outline for the effective use of RNAi against HIV. This includes a new strategy to prevent the emergence of resistant variants by including additional shRNAs that are directed against the most likely escape mutants.
HIV-1 escapes from a single shRNA Two recent studies addressed the potency and durability of anti-HIV RNAi approaches. A short hairpin RNA (shRNA) against the tat gene was expressed from an H1promoter in an adeno-associated virus vector (Boden et al, 2003a). Potent inhibition of HIV-1 replication was scored, but an escape virus appeared in prolonged cultures. Similar results were described for a shRNA against the Nef gene using a retroviral vector (Das et al, 2004). The latter study described 7 independent HIV-1 escape variants. These studies convincingly demonstrate that inhibition is potent and sequence-specific, but also that HIV-1 is able to escape from the inhibitory action of a single shRNA. Boden et al described a single escape virus with a point mutation in the target sequence, and Das et al (2004) described a multitude of escape routes (point mutation, double point mutation, partial or complete deletion of the target sequence). The possibility of virus escape seems even more apparent with the recent demonstration that HIV-1 can also gain resistance by a point mutation outside the target sequence (Westerhout et al, 2005). This mutation was demonstrated to change local RNA folding, such that the target sequence becomes inaccessible to the RNAi machinery. The emergence of resistant virus variants poses a serious problem for using RNA interference in a therapeutic setting.
Counteracting viral escape with multiple shRNAs One strategy to counteract viral escape is to use multiple shRNAs targeting different conserved regions of HIV-1 (Haasnoot et al, 2003;Stevenson, 2003;Hannon and Rossi, 2004;Berkhout, 2004;Shankar et al, 2005). Conserved targets in the HIV-1 RNA genome will not allow for a deletion-based escape route, as occurred with the shRNA targeting the non-essential Nef gene sequences (Das et al, 2004). One can try to estimate the chance of escape with one versus multiple shRNAs. First, we make the assumption that deletion is not an option for the virus; only point mutations are allowed to occur. In principle, a single point mutation can make the virus insensitive to RNAi. The error rate of the reverse transcriptase of HIV-1 is 3 x 10 -5 (Mansky, 1996), consequently the chance of viral escape for a 19 nucleotide target in a single infection is 19 x (3 x 10 -5 ) = 5.7 x 10 -4 . Studies in the field of drug resistance indicate that an untreated HIV infected individual has a virus population size of 10 4 to 10 5 . This means that for each shRNA, several potential escape variants are already present before the start of therapy. Thus, the emergence of a drug resistant variant seems inevitable when a single shRNA is used. When multiple shRNAs (N) are used simultaneously, the likelihood of obtaining a drug resistant variant drops exponentially with the number of shRNAs (5.7 x 10 -4 ) N . If we assume that there is already resistance to at least one of the shRNAs used in a patient, than the chance of a resistant variant emerging is (5.7 x 10 -4 ) N-1 . For instance, if four shRNAs are used simultaneously, the chance of escape is 1.9 x 10 -10 . Although this chance seems remote given the average viral load in a patient, it cannot be excluded that multi-shRNA resistant mutants can evolve through recombination.
That resistance can evolve, even after prolonged in vitro culturing, indicates that suppression of HIV-1 replication is not absolute when a single shRNA is used. Recently, it was shown that inhibition of a target gene is increased proportionate to the shRNA expression level. This result was obtained by combining multiple expression cassettes encoding the same shRNA in a single vector (Gonzalez et al, 2005). In addition, the combination of two different shRNAs resulted in effective and simultaneous inhibition of two targets, while their individual activity was maintained Schubert et al, 2005a). These results indicate that, when a multiple shRNA strategy would be used against HIV-1, the magnitude of inhibition will increase, perhaps approaching complete inhibition, combined with a severely reduced chance of escape.

