MicroRNAs Form Triplexes with Double Stranded DNA at Sequence-Specific Binding Sites; a Eukaryotic Mechanism via which microRNAs Could Directly Alter Gene Expression.

MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA) and typically down-regulating their stability or translation. Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence (i.e., NMR, FRET, SPR) that purine or pyrimidine-rich microRNAs of appropriate length and sequence form triple-helical structures with purine-rich sequences of duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show that several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 × 10(-16)) for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. This work has thus revealed a new mechanism by which microRNAs could interact with gene promoter regions to modify gene transcription.

A novel approach for the analysis of T-cell reconstitution by using a T-cell receptor beta-based oligonucleotide microarray in hematopoietic stem cell transplantation.

OBJECTIVE: Analysis of T-cell population diversity is important to hematopoietic stem cell transplantation (HSCT). The millions of specificities in T-cell receptor (TCR) hypervariable complementarity- determining region 3 (CDR3) precludes detection of all T-cell populations by antibody-based flow cytometry. An alternative method, the TCR CDR3 spectratyping assay, involves multiple polymerase chain reaction (PCR) analyses and is interpreted only qualitatively. In this study, we designed the first TCRbeeta-based oligonucleotide microarray and investigated its specificity, clonality discrimination, sensitivity of detection, and feasibility for monitoring T-cell population diversity in HSCT. MATERIALS AND METHODS: The array contains 27 TCR Vbeta probes and 13 Jbeta probes. TCRbeta repertoire diversity was detected with single PCR, microarray hybridization system, and Spotfire analysis software. RESULTS: TCRO-based microarray provides specific sequence-based information and can distinguish T-cell monoclonal expansion within a polyclonal population. We successfully used this microarray to quantitatively and qualitatively analyze T-cell population diversity in recipients of hematopoietic stem cell transplants. CONCLUSION: This success suggests broad potential applications of the microarray for use in many other areas, including anti-tumor immunity, vaccination, autoimmunity, infectious diseases, and leukemia. By providing a single PCR-based assay to quantify multiple T-cell populations in parallel, this device will allow clinicians and researchers to rapidly perform high-throughput surveys.

Arf induces p53-dependent and -independent antiproliferative genes.

The tumor suppressor p19(Arf) (p14(ARF) in humans), encoded by the Ink4a/Arf locus, is mutated, deleted, or silenced in many forms of cancer. p19(Arf) induces growth arrest by antagonizing the activity of the p53-negative regulator, Mdm2, thereby inducing a p53 transcriptional response. p19(Arf) can also inhibit cell cycle progression of mouse embryo fibroblasts lacking Cip1 or lacking both Mdm2 and p53, although in the absence of p53, arrest occurs more slowly. Profiling with high-density oligonucleotide GeneChips and cDNA microarrays was used to interrogate mouse genes, the expression of which was induced or suppressed by a conditionally regulated Arf gene. Cluster analysis of temporal gene expression patterns and validation of the results by RNA analysis identified Arf-responsive genes whose induction was both p53-dependent and -independent. The latter included four members of the B-cell translocation gene family (Btg1, Btg2, Btg3, and Tob1) that were demonstrated to inhibit cell proliferation in primary mouse embryo fibroblasts expressing or lacking functional p53. Together, the results indicate that p19(Arf) induces a broad spectrum of proteins that likely act in concert to arrest cell proliferation.