Jaeseung Yoon

Regulation of vascular endothelial growth factor signaling by miR-200b

Vascular endothelial growth factor (VEGF) signaling plays an important role in angiogenesis. In the VEGF signaling pathway, the key components are VEGF and its receptors, Flt-1 and KDR. In this study, we show that transfection of synthetic miR-200b reduced protein levels of VEGF, Flt-1, and KDR. In A549 cells, miR-200b targeted the predicted binding sites in the 3′-untranslated region (3′-UTR) of VEGF, Flt-1, and KDR as revealed by a luciferase reporter assay. When transfected with miR-200b, the ability of HUVECs to form a capillary tube on Matrigel and VEGF-induced phosphorylation of ERK1/2 were significantly reduced. Taken together, these results suggest that miR-200b negatively regulates VEGF signaling by targeting VEGF and its receptors and that miR-200b may have therapeutic potential as an angiogenesis inhibitor.

Induction of growth arrest by miR-542-3p that targets survivin

Survivin is a protein which functions as a mitotic regulator as well as apoptosis inhibitor. In this study, we show that introduction of synthetic miR‐542‐3p mimetic reduced both mRNA and protein levels of survivin. In A549 cells, luciferase reporter assay revealed that miR‐542‐3p targeted predicted binding sites in the 3′‐untranslated region (3′‐UTR) of survivin. We also demonstrate that ectopic expression of miR‐542‐3p inhibited cell proliferation by inducing Gap 1 (G1) and Gap 2/Mitosis (G2/M) cell cycle arrest. Collectively, these results suggest that survivin is a direct target of miR‐542‐3p and growth inhibition by miR‐542‐3p may have a potential utility as an anti‐cancer therapy.

Molecular characterization of a gene encoding the Drosophila melanogaster phospholipase A2.

A gene encoding Drosophila melanogaster secretory phospholipase A2 (sPLA2) has been cloned and characterized(.) The coding region of the sPLA2 gene was interrupted by a short intron, and codes for a signal peptide of 18 amino acids, followed by a mature protein of 168 amino acids, containing the structural features of group III sPLA2. From a Northern blot analysis, about a 1.0-kb Drosophila sPLA2 transcript was found to be expressed throughout its development and in both the adult bodies and heads. The recombinant Drosophila sPLA2 expressed and purified in Escherichia coli was found to be Ca(+2)-dependent and maximally active at pH 5.

Efficient Selection of Stable Chinese Hamster Ovary (CHO) Cell Lines for Expression of Recombinant Proteins by Using Human Interferon β SAR Element

We describe the development of an efficient expression system suitable for the stable expression of recombinant genes in Chinese hamster ovary (CHO) cells using the human interferon β SAR element. The insertion of two copies of the human interferon β SAR element at the 5′ and 3′ flanking regions of the β‐galactosidase reporter gene increased the frequency of β‐galactosidase positive colonies by up to 75% and enhanced β‐galactosidase expression by 15‐ to 20‐fold after G418 selection or 30‐ to 40‐fold at the initial stage of the MTX selection procedure. Deletion analysis showed that the whole DNA regions of the human interferon β SAR element are required for β‐galactosidase expression enhancement. The developed expression system was also highly effective at enhancing the stable expression of two therapeutically important proteins, namely, erythropoietin (EPO) and hepatocyte growth factor (HGF). We isolated stable colonies with expression levels of 47 μg/106 cells/day for EPO and 13 μg/106 cells/day for HGF, suggesting that the developed expression system based on the human β SAR element is suitable for expressing high levels of recombinant proteins in CHO cells.

The DNA replication-related element (DRE)–DRE-binding factor (DREF) system may be involved in the expression of the Drosophila melanogaster TBP gene

The TATA box binding protein (TBP) is a general transcription factor required for initiation by all three eukaryotic RNA polymerases. Previously, we found that the promoter region of the Drosophila melanogaster TBP gene contains three sequences similar to the DNA replication‐related element (DRE) (5′‐TATCGATA). In the present study, we found that the DRE‐like sequences are also present in the promoter of the Drosophila virilis TBP gene, suggesting a role for these sequences in TBP expression. Band mobility shift assays revealed that oligonucleotides containing sequences similar to the DRE of D. melanogaster TBP gene promoter form specific complexes with a factor in a Kc cell nuclear extract and with recombinant DRE‐binding factor (DREF). Furthermore, these complexes were either supershifted or diminished by monoclonal antibodies to DREF. Transient luciferase assays demonstrated that induction of mutations in two DRE‐related sequences at positions −223 and −63 resulted in an extensive reduction of promoter activity. Thus, the DRE–DREF system appears to be involved in the expression of the D. melanogaster TBP gene.

Direct transcriptional regulation of Six6 is controlled by SoxB1 binding to a remote forebrain enhancer.

