Gamma-retroviruses and lentiviruses integrate non-randomly in mammalian genomes, with specific preferences for active chromatin, promoters and regulatory regions. Gene transfer vectors derived from gamma-retroviruses target at high frequency genes involved in the control of growth, development and differentiation of the target cell, and may induce insertional tumors or pre-neoplastic clonal expansions in patients treated by gene therapy. The gene expression program of the target cell is apparently instrumental in directing gamma-retroviral integration, although the molecular basis of this phenomenon is poorly understood. We report a bioinformatic analysis of the distribution of transcription factor binding sites (TFBSs) flanking >4,000 integrated proviruses in human hematopoietic and non-hematopoietic cells. We show that gamma-retroviral, but not lentiviral vectors, integrate in genomic regions enriched in cell-type specific subsets of TFBSs, independently from their relative position with respect to genes and transcription start sites. Analysis of sequences flanking the integration sites of Moloney leukemia virus (MLV)- and human immunodeficiency virus (HIV)-derived vectors carrying mutations in their long terminal repeats (LTRs), and of HIV vectors packaged with an MLV integrase, indicates that the MLV integrase and LTR enhancer are the viral determinants of the selection of TFBS-rich regions in the genome. This study identifies TFBSs as differential genomic determinants of retroviral target site selection in the human genome, and suggests that transcription factors binding the LTR enhancer may synergize with the integrase in tethering retroviral pre-integration complexes to transcriptionally active regulatory regions. Our data indicate that gamma-retroviruses and lentiviruses have evolved dramatically different strategies to interact with the host cell chromatin, and predict a higher risk in using gamma-retroviral vs. lentiviral vectors for human gene therapy applications.

Aim: To extensively explore microRNA expression profiles in lung carcinoids in correlation with clinical and pathological features. Methods: A PCR-based array was employed in the screening phase to analyze 752 microRNAs in a discovery set of 12 lung carcinoids, including 6 typical (3 with lymph node metastasis) and 6 atypical (3 with lymph node metastasis). The results were validated by means of real-time PCR in 37 carcinoids, including 22 typical (4 with lymph node metastasis) and 15 atypical (7 with lymph node metastasis), and 19 high-grade neuroendocrine carcinomas. Results: Unsupervised cluster analysis segregated the pilot cases into 3 distinct groups. Twenty-four microRNAs were differentially regulated in atypical versus typical carcinoids, and 29 in metastatic versus nonmetastatic cases. Eleven microRNAs were selected for validation. All but 1 were significantly different among lung neuroendocrine tumor histotypes. Moreover, 5 (miR-129-5p, miR-409-3p, miR-409-5p, miR-185 and miR-497) were significantly upregulated in typical compared to atypical carcinoids. MiR-409-3p, miR-409-5p and miR-431-5p were also significantly downregulated in carcinoids metastatic to the lymph nodes. Predictive in silico analysis of specific target genes showed that these 3 latter microRNAs linked to metastatic potential are implicated in several cellular functions and highlighted several novel genes which may be worth exploring. Conclusions: Our findings demonstrate that lung carcinoids have distinct microRNA expression profiles as compared to high-grade neuroendocrine carcinomas and that specific microRNAs might have potential implications as diagnostic tools or clinical biomarkers.

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We have investigated the genomic organization, the occurrence of alternative splicing and the differential expression of the zebrafish disabled1 (dab1) gene. Dab1 is a key effector of the Reelin pathway, which regulates neuronal migration during brain development in vertebrates. The coding region of the zebrafish dab1 gene spans over 600 kb of genomic DNA and is composed of 15 exons. Alternative splicing in a region enriched for tyrosine residues generates at least three different isoforms. These isoforms are developmentally regulated and show differential tissue expression. Comparison with mouse and human data shows an overall conservation of the genomic organization with different alternative splicing events generating species-specific isoforms. Because these alternative splicing events give rise to isoforms with different numbers of phosphorylateable tyrosines, we speculate that alternative splicing of the dab1 gene in zebrafish and in other vertebrates regulates the nature of the cellular response to the Reelin signal.

Supplementary data are available at Bioinformatics online.