Regulatory Circuit of Human MicroRNA Biogenesis

miRNAs (microRNAs) are a class of endogenous small RNAs that are thought to negatively regulate protein production. Aberrant expression of many miRNAs is linked to cancer and other diseases. Little is known about the factors that regulate the expression of miRNAs. We have identified numerous regulatory elements upstream of miRNA genes that are likely to be essential to the transcriptional and posttranscriptional regulation of miRNAs. Newly identified regulatory motifs occur frequently and in multiple copies upstream of miRNAs. The motifs are highly enriched in G and C nucleotides, in comparison with the nucleotide composition of miRNA upstream sequences. Although the motifs were predicted using sequences that are upstream of miRNAs, we find that 99% of the top-predicted motifs preferentially occur within the first 500 nucleotides upstream of the transcription start sites of protein-coding genes; the observed preference in location underscores the validity and importance of the motifs identified in this study. Our study also raises the possibility that a considerable number of well-characterized, disease-associated transcription factors (TFs) of protein-coding genes contribute to the abnormal miRNA expression in diseases such as cancer. Further analysis of predicted miRNA–protein interactions lead us to hypothesize that TFs that include c-Myb, NF-Y, Sp-1, MTF-1, and AP-2α are master-regulators of miRNA expression. Our predictions are a solid starting point for the systematic elucidation of the causative basis for aberrant expression patterns of disease-related (e.g., cancer) miRNAs. Thus, we point out that focused studies of the TFs that regulate miRNAs will be paramount in developing cures for miRNA-related diseases. The identification of the miRNA regulatory motifs was facilitated by a new computational method, K-Factor. K-Factor predicts regulatory motifs in a set of functionally related sequences, without relying on evolutionary conservation

Investigation of miRNA Biology by Bioinformatic Tools and Impact of miRNAs in Colorectal Cancer—Regulatory Relationship of c-Myc and p53 with miRNAs

MicroRNAs (miRNAs) are a class of small non-coding RNAs that mediate gene expression at the posttranscriptional and translational levels and have been demonstrated to be involved in diverse biological functions. Mounting evidence in recent years has shown that miRNAs play key roles in tumorigenesis due to abnormal expression of and mutations in miRNAs. High throughput miRNA expression profiling of several major tumor types has identified miRNAs associated with clinical diagnosis and prognosis of cancer treatment. Previously our group has discovered a novel regulatory relationship between tumor suppressor gene p53 with miRNAs expression and a number of miRNA promoters contain putative p53 binding sites. In addition, others have reported that c-myc can mediate a large number of miRNAs expression. In this review, we will emphasize algorithms to identify mRNA targets of miRNAs and the roles of miRNAs in colorectal cancer. In particular, we will discuss a novel regulatory relationship of miRNAs with tumor suppressor p53 and c-myc. miRNAs are becoming promising novel targets and biomarkers for future cancer therapeutic development and clinical molecular diagnosis

Prioritization of candidate cancer genes—an aid to oncogenomic studies

The development of techniques for oncogenomic analyses such as array comparative genomic hybridization, messenger RNA expression arrays and mutational screens have come to the fore in modern cancer research. Studies utilizing these techniques are able to highlight panels of genes that are altered in cancer. However, these candidate cancer genes must then be scrutinized to reveal whether they contribute to oncogenesis or are coincidental and non-causative. We present a computational method for the prioritization of candidate (i) proto-oncogenes and (ii) tumour suppressor genes from oncogenomic experiments. We constructed computational classifiers using different combinations of sequence and functional data including sequence conservation, protein domains and interactions, and regulatory data. We found that these classifiers are able to distinguish between known cancer genes and other human genes. Furthermore, the classifiers also discriminate candidate cancer genes from a recent mutational screen from other human genes. We provide a web-based facility through which cancer biologists may access our results and we propose computational cancer gene classification as a useful method of prioritizing candidate cancer genes identified in oncogenomic studies

Genome-Wide Survey of MicroRNA - Transcription Factor Feed-Forward Regulatory Circuits in Human

In this work, we describe a computational framework for the genome-wide identification and characterization of mixed transcriptional/post-transcriptional regulatory circuits in humans. We concentrated in particular on feed-forward loops (FFL), in which a master transcription factor regulates a microRNA, and together with it, a set of joint target protein coding genes. The circuits were assembled with a two step procedure. We first constructed separately the transcriptional and post-transcriptional components of the human regulatory network by looking for conserved over-represented motifs in human and mouse promoters, and 3'-UTRs. Then, we combined the two subnetworks looking for mixed feed-forward regulatory interactions, finding a total of 638 putative (merged) FFLs. In order to investigate their biological relevance, we filtered these circuits using three selection criteria: (I) GeneOntology enrichment among the joint targets of the FFL, (II) independent computational evidence for the regulatory interactions of the FFL, extracted from external databases, and (III) relevance of the FFL in cancer. Most of the selected FFLs seem to be involved in various aspects of organism development and differentiation. We finally discuss a few of the most interesting cases in detail.Comment: 51 pages, 5 figures, 4 tables. Supporting information included. Accepted for publication in Molecular BioSystem