Target site selection
During the HIV-1 life cycle, there are several steps at which RNA interference may inhibit replication ( Figure  1A). Upon virus infection, the incoming RNA genome is a potential target. There is controversy with regards to targeting of the incoming HIV-1 genome with RNAi. Several publications report RNAi-mediated degradation of the RNA genome during infection in cells transfected with siRNAs (Capodici et al, 2002;Coburn and Cullen, 2002;Jacque et al, 2002), while others reported no effect at all (Surabhi and Gaynor, 2002;Hu et al, 2002). These contradicting results may perhaps be explained by differences in target accessibility in the context of the packaged RNA genome, since viral and/or cellular proteins may protect some regions, but not all. Variations in experimental conditions, e.g., the cell types used, differences in transfection efficiency, variable amounts of siRNAs or infectious HIV-1 virus used, may also have caused these differences. Recently, it was shown that cells expressing shRNAs from an integrated lentiviral vector effectively blocked integration of the DNA provirus at low multiplicity of infection; this effect was overcome with a high dose of virus (Joshi et al, 2005). This result shows, in a relevant gene therapy setting, After virus infection, the HIV-1 core with the RNA genome will be released into the cytoplasm. The incoming RNA genome represents a desirable target, since destruction of the genome (route 1) prevents reverse transcription and integration of the provirus. After integration of the provirus, viral messenger RNAs are expressed. Viral mRNAs can potentially be degraded in the nucleus (route 2) or in the cytoplasm (route 3), preventing viral protein production and virus particle assembly and release. (B) The proviral DNA genome. Early gene expression results in fully spliced mRNAs encoding for Tat, Rev and Nef. Late gene expression results in partially spliced mRNAs, encoding for Env, Vif, Vpr, and unspliced mRNA for Gag and Gag-Pol proteins or as the genomic RNA. that the incoming HIV-1 genome can be subject to RNAimediated degradation.
Late in infection, viral mRNAs are transcribed from the integrated provirus. The early viral transcripts are processed, resulting predominantly in spliced transcripts in the cytoplasm ( Figure 1B). Once sufficient Rev protein is made, partially spliced messengers and unspliced genomic transcripts will be exported out of the nucleus into the cytoplasm. It is generally believed that RNA interference is a cytoplasmic phenomenon (Meister and Tuschl, 2004;Shankar et al, 2005). Since it may be beneficial to block early viral gene expression, one may prefer HIV-1 target sequences that are present in (all) early transcripts (Purcell and Martin, 1993). For instance, down-regulation of Tat and Rev protein expression may block virus production early on. In the absence of Tat protein, no induction of the viral LTR promoter occurs and without Rev the shift towards unspliced RNA cannot be made. In contrast, targeting sequences present exclusively in late unspliced mRNAs will allow for the production of early spliced mRNAs and early viral proteins.
It has been shown that siRNAs targeting early transcripts down-regulate all transcripts after infection with HIV-1 (Coburn and Cullen, 2002;Lee et al, 2003). Contrary to expectation, an siRNA targeting a gag sequence that is exclusively present in unspliced, late mRNA gave a similar result (Novina et al, 2002). Although this result may reflect the combined effect of the destruction of the incoming genome and the targeting of viral messengers, it may also hint at the possibility that viral transcripts are cleaved by RNAi in the nucleus prior to splicing. This option was previously discarded because RNAi was considered to be an exclusively cytoplasmic event (Novina et al, 2002). Supporting the concept of nuclear targeting of HIV-1 mRNAs, both nuclear and cytoplasmic viral RNA was downregulated by a lentiviral shRNA against the late vpu transcript (Chang et al, 2005). It was recently shown that RNA interference can effectively degrade mRNAs that are localized in the nucleus of mammalian cells (Robb et al, 2005). Similar results were obtained with shRNAs delivered by a lentiviral vector (Langlois et al, 2005). These studies suggest that RNAi may also occur in the nucleus, thus explaining the results obtained with siRNAs that target late viral transcripts.
In addition to the degradation of RNA, which is the focal point of this review, two other functions of RNA interference may also contribute to inhibition of HIV-1 replication. First, the RNA interference silencing complex may silence mRNA translation. This process is less sensitive to sequence variation since perfect basepairing of siRNA and mRNA is not required. Second, inhibition can also occur through transcriptional silencing mediated through chromatin remodeling (Morris et al, 2004;Kawasaki and Taira, 2005). This would prevent the expression of viral transcripts. It was recently shown that targeting of the HIV-1 LTR promoter region results in sustained inhibition (Suzuki et al, 2005). Interestingly, methylation of CpG motifs was observed in and surrounding the siRNA target regions, implicating that transcriptional silencing suppressed HIV-1 replication.
Since there is support for shRNA induced targeting of the incoming viral RNA genome and also suggestive evidence  that viral mRNAs may be degraded prior to splicing in the nucleus, we propose that target sites can be chosen throughout the HIV-1 genome. Selection can therefore be based solely on conservedness of the target sequence in different HIV-1 isolates. We have listed all 42 published siRNAs and shRNAs that were successfully used to inhibit HIV-1 production or replication (Table 1). It is important to note that each study used different ways to score inhibition of HIV-1 production, and in a few cases HIV-1 replication, such that a direct comparison is not possible. There is ample evidence that effective siRNAs against HIV-1 can be transformed into effective shRNAs (Coburn and Cullen, 2002;Jacque et al, 2002;Han et al, 2004;Das et al, 2004;Nishitsuji et al, 2004;Boden et al, 2004a;Lee et al, 2005). We aligned all 42 RNAi target sequences with 538 HIV-1 full genome sequences available from the Los Alamos National Laboratory database using the PrimaAlign tool available from their website (http://www.hiv.lanl.gov). We calculated the percentage of viral isolates that show complete similarity to the published target sequence ( Table 1). Nine of the 42 target sequences are conserved in at least half of known isolates, which represent the best candidates for the multiple shRNA approach.
Counteracting viral escape with a 2 nd generation of shRNAs Although, the Nef gene target (Jacque et al, 2002;Das et al, 2004) is not highly conserved (Table 1) and, therefore, not perfectly suited for an RNAi based therapy, the extensive set of viral escape variants described for this RNAi treatment (Das et al, 2004) provided us with a tool to test a different strategy to prevent viral escape. The novel approach is to include a second generation of shRNAs that target the most likely escape variants ( Figure 2A). We constructed pSUPER vectors (Brummelkamp et al, 2002) expressing shRNAs directed against the mutated Nef sequences of the escape viruses ( Figure 2B). HIV-1 molecular clones, based on pLAI (Peden et al, 1991), with one or two nucleotide substitutions in the target ( Figure 2B), were co-transfected with a control pSUPER shLUC vector (Figure 2C, black bars) and relative virus production, as measured by Gag CA-p24 production, was set at 100%. Cotransfection of these molecular clones with the shNEF-wt plasmid showed strong inhibition of wild-type pLAI, but not of the variants with a mutated target site, thus confirming the sequence specificity of RNAi ( Figure 2C, grey bars). All pLAI variants, except the wild-type, were cotransfected with the matching shRNA ( Figure 2C, white bars). The R3 and R3' escape mutants are potently inhibited by their matching shRNA and the level of inhibition is comparable to the original situation of the wild-type virus with the wild-type shRNA. For the R4 escape mutant, inhibition is improved with the matching shRNA (white bar) compared to the wild-type shRNA (grey bar), although inhibition is only partially restored.
R6 is a special escape mutant because the single nucleotide substitution imposes an altered RNA folding that occludes the target site (Westerhout et al, 2005). Therefore, RNAi is not rescued with the matching shRNA. For the R9 mutant, the matching shRNA is not effective at all. These results show that it is possible to reconstitute at least some level of inhibition, and in most cases restore complete in-hibition, with a second generation of shRNAs aimed at escape variants.
We can apply this concept to the effective and conserved targets of HIV-1 listed in Table 1. One can empirically determine which target site mutations result in replication competent escape variants by studying virus escape in cell culture infections. For the targets with a conserved sequence, we assume that most point mutations will affect virus replication. A conserved sequence may have multiple functions, e.g., multiple overlapping reading frames and RNA or DNA signals that code for splicing, packaging or promoter functions. Because it is likely that multiple mutations in the target sequence will result in impairment of its function(s), a first assumption is that only a single point mutation is allowed. A second assumption is that only silent codon changes can occur. For example, the conserved tat/rev sequence (Surabhi and Gaynor, 2002) encodes two overlapping reading frames (Figure 3). There are 9 silent codon changes possible for each individual open reading frame, but only at position 7 and 19 these changes are silent in both reading frames ( Figure 3). In both cases, a silent variation at the first nucleotide position of an Arginine (R) codon in tat coincides with a silent change of a third nucleotide of a rev codon. A combination of three shRNAs targeting the wt, A7C and A19C sequences may prevent viral escape.
In general, this strategy may be applied to all conserved HIV-1 targets. It is important to note that the success of this approach depends upon several criteria. First, only a limited number of escape routes should be possible, otherwise too many second generation shRNAs have to be designed. Second, the new shRNA should be capable of inducing RNAi. For instance, shNEF-wt partially inhibits the escape mutant LAI-R9 but the adapted shNEF-R9 failed to silence LAI-R9 at all, indicating that this particular shRNA is inactive ( Figure 2C). Finally, the target site should remain accessible (Westerhout et al, 2005;Schubert et al, 2005b). The R6 escape variant provides an example where any shRNA is likely to be ineffective because this nucleotide substitution induced a change of the RNA structure such that the target sequence is inaccessible (Westerhout et al, 2005).

