Genome-wide identification of grain filling genes regulated by the OsSMF1 transcription factor in rice

Kim, Joung Sug,Chae, Songhwa,Jun, Kyong Mi,Pahk, Yoon-Mok,Lee, Tae-Ho,Chung, Pil Joong,Kim, Yeon-Ki,Nahm, Baek Hie Springer US 2017 Rice Vol.10 No.-

<P><B>Background</B></P><P>Spatial- and temporal-specific expression patterns are primarily regulated at the transcriptional level by gene promoters. Therefore, it is important to identify the binding motifs of transcription factors to better understand the networks associated with embryogenesis.</P><P><B>Results</B></P><P>Here, we used a protein-binding microarray (PBM) to identify the binding motifs of OsSMF1, which is a basic leucine zipper transcription <B>f</B>actor involved in the regulation of rice <B>s</B>eed <B>m</B>aturation. <I>OsSMF1</I> (previously called <I>RISBZ1 or OsbZIP58</I>) is known to interact with GCN4 motifs (TGA(G/C)TCA) to regulate seed storage protein synthesis, and it functions as a key regulator of starch synthesis. Quadruple 9-mer-based PBM analysis and electrophoretic mobility shift assay revealed that OsSMF1 bound to the GCN4 (TGA(G/C)TCA), ACGT (CCACGT(C/G)), and ATGA (GGATGAC) motifs with three different affinities. We predicted 44 putative OsSMF1 target genes using data obtained from both the PBM and RiceArrayNet. Among these putative target genes, 18, 21, and 13 genes contained GCN4, ACGT, and ATGA motifs within their 1-kb promoter regions, respectively. Among them, six genes encoding major grain filling proteins and transcription factors were chosen to confirm the activation of their expression in vivo. OsSMF1 was shown to bind directly to the promoters of Os03g0168500 (GCN4 motif), patatin-like gene (GCN4 motif), α-globulin (ACGT motif), rice prolamin box-binding factor (RPBF) (ATGA motif), and ONAC024 (GCN4 and ACGT motifs) and to regulate their expression.</P><P><B>Conclusions</B></P><P>The results of this study suggest that <I>OsSMF1</I> is one of the key transcription factors that functions in a wide range of seed developmental processes with different specific binding affinities for the three DNA-binding motifs.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (doi:10.1186/s12284-017-0155-4) contains supplementary material, which is available to authorized users.</P>

Development of Oryza sativa Alternative Spliced Transcripts Detecting Microarray

Songhwa Chae,Kyong-Mi Jun,Joung Sug Kim,Baek-Hie Nahm,Yeon-Ki Kim 한국육종학회 2015 한국육종학회 심포지엄 Vol.2015 No.07

Expression profiling was conducted with the Oryza sativa alternative splicing detecting microarray v.4 (OsASDM). Probe features are designed based on rice genome IRGSP_1.0 (http://rapdb.dna.affrc.go.jp/ ). The genome contains 37,868 genes. Among these 5,254 genes have alternative spliced sites, 11,938 transcripts. In the microarray, a total of 41,953 transcripts are covered from all the loci and 9112 alternative spliced transcripts. Four 60-nt long probes were designed from each transcript starting 60 bp ahead the end of stop codon and with shifting 30 bp so 4 probes cover 150 bp in the 3’ region of the gene. Genes from chloroplast (123) and mitochondria (74) and selection markers such as gfp, gus, hyg, bar, and kan are included. In total, he 125,956 probes were designed. To find organ specific transcripts RNA was prepared from leaf, root, panicle at 1 cm (P1cm). The signal intensity files were analyzed with limma package. Background correction and normalization were performed with libraries in the package. 13,486 genes are organ specific and 1,856 transcripts are alternatively spliced. Transcripts that specifically alternatively spliced in leaf are Os02t0197600-02_UE; Chlorophyll a-b binding protein 8, Os11t0707000-01_UE; Ribulose bisphosphate carboxylase/oxygenase, Os12t0291100-01_UE; ribulose 1,5-bisphosphate carboxylase small subunit. Transcripts that specifically alternatively spliced in root are Os03t0669100-02_UE; Deoxyuridine 5’-triphosphate nucleotidohydrolase, Transcripts that specifically alternatively spliced in tissues at P1cm are Os11t0210300-02_UE; Alcohol dehydrogenase 1, Os04t0631200-02_UE; Xyloglucan endotransglycosylase. Os03t0669100-02_UE ; Deoxyuridine 5’-triphosphate nucleotidohydrolase, Os11t0210300-02_UE ; Alcohol dehydrogenase 1, Os04t0631200-02_UE; Xyloglucan endotransglycosylase. These results show that OsASDM could be used to find alternatively spliced gene at ease.

