Transcriptome Dynamics and Potential Roles of Sox6 in the Postnatal Heart.

The postnatal heart undergoes highly coordinated developmental processes culminating in the complex physiologic properties of the adult heart. The molecular mechanisms of postnatal heart development remain largely unexplored despite their important clinical implications. To gain an integrated view of the dynamic changes in gene expression during postnatal heart development at the organ level, time-series transcriptome analyses of the postnatal hearts of neonatal through adult mice (P1, P7, P14, P30, and P60) were performed using a newly developed bioinformatics pipeline. We identified functional gene clusters by principal component analysis with self-organizing map clustering which revealed organized, discrete gene expression patterns corresponding to biological functions associated with the neonatal, juvenile and adult stages of postnatal heart development. Using weighted gene co-expression network analysis with bootstrap inference for each of these functional gene clusters, highly robust hub genes were identified which likely play key roles in regulating expression of co-expressed, functionally linked genes. Additionally, motivated by the role of the transcription factor Sox6 in the functional maturation of skeletal muscle, the role of Sox6 in the postnatal maturation of cardiac muscle was investigated. Differentially expressed transcriptome analyses between Sox6 knockout (KO) and control hearts uncovered significant upregulation of genes involved in cell proliferation at postnatal day 7 (P7) in the Sox6 KO heart. This result was validated by detecting mitotically active cells in the P7 Sox6 KO heart. The current report provides a framework for the complex molecular processes of postnatal heart development, thus enabling systematic dissection of the developmental regression observed in the stressed and failing adult heart.

Induction of ATF3 gene network by triglyceride-rich lipoprotein lipolysis products increases vascular apoptosis and inflammation.

OBJECTIVE: Elevation of triglyceride-rich lipoproteins (TGRLs) contributes to the risk of atherosclerotic cardiovascular disease. Our work has shown that TGRL lipolysis products in high physiological to pathophysiological concentrations cause endothelial cell injury; however, the mechanisms remain to be delineated. APPROACH AND RESULTS: We analyzed the transcriptional signaling networks in arterial endothelial cells exposed to TGRL lipolysis products. When human aortic endothelial cells in culture were exposed to TGRL lipolysis products, activating transcription factor 3 (ATF3) was identified as a principal response gene. Induction of ATF3 mRNA and protein was confirmed by quantitative reverse-transcription polymerase chain reaction and Western blot respectively. Immunofluorescence analysis showed that ATF3 accumulated in the nuclei of cells treated with lipolysis products. Nuclear expression of phosphorylated c-Jun N-terminal kinase (JNK), previously shown to be an initiator of the ATF3 signaling cascade, also was demonstrated. Small interfering RNA (siRNA)-mediated inhibition of ATF3 blocked lipolysis products-induced transcription of E-selectin and interleukin-8, but not interleukin-6 or nuclear factor-κB. c-Jun, a downstream protein in the JNK pathway, was phosphorylated, whereas expression of nuclear factor-κB-dependent JunB was downregulated. Additionally, JNK siRNA suppressed ATF3 and p-c-Jun protein expression, suggesting that JNK is upstream of the ATF3 signaling pathway. In vivo studies demonstrated that infusion of TGRL lipolysis products into wild-type mice induced nuclear ATF3 accumulation in carotid artery endothelium. ATF3(-/-) mice were resistant to vascular apoptosis precipitated by treatment with TGRL lipolysis products. Also peripheral blood monocytes isolated from postprandial humans had increased ATF3 expression as compared with fasting monocytes. CONCLUSIONS: This study demonstrates that TGRL lipolysis products activate ATF3-JNK transcription factor networks and induce endothelial cells inflammatory response.

Trip12, a HECT domain E3 ubiquitin ligase, targets Sox6 for proteasomal degradation and affects fiber type-specific gene expression in muscle cells.