Anti-AIDS gene therapy
For an effective therapy, the RNAi-triggering genes have to be transferred into the appropriate cells. Such a gene therapy protocol seems ideally suited for the treatment of individuals that are chronically infected with HIV-1 and fail on standard antiretroviral therapy. In chronically infected individuals, HIV-1 infects a significant fraction of the mature T cells each day, leading to cell killing directly by HIV-1 or indirectly by the HIV-induced immune system. In addition, HIV-1 infection may induce immune activation resulting in apoptosis in uninfected bystander cells (Herbein et al, 1998). Nevertheless, the preferential survival of even a minority of shRNA-expressing cells will result in their outgrowth over time. One could carry out an ex vivo gene therapy treatment of either the patients mature CD4+ T cells from the blood or CD34+ haematopoietic stem cells from the bone marrow, and give them back to the patient. The stem cells will proliferate into mature T  (Strayer et al, 2005). However, the introduction of shRNAs against highly conserved HIV-1 sequences may pose a problem for vector production, since these shRNAs could cross-react with critical sequences of the vector. Indeed, it has been reported that expression of shRNAs that target the vector can result in a reduction of the titer Chang et al, 2005). One may expect that when a multiple shRNA expression lentiviral vector is developed, this will become a serious issue, but solutions for this potential problem are already available. With safety in mind, lentiviral packaging plasmids were developed that are optimized for human codon usage (Kotsopoulou et al, 2000;Wagner et al, 2000), and thus quite different from the original viral sequences. We found that the reduction in titer will be resolved when these constructs are used for lentiviral vector production (Ter Brake and Berkhout, in preparation).

Interferon response
The paradigm was that only dsRNAs larger than 30 basepairs induce the interferon response, but it was recently shown this is also true for some siRNAs. This effect is largely dose-dependent and may be specific for synthetic  Figure 3. Predicting viral escape. The highly conserved tat/rev target is shown with flanking sequences. The 19 nucleotide target sequence encompasses two overlapping reading frames for tat and rev. For each reading frame the codons and amino acid sequence are shown. Numerous silent codon changes are possible in each reading frame (grey box), but a silent mutation in both reading frames is possible only at 2 positions within the target sequence (black box).
siRNAs (Myers et al, 2003;Kim et al, 2004). In addition, for systemic delivery of siRNAs in vivo, a sequence motif that interacts with a Toll like receptor was implicated in the induction of interferon (Hornung et al, 2005). However, these kind of interferon responses were associated with extracellularly added siRNAs and not intracellular siRNAs when tested in peripheral blood mononuclear cells in vitro (Sioud, 2005). We propose to induce RNAi by intracellular shRNA expression and, therefore, we expect that these undesired responses will not occur.
Since siRNA transfections clearly differ from intracellular shRNA expression, results obtained in siRNA experiments may not be relevant for our proposed therapeutic approach. Two shRNAs expressed from a lentiviral vector at a relatively high multiplicity of infection (moi) did not induce the interferon response (Anderson and Akkina, 2005). However, the presence of an AA-dinucleotide near the transcription start site of an shRNA was implicated in interferon induction (Bridge et al, 2003;Pebernard and Iggo, 2004). Interestingly, such a shRNA was a more prominent inducer when expressed from a U6 promoter compared to expression from an H1 promoter (Bridge et al, 2003). These experiments used an moi of 10, and the observed interferon response for these shRNAs was lost when an moi of 1 was used. More importantly, the majority of shRNAs did not induce an interferon response even at the high moi. Few of the published anti-HIV siRNAs and shRNAs (Table 1) contain an AA-dinucleotide near the transcription start site, and none contain the motif that interacts with the Toll like receptor. We propose that the multiplicity of infection should always be kept below 1 in a therapeutic setting. This will keep expression levels low, thereby preventing possible side effects such as translational inhibition or saturation of the miRNA or siRNA machinery because these effects are usually associated with a high concentration of siRNA molecules (Scacheri et al, 2004;Huppi et al, 2005). Anyhow, with at least nine candidate RNAi molecules available for the multi-shRNA approach, one could test for putative side effects and select non-toxic shRNAs for a clinical trial.