Long-Term Priming by Three Small Molecules Is a Promising Strategy for Enhancing Late Endothelial Progenitor Cell Bioactivities

Kim, Yeon-Ju,Ji, Seung Taek,Kim, Da Yeon,Jung, Seok Yun,Kang, Songhwa,Park, Ji Hye,Jang, Woong Bi,Yun, Jisoo,Ha, Jongseong,Lee, Dong Hyung,Kwon, Sang-Mo Korean Society for Molecular and Cellular Biology 2018 Molecules and cells Vol.41 No.6

Endothelial progenitor cells (EPCs) and outgrowth endothelial cells (OECs) play a pivotal role in vascular regeneration in ischemic tissues; however, their therapeutic application in clinical settings is limited due to the low quality and quantity of patient-derived circulating EPCs. To solve this problem, we evaluated whether three priming small molecules (tauroursodeoxycholic acid, fucoidan, and oleuropein) could enhance the angiogenic potential of EPCs. Such enhancement would promote the cellular bioactivities and help to develop functionally improved EPC therapeutics for ischemic diseases by accelerating the priming effect of the defined physiological molecules. We found that preconditioning of each of the three small molecules significantly induced the differentiation potential of $CD34^+$ stem cells into EPC lineage cells. Notably, long-term priming of OECs with the three chemical cocktail (OEC-3C) increased the proliferation potential of EPCs via ERK activation. The migration, invasion, and tube-forming capacities were also significantly enhanced in OEC-3Cs compared with unprimed OECs. Further, the cell survival ratio was dramatically increased in OEC-3Cs against $H_2O_2$-induced oxidative stress via the augmented expression of Bcl-2, a pro-survival protein. In conclusion, we identified three small molecules for enhancing the bioactivities of ex vivo-expanded OECs for vascular repair. Long-term 3C priming might be a promising methodology for EPC-based therapy against ischemic diseases.

Adequate concentration of B cell leukemia/lymphoma 3 (Bcl3) is required for pluripotency and self-renewal of mouse embryonic stem cells via downregulation of Nanog transcription

( Songhwa Kang ),( Jisoo Yun ),( Da Yeon Kim ),( Seok Yun Jung ),( Yeon Ju Kim ),( Ji Hye Park ),( Seung Taek Ji ),( Woong Bi Jang ),( Jongseong Ha ),( Jae Ho Kim ),( Sang Hong Baek ),( Sang-mo Kwon ) 생화학분자생물학회(구 한국생화학분자생물학회) 2018 BMB Reports Vol.51 No.2

B cell leukemia/lymphoma 3 (Bcl3) plays a pivotal role in immune homeostasis, cellular proliferation, and cell survival, as a co-activator or co-repressor of transcription of the NF-κB family. Recently, it was reported that Bcl3 positively regulates pluripotency genes, including Oct4, in mouse embryonic stem cells (mESCs). However, the role of Bcl3 in the maintenance of pluripotency and self-renewal activity is not fully established. Here, we report the dynamic regulation of the proliferation, pluripotency, and self-renewal of mESCs by Bcl3 via an influence on Nanog transcriptional activity. Bcl3 expression is predominantly observed in immature mESCs, but significantly decreased during cell differentiation by LIF depletion and in mESC-derived EBs. Importantly, the knockdown of Bcl3 resulted in the loss of self-renewal ability and decreased cell proliferation. Similarly, the ectopic expression of Bcl3 also resulted in a significant reduction of proliferation, and the self-renewal of mESCs was demonstrated by alkaline phosphatase staining and clonogenic single cell-derived colony assay. We further examined that Bcl3-mediated regulation of Nanog transcriptional activity in mESCs, which indicated that Bcl3 acts as a transcriptional repressor of Nanog expression in mESCs. In conclusion, we demonstrated that a sufficient concentration of Bcl3 in mESCs plays a critical role in the maintenance of pluripotency and the self-renewal of mESCs via the regulation of Nanog transcriptional activity. [BMB Reports 2018; 51(2): 92-97]

Hypoxia-dependent mitochondrial fission regulates endothelial progenitor cell migration, invasion, and tube formation