BACKGROUND: A sophisticated level of coordinated gene expression is necessary for skeletal muscle fibers to obtain their unique functional identities. We have previously shown that the transcription factor Sox6 plays an essential role in coordinating muscle fiber type differentiation by acting as a transcriptional suppressor of slow fiber-specific genes. Currently, mechanisms regulating the activity of Sox6 in skeletal muscle and how these mechanisms affect the fiber phenotype remain unknown. METHODS: Yeast two-hybrid screening was used to identify binding partners of Sox6 in muscle. Small interfering RNA (siRNA)-mediated knockdown of one of the Sox6 binding proteins, Trip12, was used to determine its effect on Sox6 activity in C2C12 myotubes using quantitative analysis of fiber type-specific gene expression. RESULTS: We found that the E3 ligase Trip12, a HECT domain E3 ubiquitin ligase, recognizes and polyubiquitinates Sox6. Inhibiting Trip12 or the 26S proteasome activity resulted in an increase in Sox6 protein levels in C2C12 myotubes. This control of Sox6 activity in muscle cells via Trip12 ubiquitination has significant phenotypic outcomes. Knockdown of Trip12 in C2C12 myotubes led to upregulation of Sox6 protein levels and concurrently to a decrease in slow fiber-specific Myh7 expression coupled with an increased expression in fast fiber-specific Myh4. Therefore, regulation of Sox6 cellular levels by the ubiquitin-proteasome system can induce identity-changing alterations in the expression of fiber type-specific genes in muscle cells. CONCLUSIONS: Based on our data, we propose that in skeletal muscle, E3 ligases have a significant role in regulating fiber type-specific gene expression, expanding their importance in muscle beyond their well-established role in atrophy.

Genome-wide mapping of Sox6 binding sites in skeletal muscle reveals both direct and indirect regulation of muscle terminal differentiation by Sox6.

BACKGROUND: Sox6 is a multi-faceted transcription factor involved in the terminal differentiation of many different cell types in vertebrates. It has been suggested that in mice as well as in zebrafish Sox6 plays a role in the terminal differentiation of skeletal muscle by suppressing transcription of slow fiber specific genes. In order to understand how Sox6 coordinately regulates the transcription of multiple fiber type specific genes during muscle development, we have performed ChIP-seq analyses to identify Sox6 target genes in mouse fetal myotubes and generated muscle-specific Sox6 knockout (KO) mice to determine the Sox6 null muscle phenotype in adult mice. RESULTS: We have identified 1,066 Sox6 binding sites using mouse fetal myotubes. The Sox6 binding sites were found to be associated with slow fiber-specific, cardiac, and embryonic isoform genes that are expressed in the sarcomere as well as transcription factor genes known to play roles in muscle development. The concurrently performed RNA polymerase II (Pol II) ChIP-seq analysis revealed that 84% of the Sox6 peak-associated genes exhibited little to no binding of Pol II, suggesting that the majority of the Sox6 target genes are transcriptionally inactive. These results indicate that Sox6 directly regulates terminal differentiation of muscle by affecting the expression of sarcomere protein genes as well as indirectly through influencing the expression of transcription factors relevant to muscle development. Gene expression profiling of Sox6 KO skeletal and cardiac muscle revealed a significant increase in the expression of the genes associated with Sox6 binding. In the absence of the Sox6 gene, there was dramatic upregulation of slow fiber-specific, cardiac, and embryonic isoform gene expression in Sox6 KO skeletal muscle and fetal isoform gene expression in Sox6 KO cardiac muscle, thus confirming the role Sox6 plays as a transcriptional suppressor in muscle development. CONCLUSIONS: Our present data indicate that during development, Sox6 functions as a transcriptional suppressor of fiber type-specific and developmental isoform genes to promote functional specification of muscle which is critical for optimum muscle performance and health.

Conditional RNA interference mediated by allosteric ribozyme.