Lentiviral vector design for multiple shRNA expression
An important step towards anti-HIV RNAi therapy is the design of a lentiviral vector that expresses multiple shRNAs. The goal is to achieve equal expression of each individual shRNA from the multiple shRNA vector, resulting in similar levels of inhibition per shRNA as compared to single shRNA constructs. In case the absolute inhibition would decrease for individual shRNAs in the multiple shRNA vector, selective pressure would be lost, and therapy would likely fail.
An obvious way to construct a multiple shRNA vector is to clone different expression cassettes with the same promoter in tandem orientation (Gonzalez et al, 2005). In the lentiviral vector context, this may present problems because recombination may result in deletion of one or multiple repeats and the intervening sequence (An and Telesnitsky, 2001;Marzio et al, 2001). A solution to this problem would be to increase the number of viral vector copies per cell, thereby ensuring the presence of at least one intact copy. However, this may be undesirable because the concentration of shRNAs will increase and introduce the risk of undesirable side effects. In addition, there will be an increased chance of insertion-induced oncogenesis and a significant increase in treatment cost.
To avoid recombination within the lentiviral vector, one could use different promoters for shRNA expression. Nine different promoters have been used to drive the expression of shRNAs, most common are the RNA polymerase III promoters U6 (Yu et al, 2002) and H1 (Brummelkamp et al, 2002), which have been routinely used in HIV-1 research (Paul et al, 2003;Boden et al, 2003a;Scherer et D   3  19  13 14  16  1 2  7  6  10  4  15  5  12  18  17  8 9  11 shRNA tat/rev Chang et al, 2005). Alternatives include the RNA polymerase II promoter for the U1 snRNA gene (Denti et al, 2004), the tRNA Val promoter (Kawasaki and Taira, 2003), the RNA polymerase I promoter (McCown et al, 2003), the polymerase II cytomegalovirus (CMV) promoter (Boden et al, 2003b;Song et al, 2004), the modified human tRNA Met promoter (Boden et al, 2003b), the VA I Adenovirus promoter (Cordelier et al, 2003) and the 7S K promoter (Koper-Emde et al, 2004). A direct comparison of each of these promoters in the lentiviral vector context will provide the information to decide which ones to combine in a multiple-shRNA viral vector.
Another interesting option to avoid promoter duplication would be to express multiple siRNAs from one expression unit as a single transcript. Two shRNAs connected with a short linker sequence can be equally effective as individual shRNAs . In addition, it may be possible to stack functional siRNAs on top of each other, with or without bulges in between. These transcripts could be designed resembling to mimic the branched microRNA precursors (Boden et al, 2004b). However, one must be aware that such complex RNA structures may result in significant problems within the context of the lentiviral vector RNA genome. Approaches like these are still in the early development phase, but could eventually lead to an effective approach for multiple siRNA expression.
When first generation shRNAs are combined with second generation shRNAs in the vector design, a shRNA repeat sequence will be present. However, the second generation shRNA differs in at least one basepair from the first generation and can be modified by introducing G-U wobble basepairs and a different loop sequence (Miyagishi et al, 2004). These modifications can limit the repeat sequence to 19 nucleotides, which contains at least one point mutation. We anticipate that such a minimal and imperfect repeat will not be problematic.

CONCLUSIONS
We have reviewed current publications involving RNAi approaches using siRNAs or shRNAs against the HIV-1 genome. Since it is known that the application of individual shRNAs can lead to viral escape, the focus of this review is on the development of a durable gene therapy that prevents viral escape. We worked out two strategies that may be employed. First, like many have suggested previously, multiple shRNAs targeting highly conserved HIV-1 sequences can be combined. This will increase inhibition, and reduce the chances of escape exponentially with each added shRNA. Second, we introduce a new strategy to counteract viral escape. When highly conserved sequences are targeted, escape options are limited. If, in addition to the wild-type shRNA, a selection of second-generation shRNAs would be included that anticipate these routes, escape may be prevented. As a proof of concept, we demonstrate that it is possible to efficiently inhibit escape mutants with such a second generation of shRNAs.
Which shRNAs should than be used for an RNAi based gene therapy? The incoming HIV-1 RNA genome represents an efficient target and viral messengers are likely to be subject to RNAi in the nucleus and cytoplasm. Therefore, we argued that target site selection can be based solely on conservedness among the different HIV-1 isolates. From all published target sequences from the HIV-1 genome, nine are conserved in at least half of all isolates. These seem the best candidates for a gene therapy based on multiple RNAi molecules. Studying escape for individual shRNAs in viral infection cultures will reveal whether second-generation shRNAs should be included. We expect that risks of interferon induction or other side effects like off-target inhibition or saturation of the miRNA machinery can be avoided in a gene therapy setting when the dosage of vector is kept low. Nevertheless, individual shRNAs and any combination of shRNAs should be tested for these putative side effects.
Recently, Sirna Therapeutics reported interim data for phase I clinical trials for treatment of age-related macular degeneration (Quinlan, 2005). No adverse or toxic effects were observed and disease stabilized in all patients. In addition, the first clinical trial involving lentiviral vectors showed no adverse effects in five patients (Manilla et al, 2005). Combined, these results pave the way for a clinical trial involving RNAi and lentiviral vectors. Indeed, John Rossi of the City of Hope Beckman Research Institute in California in collaboration with the Australian company Benitec Limited are planning a clinical trial involving a multi-RNA lentiviral vector approach in HIV-1 infected patients (Check, 2005). Their vector encodes a TAR decoy, a ribozyme against CCR5 and also a single shRNA against the HIV-1 genome Akkina et al, 2003;Li et al, 2005). The outline we presented here may form the basis for the development of the next phase in RNAi-based treatments of HIV-1 infection.


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Gain-of-Function Screen for Genes That Affect Drosophila Muscle Pattern Formation

This article reports the production of an EP-element insertion library with more than 3,700 unique target sites within the Drosophila melanogaster genome and its use to systematically identify genes that affect embryonic muscle pattern formation. We designed a UAS/GAL4 system to drive GAL4-responsive expression of the EP-targeted genes in developing apodeme cells to which migrating myotubes finally attach and in an intrasegmental pattern of cells that serve myotubes as a migration substrate on their way towards the apodemes. The results suggest that misexpression of more than 1.5% of the Drosophila genes can interfere with proper myotube guidance and/or muscle attachment. In addition to factors already known to participate in these processes, we identified a number of enzymes that participate in the synthesis or modification of protein carbohydrate side chains and in Ubiquitin modifications and/or the Ubiquitin-dependent degradation of proteins, suggesting that these processes are relevant for muscle pattern formation.