Recipe for a Busy Bee: MicroRNAs in Honey Bee Caste Determination

Social caste determination in the honey bee is assumed to be determined by the dietary status of the young larvae and translated into physiological and epigenetic changes through nutrient-sensing pathways. We have employed Illumina/Solexa sequencing to examine the small RNA content in the bee larval food, and show that worker jelly is enriched in miRNA complexity and abundance relative to royal jelly. The miRNA levels in worker jelly were 7-215 fold higher than in royal jelly, and both jellies showed dynamic changes in miRNA content during the 4(th) to 6(th) day of larval development. Adding specific miRNAs to royal jelly elicited significant changes in queen larval mRNA expression and morphological characters of the emerging adult queen bee. We propose that miRNAs in the nurse bee secretions constitute an additional element in the regulatory control of caste determination in the honey bee.The research was supported by National Sciences Foundation of China Grant No.30630040; National Key Basic Research & Development Program 973 under Grant Nos. 2009CB825401 and 2007CB946901 to RSC, the earmarked fund for Modern Agro-industry Technology Research System (No. CARS-45-KXJ3), and a grant of the National Natural Science Foundation of China (NSFC 30571409) to SKS, and the Nature and Science Foundation Commission of Zhejiang Province (R3080306) to SKS. Zhang was supported by the Australian Research Council through the ARC Centre of Excellence in Vision Science (CE0561903). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

NF-Y substitutes H2A-H2B on active cell-cycle promoters: recruitment of CoREST-KDM1 and fine-tuning of H3 methylations

The CCAAT box is a frequent promoter element, as illustrated by bioinformatic analysis, and it is bound by NF-Y, a trimer with H2A-H2B-like subunits. We developed a MNase I-based ChIP protocol on homogeneous cell populations to study cell-cycle promoters at the single nucleosome level. We analyzed histone methylations and the association of enzymatic activities. Two novel results emerged: (i) H3-H4 are present on core promoters under active conditions, with the expected cohort of ‘positive’ modifications; H2A-H2B are removed and substituted by NF-Y. Through the use of a dominant negative mutant we show that NF-Y is important for H3K36me3 deposition and for elongation, not recruitment of Pol II; (ii) H3K4 methylations are highly dynamic and H3K4me1 is a crucial positive mark. Functional siRNA inactivation and treatment with Tranylcypromine determined that KDM1 (LSD1) plays a positive role in transcription, specifically of G2/M genes. It requires CoREST, which is recruited on active promoters through direct interactions with NF-Y. These data are the first in vivo indication of a crucial interplay between core histones and ‘deviant’ histone-fold such as NF-Y, leading to fine-tuning of histone methylations

microRNA profiling in Epstein–Barr virus-associated B-cell lymphoma

The Epstein–Barr virus (EBV) is an oncogenic human Herpes virus found in ∼15% of diffuse large B-cell lymphoma (DLBCL). EBV encodes miRNAs and induces changes in the cellular miRNA profile of infected cells. MiRNAs are small, non-coding RNAs of ∼19–26 nt which suppress protein synthesis by inducing translational arrest or mRNA degradation. Here, we report a comprehensive miRNA-profiling study and show that hsa-miR-424, -223, -199a-3p, -199a-5p, -27b, -378, -26b, -23a, -23b were upregulated and hsa-miR-155, -20b, -221, -151-3p, -222, -29b/c, -106a were downregulated more than 2-fold due to EBV-infection of DLBCL. All known EBV miRNAs with the exception of the BHRF1 cluster as well as EBV-miR-BART15 and -20 were present. A computational analysis indicated potential targets such as c-MYB, LATS2, c-SKI and SIAH1. We show that c-MYB is targeted by miR-155 and miR-424, that the tumor suppressor SIAH1 is targeted by miR-424, and that c-SKI is potentially regulated by miR-155. Downregulation of SIAH1 protein in DLBCL was demonstrated by immunohistochemistry. The inhibition of SIAH1 is in line with the notion that EBV impedes various pro-apoptotic pathways during tumorigenesis. The down-modulation of the oncogenic c-MYB protein, although counter-intuitive, might be explained by its tight regulation in developmental processes