Conditional RNA interference (RNAi) enables spatial and/or temporal control over gene silencing. The currently available methods require coexpression of engineered proteins and/or modified promoters which may limit their applications. We designed a novel RNA architecture that combines a drug-inducible allosteric ribozyme with a microRNA precursor analogue that allows chemical induction of RNAi in mammalian cells. The compact and highly modular RNA design should facilitate the construction of conditional RNAi systems that can sense and respond to a variety of molecules recognized by RNA aptamers to regulate virtually any desired genes sensitive to RNAi.

Modulating endogenous gene expression of mammalian cells via RNA-small molecule interaction.

RNA interference (RNAi) has emerged as a powerful technology to silence arbitrary genes by designing small RNA constructs based on the targeted messenger RNA sequences. We recently developed a small molecule-controlled RNAi gene switch that combined the molecular recognition by in vitro selected RNA aptamers with versatile gene silencing by small interfering RNAs, and demonstrated for the first time, posttranscriptional modulation of RNAi through direct RNA-small molecule interaction. In this report, we describe the first application of this technology to regulate an endogenous gene in mammalian cells. As a proof-of-concept demonstration we chose to modulate expression of albumin-serum protein produced by the liver. We designed and constructed a theophylline aptamer-fused short hairpin RNA (shRNA) expression vector targeting albumin mRNA in hepatic (HepG2) cells. Transfection of HepG2 cells with the aptamer-shRNA expression vector allowed to control albumin gene expression by adding theophylline into the culture media in dose dependent fashion.

Artificial control of gene expression in mammalian cells by modulating RNA interference through aptamer-small molecule interaction.

Recent studies have uncovered extensive presence and functions of small noncoding RNAs in gene regulation in eukaryotes. In particular, RNA interference (RNAi) has been the subject of significant investigations for its unique role in post-transcriptional gene regulation and utility as a tool for artificial gene knockdown. Here, we describe a novel strategy for post-transcriptional gene regulation in mammalian cells in which RNAi is specifically modulated through RNA aptamer-small molecule interaction. Incorporation of an RNA aptamer for theophylline in the loop region of a short hairpin RNA (shRNA) designed to silence fluorescent reporter genes led to dose-dependent inhibition of RNAi by theophylline. shRNA cleavage experiments using recombinant Dicer demonstrated that theophylline inhibited cleavage of an aptamer-fused shRNA by Dicer in vitro. Inhibition of siRNA production by theophylline was also observed in vivo. The results presented here provide the first evidence of specific RNA-small molecule interaction affecting RNAi, and a novel strategy to regulate mammalian gene expression by small molecules without engineered proteins.

Functional analysis of two solanesyl diphosphate synthases from Arabidopsis thaliana.

Solanesyl diphosphate (SPP) is regarded as the precursor of the side-chains of both plastoquinone and ubiquinone in Arabidopsis thaliana. We previously analyzed A. thaliana SPP synthase (At-SPS1) (Hirooka et al., Biochem. J., 370, 679-686 (2003)). In this study, we cloned a second SPP synthase (At-SPS2) gene from A. thaliana and characterized the recombinant protein. Kinetic analysis indicated that At-SPS2 prefers geranylgeranyl diphosphate to farnesyl diphosphate as the allylic substrate. Several of its features, including the substrate preference, were similar to those of At-SPS1. These data indicate that At-SPS1 and At-SPS2 share their basic catalytic machinery. Moreover, analysis of the subcellular localization by the transient expression of green fluorescent protein-fusion proteins showed that At-SPS2 is transported into chloroplasts, whereas At-SPS1 is likely to be localized in the endoplasmic reticulum in the A. thaliana cells. It is known that the ubiquinone side-chain originates from isopentenyl diphosphate derived from the cytosolic mevalonate pathway, while the plastoquinone side-chain is synthesized from isopentenyl diphosphate derived from the plastidial methylerythritol phosphate pathway. Based on this information, we propose that At-SPS1 contributes to the biosynthesis of the ubiquinone side-chain and that At-SPS2 supplies the precursor of the plastoquinone side-chain in A. thaliana.