Introduction
Whole genome sequences of many animals are now known, including those of Caenorhabditis elegans, human, mouse, and Drosophila melanogaster (see for example [1][2][3][4]). The task now facing biologists is to discover the functions of the annotated genes within the genomes. For some organisms, such as C. elegans, it is possible to adopt a systematic approach to ablate gene function by, for example, the RNA interference technique [5,6]. For Drosophila melanogaster, a widespread analysis of gene function has been undertaken by systematic EMS mutagenesis and transposon tagging approaches using the P-element [7,8]. However, since two-thirds of genes in D. melanogaster and in C. elegans cause only subtle or even unscorable mutant phenotypes [9], a complementary approach was used. This approach is based on conditional overexpression of genes in order to generate gain-offunction phenotypes. It involves upstream activating sequences (UASs) of yeast provided through a recombinant transposon insertion, termed EP-element [10]. The inserted UAS can be used to transcriptionally activate an endogenous gene next to the insertion site by the transgene-dependent expression of the yeast transcription factor GAL4 under the control of a constitutively active promoter or spatiotemporally regulated enhancer elements [10,11].
Here we describe a newly generated EP-element library composed of more than 3,700 unique insertion sites and their location within the D. melanogaster genome. We employed this and previously constructed EP-element libraries for a systematic gain-of-function screen to identify gene activities that interfere with the proper development of the segmentally repeated muscle pattern. We designed a GAL4driver to express endogenous genes in single epidermal cell rows, one anterior and one posterior to the embryonic engrailed expression domain [12], asking whether misexpression of genes in these locations alters the identity and/or the spatial cues of cells and thereby interferes with the genetically controlled migration and pathfinding ability of myotubes, as well as their anchoring properties (reviewed in [13]).
Muscle pattern formation is a stereotyped and segmentally repeated developmental process. Once muscle founder cells are born and determined, they grow by fusion with undetermined muscle cells. The resulting myotubes extend via the growth of cone-like tips along the inner surface of epidermal cells (reviewed in [13]), which serve as a migration substrate towards a distinct set of tendon-like segment border cells, termed apodemes, to which myotubes finally attach [13,14]. stripe, which encodes a zinc-finger-type transcription factor, is essential for apodeme cell formation at the segment borders [15]. In stripe mutants, myotubes soon fail to be properly guided, indicating that developing apodeme cells not only serve as attachment sites but also provide guiding cues for the migrating myotubes [15,16]. In addition, myotube guidance is also controlled by the myotube-expressed gene grip [17], by Slit/Robo signaling [18,19], and the attachment to apodemes involves the atypical receptor tyrosine kinase Derailed [20] as well as fibroblast growth factor signaling activity [21]. These and other results [13] have established that myotube guidance and attachment are controlled by interactions between epidermal and muscle cells and that interfering with their interactions causes scorable effects on the stereotyped muscle pattern. Here, we describe a systematic gain-of-function screen towards identifying gene activities that can interfere with the formation of the proper muscle pattern in the D. melanogaster embryo by using the specially designed UAS/GAL4 misexpression system.

Generation of an EP-Element Insertion Collection
We generated a novel EP-element collection for D. melanogaster. It contains single insertions bearing GAL4dependent UASs of yeast at their ends [22,23]. Genes properly oriented with respect to the UAS sequences can therefore be conditionally expressed via transgene-derived GAL4 activity [10].
Of more than 13,800 individual EP-element lines initially generated, the insertion sites of more than 11,700 individual lines were determined by a combined PCR/sequencing approach [22]. Among these insertion sites, we identified a total of 3,707 unique EP-element insertion sites within the D. melanogaster genome. Their location and the orientation of the EP-elements are summarized in Tables S1 and S2. Using this collection, roughly a quarter of the D. melanogaster genes [2] can be activated by transgene-derived GAL4 activity that is driven by constitutively active promoters or cell-specific enhancer elements [11]. In addition, a sizable portion of the EP-elements are in reverse orientation with respect to genes (that is, there are no other annotated transcription units within a range of 10 kb of genomic DNA [2]), suggesting that the activity of these genes would likely be knocked down in response to transgene-derived GAL4 activation (see Tables S1  and S2).

Generation of a GAL4-Driver Line Causing Epidermal Stripe Expression
In order to perform a large-scale gain-of-function screen for gene activities that interfere with D. melanogaster muscle pattern formation during embryogenesis, we designed a GAL4-driver that allowed the misexpression of EP-targeted genes in presumptive apodeme precursors at the segment border and in an ectopic array of intrasegmental cell rows within the epidermis of the embryo. We made use of the sr239 enhancer element of the stripe gene, which drives gene expression in a single cell row posterior to the engrailed expression domain. These cells correspond to dorsal and lateral apodeme precursor cells at a midstage of embryogenesis (stage 12) [12]. We fused this element with the GAL4 coding region to express UAS-targeted genes in a subset of apodeme cells. In addition, we used the sr239Dpan enhancer [12], termed srmod, to drive GAL4 expression in apodeme cells and in a subset of epidermal cells anterior to the engrailed expression domain ( Figure 1A and 1B) that serve as a migration substrate for myotubes. We expected these tools to facilitate the identification of genes whose activities interfere with processes such as myotube guidance or muscle attachment when expressed in response to one or both of the GAL4-drivers.

Screening for Genes That Interfere with Muscle Pattern Formation
To activate misexpression of endogenous EP-targeted genes, we crossed females bearing the srmodGAL4-driver with male individuals from about 4,500 lines of the new (Table S1) and a previous EP-element collection [10] and asked whether the misexpression causes a lethal phenotype, knowing that impairing the stereotyped muscle pattern prevents the hatching of fully differentiated larvae from the egg shell [15,16]. In the next step, we examined whether muscle pattern defects can be observed after staining of the fully differentiated but unhatched embryos with anti-Myosin heavy chain (MHC) antibodies. To distinguish between interfering gene activities that were induced in the segment border apodeme cells and those that were derived from the intervening epidermal cells, we performed corresponding crosses using srGAL4-bearing instead of srmodGAL4-bearing females.
We identified an initial set of 78 EP-element lines (1.7%) that caused a specific srmodGAL4-dependent muscle pattern phenotypes. To confirm that the observed phenotypes in the embryonic muscle pattern originated from misexpression of a given gene, we tested whether (i) the phenotype could be reverted by the precise excision of the EP-element, (ii) the potential target gene was expressed in a GAL4-dependent fashion (this was tested using in situ hybridization or antibody staining for product detection), (iii) the muscle pattern defects also occurred by over-expression of corresponding cDNA from UAS-dependent transgenes, or (iv) whether misexpression of the same transcription unit by a different EP-element insertion caused a similar phenotype.
The strength and penetrance of the misexpression muscle pattern phenotypes were variable (compare Figure 1C and 1D with 1E-1J). We found embryos in which only single

