Noriko Ninomiya

Induction of microRNAs, mir-155, mir-222, mir-424 and mir-503, promotes monocytic differentiation through combinatorial regulation

Acute myeloid leukemia (AML) involves a block in terminal differentiation of the myeloid lineage and uncontrolled proliferation of a progenitor state. Using phorbol myristate acetate (PMA), it is possible to overcome this block in THP-1 cells (an M5-AML containing the MLL-MLLT3 fusion), resulting in differentiation to an adherent monocytic phenotype. As part of FANTOM4, we used microarrays to identify 23 microRNAs that are regulated by PMA. We identify four PMA-induced microRNAs (mir-155, mir-222, mir-424 and mir-503) that when overexpressed cause cell-cycle arrest and partial differentiation and when used in combination induce additional changes not seen by any individual microRNA. We further characterize these pro-differentiative microRNAs and show that mir-155 and mir-222 induce G2 arrest and apoptosis, respectively. We find mir-424 and mir-503 are derived from a polycistronic precursor mir-424-503 that is under repression by the MLL-MLLT3 leukemogenic fusion. Both of these microRNAs directly target cell-cycle regulators and induce G1 cell-cycle arrest when overexpressed in THP-1. We also find that the pro-differentiative mir-424 and mir-503 downregulate the anti-differentiative mir-9 by targeting a site in its primary transcript. Our study highlights the combinatorial effects of multiple microRNAs within cellular systems.

Clusters of internally primed transcripts reveal novel long noncoding RNAs.

Non-protein-coding RNAs (ncRNAs) are increasingly being recognized as having important regulatory roles. Although much recent attention has focused on tiny 22- to 25-nucleotide microRNAs, several functional ncRNAs are orders of magnitude larger in size. Examples of such macro ncRNAs include Xist and Air, which in mouse are 18 and 108 kilobases (Kb), respectively. We surveyed the 102,801 FANTOM3 mouse cDNA clones and found that Air and Xist were present not as single, full-length transcripts but as a cluster of multiple, shorter cDNAs, which were unspliced, had little coding potential, and were most likely primed from internal adenine-rich regions within longer parental transcripts. We therefore conducted a genome-wide search for regional clusters of such cDNAs to find novel macro ncRNA candidates. Sixty-six regions were identified, each of which mapped outside known protein-coding loci and which had a mean length of 92 Kb. We detected several known long ncRNAs within these regions, supporting the basic rationale of our approach. In silico analysis showed that many regions had evidence of imprinting and/or antisense transcription. These regions were significantly associated with microRNAs and transcripts from the central nervous system. We selected eight novel regions for experimental validation by northern blot and RT-PCR and found that the majority represent previously unrecognized noncoding transcripts that are at least 10 Kb in size and predominantly localized in the nucleus. Taken together, the data not only identify multiple new ncRNAs but also suggest the existence of many more macro ncRNAs like Xist and Air.

Gene organization in rice revealed by full-length cDNA mapping and gene expression analysis through microarray.

Rice (Oryza sativa L.) is a model organism for the functional genomics of monocotyledonous plants since the genome size is considerably smaller than those of other monocotyledonous plants. Although highly accurate genome sequences of indica and japonica rice are available, additional resources such as full-length complementary DNA (FL-cDNA) sequences are also indispensable for comprehensive analyses of gene structure and function. We cross-referenced 28.5K individual loci in the rice genome defined by mapping of 578K FL-cDNA clones with the 56K loci predicted in the TIGR genome assembly. Based on the annotation status and the presence of corresponding cDNA clones, genes were classified into 23K annotated expressed (AE) genes, 33K annotated non-expressed (ANE) genes, and 5.5K non-annotated expressed (NAE) genes. We developed a 60mer oligo-array for analysis of gene expression from each locus. Analysis of gene structures and expression levels revealed that the general features of gene structure and expression of NAE and ANE genes were considerably different from those of AE genes. The results also suggested that the cloning efficiency of rice FL-cDNA is associated with the transcription activity of the corresponding genetic locus, although other factors may also have an effect. Comparison of the coverage of FL-cDNA among gene families suggested that FL-cDNA from genes encoding rice- or eukaryote-specific domains, and those involved in regulatory functions were difficult to produce in bacterial cells. Collectively, these results indicate that rice genes can be divided into distinct groups based on transcription activity and gene structure, and that the coverage bias of FL-cDNA clones exists due to the incompatibility of certain eukaryotic genes in bacteria.