Kim, Da Yeon,Jung, Seok Yun,Kim, Yeon Ju,Kang, Songhwa,Park, Ji Hye,Ji, Seung Taek,Jang, Woong Bi,Lamichane, Shreekrishna,Lamichane, Babita Dahal,Chae, Young Chan,Lee, Dongjun,Chung, Joo Seop,Kwon, Sa The Korean Society of Pharmacology 2018 The Korean Journal of Physiology & Pharmacology Vol.22 No.2

Tumor undergo uncontrolled, excessive proliferation leads to hypoxic microenvironment. To fulfill their demand for nutrient, and oxygen, tumor angiogenesis is required. Endothelial progenitor cells (EPCs) have been known to the main source of angiogenesis because of their potential to differentiation into endothelial cells. Therefore, understanding the mechanism of EPC-mediated angiogenesis in hypoxia is critical for development of cancer therapy. Recently, mitochondrial dynamics has emerged as a critical mechanism for cellular function and differentiation under hypoxic conditions. However, the role of mitochondrial dynamics in hypoxia-induced angiogenesis remains to be elucidated. In this study, we demonstrated that hypoxia-induced mitochondrial fission accelerates EPCs bioactivities. We first investigated the effect of hypoxia on EPC-mediated angiogenesis. Cell migration, invasion, and tube formation was significantly increased under hypoxic conditions; expression of EPC surface markers was unchanged. And mitochondrial fission was induced by hypoxia time-dependent manner. We found that hypoxia-induced mitochondrial fission was triggered by dynamin-related protein Drp1, specifically, phosphorylated DRP1 at Ser637, a suppression marker for mitochondrial fission, was impaired in hypoxia time-dependent manner. To confirm the role of DRP1 in EPC-mediated angiogenesis, we analyzed cell bioactivities using Mdivi-1, a selective DRP1 inhibitor, and DRP1 siRNA. DRP1 silencing or Mdivi-1 treatment dramatically reduced cell migration, invasion, and tube formation in EPCs, but the expression of EPC surface markers was unchanged. In conclusion, we uncovered a novel role of mitochondrial fission in hypoxia-induced angiogenesis. Therefore, we suggest that specific modulation of DRP1-mediated mitochondrial dynamics may be a potential therapeutic strategy in EPC-mediated tumor angiogenesis.

Characterization of tissue-specific gene promoters in various organs and stage of reproductive development in rice

Songhwa Chae,Joung Sug Kim,Kyong-Mi Jun,Yeon-Ki Kim,Baek-hie Nahm 한국육종학회 2013 한국육종학회 심포지엄 Vol.2013 No.07

Rice is an important model species and one of the most staple crops of the world. The use of rice appropriate promoters suitable for a specific target transgene is important for the control of spatial and temporal transgene expression. To isolate rice tissue-specific promoters, we exploited the potential of whole genome microarrays in 17 stages: callus, germinating seed, leaf, root, the size of the panicles before heading (1, 3, 5, 8, 10, 15, 20, and 22 cm), and the number of days after pollination (1, 3, 5, 11, 21 DAP) using a 300 K Rice Genome Microarray, covering 31,439 genes of the rice. Eight candidate genes for tissue-specific expression were selected in various organs and stage of reproductive development in rice: Histone H4 for constitutive expression, Dehydrin DHN1 for callus-specific expression, germinating seed-specific hypothetical protein, root-specific hypothetical protein, DNA topoisomerase and Retinoblastoma for expression at panicles before heading, heading-specific profiling, and invertase for expression at seed after pollination. Promoter regions of the selected genes were isolated and fused to the β-glucoronidase (GUS) reporter gene, and the constructs were introduced into rice plants. These promoters are highly active in the tissue-specific manner of rice and can be useful for the spatial and temporal enhancement of target gene(s).

Genome-scale analysis and comparison of gene expression profile in stage of panicle and seed development in rice

Songhwa Chae,Joung Sug Kim,Kyong-Mi Jun,Byung Hyun Dang,Yeon-Ki Kim,Baek-hie Nahm 한국육종학회 2012 한국육종학회 심포지엄 Vol.2012 No.07