Synopsis
Muscle pattern formation during embryogenesis requires the activity of a distinct network of genes. In the model organism Drosophila, this process involves the determination of stem-cell-like muscle founder cells, their differentiation, and their attraction to tendon-like epidermal cells, termed apodemes, to which the muscles attach. In order to systematically identify genes involved in these processes, a collection of fruit fly strains was generated that can be used for the ectopic expression of more than 3,700 individual fruit fly genes in a spatiotemporally restricted manner. In order to address muscle pattern formation, the collection was used to express the genes in the developing apodemes and in a series of distinct epidermal cells that serve as migration substrate for developing muscles towards the apodemes. In addition to already known factors, some 60 novel gene activities were found to interfere under these circumstances with the formation of the muscle pattern. In addition to providing a most valuable tool for the Drosophila community of researchers, the results provide a framework for a detailed analysis of the gene network and insight into molecular mechanisms underlying embryonic muscle pattern formation. muscle fibers were abnormally attached to apodemes ( Figure  1E and 1F), embryos in which most muscles of the dorsal and lateral region of the embryo were abnormally shaped and attached to ectopic epidermal sites ( Figure 1G), and embryos in which the early processes of myogenesis were aberrant, as concluded from impaired myoblast fusions ( Figure 1H) and muscle misdetermination ( Figure 1I and 1J). The different defects suggest that activities derived from the misexpressed genes can interfere with cell determination as well as guiding and targeting events during muscle pattern formation. In some cases, the defects observed were not restricted to dorsal and lateral muscles but also included muscles in the ventral region of the embryo, where few epidermal cells express srmod-dependent GAL4 activity (not shown).

Gene Activities That Interfere with Muscle Growth and Attachment
Of the initially identified 78 EP-element insertions, 66 GAL4-driven transcription units could be unambiguously identified to be the cause of the gain-of-function phenotypes (Table S3). Of those, ten transcription units were expressed in anti-sense orientation, implying that misexpression of transcripts in reverse orientation is likely to cause a knock-down phenotype. Analysis of the expression patterns of some of the anti-sense-tagged candidates indicated that the transcripts are expressed ubiquitously or accumulate at the segment border (see below). Thus, GAL4-driven misexpression may result in reduced gene activity. Fifty-six transcription units were in sense orientation, suggesting misexpression phenotypes in response to GAL4-drivers. Computer assisted analysis of the products of the targeted transcription units revealed that many of the candidates with known or predicted functions encode for membrane-associated or -secreted factors as well as for components known to be involved in protein modification and degradation ( Figure 2).
Genes coding for membrane associated and secreted factors. Thirteen genes encode proteins that contain diagnostic domains for membrane association or secretion. This group includes Tetraspanin Tsp42Ee (CG10106), one protein with three transmembrane domains (CG9030), and five factors with a single transmembrane domain that is typical for receptor-type proteins. This last group includes Toll, a receptor that participates in dorsoventral patterning of the embryo and innate immune response, and Syndecan (Sdc), a heparan sulfate proteoglycan (HSPG) that participates in Slit/ Robo signaling [24,25]. Furthermore, membrane-associated factors were identified including CG33207/Pxb, which functions as an attenuator for hedgehog signaling [26], and the polychaetoid protein, a guanylate kinase at the adherens junctions that participates in JNK signaling [27].
The identification of a subset of transmembrane proteins in which two out of four proteins (Sdc and Toll) are already known to participate in muscle pattern formation [25,28] provides trust that other identified genes that code for membrane associated and secreted factors with unknown functions may also take part in the process. These uncharacterized factors include CG14052, CG6301, and CG17368, which encode small proteins containing an N-terminal signal peptide, implying that they represent secreted factors for which functions need to be established.
Protein modification and degradation. A total of eight genes encode for factors involved in protein modification and degradation. Three genes encode components of the Ubiquitin pathway including uba1 and effete, which encode E1 and E2 enzymes, respectively, as well as CG11033, which codes for an uncharacterized F-Box protein. F-Box proteins are required for target protein binding and for Ubiquitin transfer by the E2/E3 complex. Both effete and uba1 have been shown to participate in neurogenesis [29,30]. uba1 was initially found in a gain-of-function screen for genes involved in motor axon guidance [31].
Of the subset of proteins modifying enzymes, five play a role in modifying carbohydrate side chains of peptides. Of those, sulfateless encodes a heparan sulfate-glucosamine-Nsulfotransferase required for Decapentaplegic, Hedgehog, and fibroblast growth factor signaling [32]. The finding of a HSPG-modifying enzyme is consistent with the concurrent identification of the HSPG Sdc (see above), already known to affect muscle guidance [25,33]. We identified also a second sulfotransferase (CG32629/CG32632 fusion) and two genes that code for proteins that modify extracellular carbohydrates (CG31973 and gnbp3). The finding of several enzymes involved in carbohydrate side chain synthesis and modification suggests that they play a role not only in axon guidance [34] but also in muscle guidance and/or apodeme targeting. In addition, the identification of several Ubiquitin pathway components implicated in protein degradation suggests a role also of this process in muscle pattern formation.
Transcription factors and RNA binding proteins. Only six potential or known DNA or RNA binding factors were identified. This result suggests that only a comparatively small number of transcription factors can interfere with the functional development of apodeme cells in a manner recently shown for the zinc finger protein encoded by stripe [15,16]. Interestingly, the identified transcription factors also include two zinc finger proteins, encoded by schnurri and escargot (esg), that have been shown to act in the formation of the tracheal system [35][36][37]. Esg is involved in Cadherinmediated adhesion [37]. Thus, its misexpression may cause abnormal adhesion of muscles when esg-expressing epidermal cells are provided as a substrate. schnurri activity is required to properly mediate TGFb signaling [35]. Its ectopic expression may therefore cause an improper signaling read- The affiliation of the genes products is indicated by the color and the size of the fragments represents the quantitative distribution. cytsk., cytoskeleton; nucl. acid bdg., nucleic acid binding; prot. mod. þ degrade., protein modification or degradation; secr. þ membrane assoc., secreted or membrane-associated factors; transp. þ carrier, transporter or carrier; unknown fct., unknown function. DOI: 10.1371/journal.pgen.0010055.g002 out that impairs myotube outgrowth and/or muscle attachment.
Cytoskeleton factors. Three genes code for known cytoskeleton binding proteins such as Katanin80, a WD40 domain microtubule binding protein of the Katanin complex involved in micotubule severing. In addition, we found chickadee, which was identified twice by independent EPelement insertions in this screen. chickadee protein is involved in Actin filament organization and contains a phosphatidylinositol-4,5-bisphophate binding motif. This motif is noteworthy with respect to rdgBb, which codes for a phosphatidylinositol transfer protein coupling phosphatidylinositol delivery and phosphatidylinositol-4,5-bisphophate synthesis relevant for cell-cell signaling processes (reviewed in [38]) and which was also identified in the screen.
Factors involved in cell cycle control and biosynthesis. Seven factors involved in central steps of biosynthesis were identified. They include the ribosomal protein RpL18A and the polyadenylation binding protein Pabp2. In addition, cell cycle control genes such as the D. melanogaster CDC25 homolog twine and two cycline genes were found. Interference of overexpressed general biosynthesis factors and cell cycle control genes can be explained if they would alter proper epidermal cell differentiation, patterns of cell death, and/or patterns of cell divisions. In these cases, gene expression could impair processes required to maintain or generate properly differentiated epidermal cells that serve as substrate for the outgrowing myotubes or provide spatial cues relevant for this process.