Six6, a sine oculis homeobox protein, plays a crucial and conserved role in the development of the forebrain and eye. To understand how the expression of Six6 is regulated during embryogenesis, we screened ~250 kb of genomic DNA encompassing the Six6 locus for cis-regulatory elements capable of directing reporter gene expression to sites of Six6 transcription in transgenic mouse embryos. Here, we describe two novel enhancer elements, that are highly conserved in vertebrate species and whose activities recapitulate Six6 expression in the ventral forebrain and eye, respectively. Cross-species comparisons of the Six6 forebrain enhancer sequences revealed highly conserved binding sites matching the consensus for homeodomain and SoxB1 transcription factors. Deletion of either of the binding sites resulted in loss of the forebrain enhancer activity in the ventral forebrain. Moreover, our studies show that members of the SoxB1 family, including Sox2 and Sox3, are expressed in the overlapping region of the ventral forebrain with Six6 and can bind to the Six6 forebrain enhancer. Loss of function of SoxB1 genes in vivo further emphasizes their role in regulating Six6 forebrain enhancer activity. Thus, our data strongly suggest that SoxB1 transcription factors are direct activators of Six6 expression in the ventral forebrain.

Inhibition of cell proliferation and migration by miR-509-3p that targets CDK2, Rac1, and PIK3C2A.

CDK2 is a key regulator of cell cycle progression. In this study, we screened for miRNAs targeting CDK2 using a luciferase-3′-untranslated region reporter assay. Among 11 hit miRNAs, miR-509-3p reduced CDK2 protein levels and significantly inhibited cancer cell growth. Microarray, Western blotting, and luciferase reporter analyses revealed additional targets of miR-509-3p, including Rac1 and PIK3C2A. Overexpression of miR-509-3p induced G1 cell-cycle arrest and inhibited colony formation and migration. RNAi experiments indicated that the growth-inhibitory effects of miR-509-3p may occur through down-regulation of CDK2, Rac1, and PIK3C2A. Targeting of multiple growth regulatory genes by miR-509-3p may contribute to effective anti-cancer therapy.

Drosophila G9a is implicated in germ cell development

In Drosophila ovaries, germline stem cells (GSCs) divide asymmetrically in the germaria to produce daughter GSCs and cystoblasts. Single cystoblasts differentiate to form germline cysts with 16 germline cells, all of which are connected by the fusome, a vesiculated structure critical for oocyte specification. We here show that histone H3K9 methyltransferase dg9a is associated with spectrosome/fusome formation in the germarium; dG9a13414 mutant ovaries have disorganized spectrosome/fusome in about half the germaria, with reduced levels of hu‐li tai shao and α‐SPECTRIN proteins. We found that the amount of germline cells within cysts was reduced and that oocyte determination often failed in egg chambers of the dG9a13414 mutant ovaries. These results suggest that a mutation in dG9a gene gives rise to anomalous spectrosome/fusome structures, which in turn lead to faulty germ‐cell development in Drosophila ovaries.

Ascl1 and Helt act combinatorially to specify thalamic neuronal identity by repressing Dlxs activation.

The mammalian thalamus is an essential diencephalic derivative that plays unique roles in processing and relaying sensory and motor information to and from the cerebral cortex. The profile of transcription factors and lineage tracing experiments revealed a spatiotemporal relationship between diencephalic progenitor domains and discrete differentiated neurons contributing to thalamic nuclei. However, the precise molecular mechanisms by which heterogeneous thalamic neurons become specified and assemble into distinct thalamic nuclei are still poorly understood. Here, we show that a combinatorial interaction between the bHLH transcription factors Ascl1 and Helt is required for acquiring thalamic progenitor identity. Surprisingly, in the combined absence of Ascl1 and Helt, rostral thalamic progenitors (TH-R) adopt a molecular profile of a more rostral diencephalic derivative, the prethalamus. Furthermore, we show that the prethalamic factors Dlxs upregulated by Ascl1/Helt deficiency play unique roles in regulating thalamic progenitor specification, and that derepression of Dlx2 and Dlx5 suppress generation of TH-R neurons. Taken together, our results suggest a model whereby the combined activity of two distinct bHLH factors plays a key role in the development of discrete classes of thalamic interneurons.

Characterization of the Drosophila melanogaster retinin gene encoding a cornea-specific protein

Two‐dimensional analysis of head extracts from Drosophila melanogaster identified the four eye‐specific protein spots corresponding to the retinin protein. The retinin protein spots were specifically stained with phosphoprotein‐specific dye, suggesting that the retinin protein undergoes post‐translational modification by phosphorylation. Northern blot analysis showed that the retinin gene begins to be expressed during the late stage of puparium formation during development. Analysis of the N‐terminal sequence and expression of the retinin gene in S2 suggest that retinin is a secretory protein. Transgenic flies with knockdown expression of the retinin gene by RNA interference (RNAi) were established. However, no significant phenotypic changes in eye structure or phototransduction were observed in the transgenic flies. Western blot analysis and immunohistochemical studies of D. melanogaster eyes suggest that retinin is a cornea‐specific protein.