Identification of region-specific transcription factor genes in the adult mouse brain by medium-scale real-time RT-PCR

We established a medium‐scale real‐time RT‐PCR system focusing on transcription factors and applied it to their expression profiles in the adult mouse 11 brain regions (http://genome.gsc.riken.jp/qRT‐PCR/). Almost 90% of the examined genes showed significant expression in at least one region. We successfully extracted 179 region‐specific genes by clustering analysis. Interestingly, the transcription factors involved in the development of the pituitary were still expressed in the adult pituitary, suggesting that they also play important roles in maintenance of the pituitary. These results provide unique molecular markers that may account for the molecular basis of the unique functions of specific brain regions.

Establishment of single-cell screening system for the rapid identification of transcriptional modulators involved in direct cell reprogramming

Combinatorial interactions of transcription modulators are critical to regulate cell-specific expression and to drive direct cell reprogramming (e.g. trans-differentiation). However, the identification of key transcription modulators from myriad of candidate genes is laborious and time consuming. To rapidly identify key regulatory factors involved in direct cell reprogramming, we established a multiplex single-cell screening system using a fibroblast-to-monocyte transition model. The system implements a single-cell ‘shotgun-transduction’ strategy followed by nested-single-cell-polymerase chain reaction (Nesc-PCR) gene expression analysis. To demonstrate this, we simultaneously transduced 18 monocyte-enriched transcription modulators in fibroblasts followed by selection of single cells expressing monocyte-specific CD14 and HLA-DR cell-surface markers from a heterogeneous population. Highly multiplex Nesc-PCR expression analysis revealed a variety of gene combinations with a significant enrichment of SPI1 (86/86) and a novel transcriptional modulator, HCLS1 (76/86), in the CD14+/HLA-DR+ single cells. We could further demonstrate the synergistic role of HCLS1 in regulating monocyte-specific gene expressions and phagocytosis in dermal fibroblasts in the presence of SPI1. This study establishes a platform for a multiplex single-cell screening of combinatorial transcription modulators to drive any direct cell reprogramming.

Development of a high-throughput method for the systematic identification of human proteins nuclear translocation potential

BackgroundImportant clues to the function of novel and uncharacterized proteins can be obtained by identifying their ability to translocate in the nucleus. In addition, a comprehensive definition of the nuclear proteome undoubtedly represents a key step toward a better understanding of the biology of this organelle. Although several high-throughput experimental methods have been developed to explore the sub-cellular localization of proteins, these methods tend to focus on the predominant localizations of gene products and may fail to provide a complete catalog of proteins that are able to transiently locate into the nucleus.ResultsWe have developed a method for examining the nuclear localization potential of human gene products at the proteome scale by adapting a mammalian two-hybrid system we have previously developed. Our system is composed of three constructs co-transfected into a mammalian cell line. First, it contains a PCR construct encoding a fusion protein composed of a tested protein, the PDZ-protein TIP-1, and the transactivation domain of TNNC2 (referred to as ACT construct). Second, our system contains a PCR construct encoding a fusion protein composed of the DNA binding domain of GAL4 and the PDZ binding domain of rhotekin (referred to as the BIND construct). Third, a GAL4-responsive luciferase reporter is used to detect the reconstitution of a transcriptionally active BIND-ACT complex through the interaction of TIP-1 and rhotekin, which indicates the ability of the tested protein to translocate into the nucleus. We validated our method in a small-scale feasibility study by comparing it to green fluorescent protein (GFP) fusion-based sub-cellular localization assays, sequence-based computational prediction of protein sub-cellular localization, and current sub-cellular localization data available from the literature for 22 gene products.ConclusionOur reporter-based system can rapidly screen gene products for their ability to be translocated to the nucleus. Large-scale applications of the system presented herein should provide invaluable information for a more complete biological atlas.