Gene Expression Patterns
We examined the expression pattern of a total of 46 of the identified genes. This criterion for validation of potential gene functions for muscle guidance and attachment control included whole mount in situ hybridization using anti-sense RNA probes prepared from respective cDNAs or genomic fragments covering parts of the identified candidates as well as information available from a D. melanogaster database [39].
The majority of genes are expressed in patterns that could not be directly correlated with muscle pattern formation. However, most were either ubiquitously expressed or they were maternally contributed, and transcripts are present in eggs and during early embryogenesis. Yet, about one-third of the genes were expressed in spatiotemporal patterns in the epidermis during the stage when myotube migration takes place. Eight of these genes were expressed in the apodeme precursor cells of wild-type embryos, including seven of the 12 genes that encode cell surface proteins or secreted factors. Examples of the gene expression pattern are shown Figure 3.
The fraction of genes that are expressed in apodeme cells at the stage when they are targeted by the muscles includes Toll, sdc, CG3563, CG13913, and gnbp3 ( Figure 3A-3J). The expression patterns of Toll and Sdc have previously been described. Toll is expressed in a subset of the developing apodeme cells and participates in muscle pattern formation [28]. Sdc is expressed in the mesoderm, the tracheal system, the axons of the central nervous system and in the differentiated apodemes. In sdc mutants, muscles fail to respect the ventral midline as a migration border, cross the border, and subsequently attach to apodemes at the other side of the midline [25]. Other genes, such as CG14713 and pxb/AT17253, are expressed in the intrasegmental region of the epidermis that is crossed by the migrating myotubes ( Figure 3K-3N).
It is noteworthy that the set of identified genes also includes genes that are normally expressed in cells of other tissues or organs whose development involves migratory processes of cells or groups of cells. These include the developing tracheal system, germ line precursor cells, the midgut, and the nervous system. Thus, although the expression patterns exclude a role for these genes during the normal process of muscle pattern formation, they could play a direct or indirect role in guiding migrating cells in regions of the wild-type embryo where they are normally expressed. Preliminary results with a gene specifically expressed in germ line precursor cells supports this proposal (G.V., unpublished data).

Gene Activities Required for Muscle Pattern Formation
Muscle pattern phenotypes of Toll, gut feeling (oda), and sulfateless mutant embryos have already been described [28,32,40]. In order to test whether other genes that were identified in the gain-of-function screen also caused a loss-offunction phenotype, we examined the muscle pattern of lossof-function mutants that were described in a context different from muscle pattern formation. Figure 4 shows two examples of the analysis, indicating that esg (compare Figure  4A with 4B) and sdc (compare Figure 4A and 4D with 4C, 4E, and 4F) loss-of-function mutant embryos develop variable muscle pattern defects that include the absence of lateral transverse muscles, loss of muscle fibers, and abnormally shaped muscles. Since sdc and esg are expressed in the epidermis, we used expression of delilah, a marker for the muscle attachment sites [41], to examine whether an altered pattern of attachment sites is a likely cause of the muscle pattern defects. No pattern defects were observed in sdc and esg mutants ( Figure 4G-4I). Thus, the muscle pattern defects observed with both the gain-of-function and loss-of-function mutants are consistent with the argument that the gene esg participates in the regulation of adhesion processes, as previously proposed for esg function during tracheal system development [37], and that sdc is required for early Slit/Robosignaling-dependent muscle guidance, as described recently ( Figure 4E; [25]). Our results also show that sdc-dependent Slit signaling serves as a muscle attractant during a late phase of muscle guidance [19], since abnormal muscle elongations are observed in fully developed but unhatched sdc mutant larvae ( Figure 4C and 4F).