Gene expression profiles can serve as a valuable reference for deciphering gene functions. We exploited the potential of whole genome microarrays to measure the temporal expression profiles of rice genes in 13 stages of reproductive development. We could profile expression of 17,676 genes in at least one of the tissues. Differential expression analysis with compare to leaf and preceding stages of development revealed reproductive stage-preferential/-specific genes. we identified 35 genes expressing specifically during panicle and seed development. The metabolic/hormonal pathways and transcription factor families playing key role in reproductive development were elucidated after overlaying the expression data on the public databases and manually curated list of transcription factors, respectively. During floral meristem differentiation (P1cm) and male meiosis (P5cm), the genes involved in jasmonic acid and gebbellin biosynthesis were significantly upregulated. F11DAP stage of seed, containing enlargement organ, exhibited enrichment of transcripts involved in starch or sucrose biosynthesis. Genes regulating auxin biosynthesis were induced during early seed development. We validated the stage-specificity of regulatory regions of two panicle-specific genes, AK072471, Os08g0538700, and AK121412, an early seed-specific gene, in transgenic rice. The data generated here provides a snapshot of the underlying complexity of the gene networks regulating rice reproductive development.

Screening of microorganisms for biodegradation of pesticide residues in soils

Songhwa Kim,Ju Hee An,Ho-Jong Ju,Jaekyeong Song 한국농약과학회 2022 한국농약과학회 학술발표대회 논문집 Vol.2022 No.4

Analysis of Genes with Alternatively Spliced Transcripts in the Leaf, Root, Panicle and Seed of Rice Using a Long Oligomer Microarray and RNA-Seq

Chae, Songhwa,Kim, Joung Sug,Jun, Kyong Mi,Lee, Sang-Bok,Kim, Myung Soon,Nahm, Baek Hie,Kim, Yeon-Ki Korean Society for Molecular and Cellular Biology 2017 Molecules and cells Vol.40 No.10

Pre-mRNA splicing further increases protein diversity acquired through evolution. The underlying driving forces for this phenomenon are unknown, especially in terms of gene expression. A rice alternatively spliced transcript detection microarray (ASDM) and RNA sequencing (RNA-Seq) were applied to differentiate the transcriptome of 4 representative organs of Oryza sativa L. cv. Ilmi: leaves, roots, 1-cm-stage panicles and young seeds at 21 days after pollination. Comparison of data obtained by microarray and RNA-Seq showed a bell-shaped distribution and a co-lineation for highly expressed genes. Transcripts were classified according to the degree of organ enrichment using a coefficient value (CV, the ratio of the standard deviation to the mean values): highly variable (CVI), variable (CVII), and constitutive (CVIII) groups. A higher index of the portion of loci with alternatively spliced transcripts in a group (IAST) value was observed for the constitutive group. Genes of the highly variable group showed the characteristics of the examined organs, and alternatively spliced transcripts tended to exhibit the same organ specificity or less organ preferences, with avoidance of 'organ distinctness'. In addition, within a locus, a tendency of higher expression was found for transcripts with a longer coding sequence (CDS), and a spliced intron was the most commonly found type of alternative splicing for an extended CDS. Thus, pre-mRNA splicing might have evolved to retain maximum functionality in terms of organ preference and multiplicity.

Development of the Tos17-insertional mutants and functional analysis of transcription factors involved in seed development

Joung Sug Kim,Songhwa Chae,Kyong-Mi Jun,Yoon Mok Pahk,Yeon-Ki Kim,Baek-hie Nahm 한국육종학회 2015 한국육종학회 심포지엄 Vol.2015 No.07

Rice, as a model system of monocotyledon plants for genomic studies, is a main staple food for over half of the world population. A rice retrotransposon, Tos17, is active during tissue culture and its ability was wildly used in insertional mutagenesis. In this study we have produced 2,000 non-GM mutants induced by Tos17 in rice. We analyzed >2,000 flanking sequences of newly transposed Tos17 copies by the adaptor-ligation PCR method. The frequencies of Tos17 insertions in the genic and intergenic regions were 60.3% and 36.6%, respectively. We also selected four Tos17 insertion mutant lines for three TF genes which can be considered to be considered to be involved in rice seed development based on expression microarray data: osrem3, osta1, osbhlh1-1, and osbhlh1-2 mutant lines. According to Quadruple 9-mer-based protein binding microarray (Q9-UPBM) experiment, we found that the OsREM3, OsTA1, and OsbHLH1 bound to the ACACCAC, CACGTG, and GTAACA motifs, respectively. In combination of Q9-UPBM, RiceArrayNet analysis, and expression microarray data, we identified 8, 20, and 9 putative target genes of OsREM3, OsTA1, and OsbHLH1, respectively. We have been screening and characterizing the mutations by extensive phenotypic analysis as well as the functional analysis of genes.