Conclusion
We identified a series of genes whose activity impairs muscle pattern formation when misexpressed in a defined pattern of epidermal cells that represent the migration substrate and/or the attachment sites for the outgrowing myotubes and muscle fibers. The 66 identified candidate genes were selected from an EP-element insertion library composed of more than 4,500 individual lines. This number suggests that about 1.5% of D. melanogaster genes can affect muscle pattern formation when expressed in cells that are contacted by myotubes or muscles. Although the screening system can certainly be regarded as artificial, it nevertheless identified genes such as Toll, gut feeling, and sulfateless that have been previously implicated in muscle pattern formation because the corresponding loss-of-function mutations cause variable muscle phenotypes [28,32,40]. In addition, it identified genes whose products are known to participate in cell migration and/or cell targeting processes in the embryo. These genes include esg and sdc [25,37], and, as shown in Figure 4, loss-of-function mutations in these two genes cause a defective muscle pattern in the embryo, indicating that the activity of these genes is essential for embryonic muscle development.
It is interesting to note that the misexpression screen identified, in addition to the HSPG Sdc, a number of other muscle-pattern-disturbing genes that code for factors known to participate in carbohydrate side chain synthesis or side chain modification. Thus, these enzymes are likely to participate in communication events between muscle and epidermal cells, processes that may also involve signaling molecules in addition to Slit [18,19]. Similarly, the independent identification of three components of the Ubiquitin system suggests that Ubiqitin modifications of proteins or their stability play a role in muscle pattern formation. The plethora of factors identified here open a new avenue towards a detailed functional analysis of processes underlying the interplay of myotubes, their epidermal migration substrate, and the specialized segmental border cells to which myofibers ultimately attach. In addition, they can be used towards developing an understanding of migratory processes in other developmental processes of D. melanogaster and, in view of the conservation of the genes identified here, possibly also in other species including mammals.

Materials and Methods
Genetics and expression detection. To generate novel EP-element integration lines we used two different EP-elements. EPg was modified to function in the female germline and contains the white þ gene as a selectable marker in white mutant individuals [23]. The second EP-element, PfMae-UAS.6.11g, contains the yellow gene as a corresponding marker [42]. More than 8,500 independent EPg insertions were generated using a jump-starter line from an EPg insert on a CyO chromosome (EPg4-38), and 5,100 independent lines were established using PfMae-UAS.6.11g. Chromosomes bearing an EP-element integration were kept either as homozygous lines or in trans to a corresponding balancer chromosome. EP-element-bearing males were crossed with srmodGAL4-bearing females, and their F1 offspring were screened for lethality. In case of lethality, candidates were crossed with srmodGAL4and srGAL4-drivers and their F1 offspring were examined after staining with anti-MHC antiserum (kindly provided by D. Kiehart) using the staining protocol previously described [12]. Anti-sense DIG-labeled RNA probes were prepared and whole mount in situ hybridization was performed as described [12].
Molecular analysis. The EPg and the pMae elements have been previously described [23,42]. The srGAL4 and the srmodGAL4 lines were obtained by cloning the KpnI and XbaI of the sr239 and sr239Dpan DNA as described in [12] into the p221 vector (kindly provided by C. Klä mbt). In order to determine the EP-element integration sites within genomic DNA, we performed inverse PCR as described on the Berkeley Drosophila Genome Project Web page (http://www.fruitfly.org/) with overnight digestion by either MaeI or Csp6I. Fragments were amplified for the 59-end of PfEP,yþg; the primers used for the pMae were 59-CAGCTGCGCTTGTTTATTTGC-39 (forward) and 59-TGGGAATTCGTTAACAGATCCAC-39 (reverse), and for the EPg were Pw new up (CAG CCG AAT TAA TTC TAG TTC CAG TGA A) and Pw new low (ACT TCG GCA CGT GAA TTA ATT TTA CTC C). The amplified DNA was sequenced and used to determine the insertion site (see Table S1). Table S1. Description of the Tested Insertion Lines The lines are ordered according to their names (Line). DG-EP denotes lines that carry the PfMae-UAS.6.11g-element whereas HD-EP stands for lines that are generated by mobilization of the modified EPg-element. The chromosome arm (Arm), orientation (Strand), and position according to D. melanogaster genomic sequence release 3 (Position) are indicated. The 59 sequence tag (forward strand) for each insertion line is listed (Sequence). Found at DOI: 10.1371/journal.pgen.0010055.st001 (607 KB XLS). Table S2. Insertion Lines Available from the Bloomington Stock Center The lines are ordered according to the name under which they will be kept in the Bloomington Stock Center (Line). The former name as used in Table 1 (Old Name) is also listed. The chromosome arm (Arm) as well as the position according to D. melanogaster genomic sequence release 4.1 (Coordinate) is shown. The next gene (Gene) with its extension (59 Gene and 39 Gene, respectively) and orientation (Strand) as well as the relative position of the insert to the gene (Position) is indicated. The 59 sequence tag for each insertion line is listed under Sequence. Found at DOI: 10.1371/journal.pgen.0010055.st002 (152 KB XLS). Table S3. Identified Candidate Genes The candidate genes are arranged into groups by their proposed biological function. For each candidate, the CG number (according to the FlyBase [http://flybase.bio.indiana.edu/]), the gene synonym, the EP number, the orientation of the expressed transcript, predicted protein domains, the biological process, the expression pattern, the criteria for the validation of candidate genes, and a description of the gain-of-function muscle phenotype are listed. The wild-type expression patterns are based on the Berkeley Drosophila Genome Project in situ expression data [39], Berkeley Drosophila Genome Project CHIPexpression data, or in situ hybridization using either genomic fragments (gen frag) or Ests. In this case the name of the Est used is listed. Abbreviations used to describe the expression are as follows: Ap, muscle attachment sites; Br, brain; Ep, epidermis; Fb, fatbody; Gc, garland cells; Go, gonads; He, heart; Hg, hindgut; mat, maternal expression; Md, early mesoderm; Mg, midgut; Ml, ventral midline; Mu, muscles; Nb, neuroblasts; Pc, pericardial cells; Sb, epidermal segment border; Sg, salivary glands; Tp, tracheal placodes; Ts, tracheal system; zyg, zygotic expression. The criteria used to validate the identified candidate genes were (1) reversion of the EP-element, (2) induction of expression, (3) similar phenotype induced by an UAS cDNA transgene, (4) additional EP-element, and (5) published data. Anti-sense candidates were only considered in the absence of a gene in sense orientation within 10 kbp downstream of the EP-element (6).