Transient RNAi induction against endogenous genes in Arabidopsis protoplasts using in vitro-prepared double-stranded RNA.

RNA interference is a powerful technique for suppressing gene functions in many eukaryotes including plants. Here we show that introduction of double-stranded RNA into Arabidopsis protoplasts leads to marked silencing of endogenous genes, as observed previously for transgenes [Biosci. Biotechnol. Biochem., 67, 2674-2677 (2003)]. This simple system should be useful for functional analysis of genes involved in fundamental cellular processes.

Transient RNA silencing of scoulerine 9-O-methyltransferase expression by double stranded RNA in Coptis japonica protoplasts.

RNAi (RNA interference, RNA silencing) is a powerful tool for functional genomics, but the construction of an RNAi vector(s) and the establishment of stable transformants are time-consuming and laborious. Here we report the transient RNAi of endogenous biosynthetic genes involved in isoquinoline alkaloid biosynthesis in Coptis japonica protoplasts. Double stranded (ds) RNA fragments of various lengths prepared from several different positions of the coding sequence of scoulerine 9-O-methyltransferase (SMT) were introduced into C. japonica protoplasts by polyethylene glycol-mediated transformation, and their effects were monitored by reverse transcription-polymerase chain reaction. Substantial silencing of SMT gene expression was obtained by the introduction of these SMT dsRNAs. A significant reduction in SMT protein levels was also observed. The potentials of this transient RNAi system to evaluate the functions of biosynthetic genes in Coptis alkaloid research are discussed.

Isolation and characterization of a cDNA encoding phytochrome A in the non-photosynthetic parasitic plant, Orobanche minor Sm.

In this study, the isolation and characterization of a phytochrome A (PHYA) homologous cDNA (OmPHYA) in the non-photosynthetic holoparasitic plant Orobanche minor are described. The present findings provide the first report of the presence of a PHYA homolog in the holoparasite. This study found that OmPHYA is of similar size to the other PHYAs of green plants and shows 72, 77, and 77% amino acid sequence identity with PHYA in Arabidopsis, potato, and tobacco respectively. The OmPHYA contains a conserved chromophore attachment cysteine at position 323. Although OmPHYA shows high sequence identity with other PHYAs in green plants, 13 amino acid substitutions located in both the N and C-terminal domains are observed (a total of 26 amino acids). OmPHYA is encoded by a single gene within the O. minor genome. The abundance of the OmPHYA transcript as well as nuclear translocation of OmphyA occurs in a light-dependent manner.

Flower color modulations of Torenia hybrida by downregulation of chalcone synthase genes with RNA interference.

Suppression of biosynthetic genes involved in flower color formation is an important approach for obtaining target flower colors. Here we report that flower color of the garden plant Torenia hybrida was successfully modulated by RNA interference (RNAi) against a gene of chalcone synthase (CHS), a key enzyme for anthocyanin and flavonoid biosynthesis. By using each of the coding region and the 3'-untranslated region of the CHS mRNA as an RNAi target, exhaustive and gene-specific gene silencing were successfully induced, and the original blue flower color was modulated to white and pale colors, respectively. Our results indicate that RNAi is quite useful for modulations of flower colors of commercially important garden plants.

A transient RNA interference assay system using Arabidopsis protoplasts.

Double-stranded RNA (dsRNA) induces sequence-specific gene silencing in eukaryotes through a process known as RNA interference (RNAi). RNAi is now used as a powerful tool for functional genomics in many eukaryotes, including plants. We herein report a dsRNA-mediated transient RNAi assay system using protoplasts from Arabidopsis mesophyll cells and suspension-cultured cells (cell line T87). Introduction of dsRNA into protoplasts led to marked silencing of target transgenes. Our assay system would provide a convenient and efficient way to induce RNAi in protoplasts of the model plant Arabidopsis thaliana.

Degradation of a peptide in pitcher fluid of the carnivorous plant Nepenthes alata Blanco.

Carnivorous plants acquire substantial amounts of nitrogen from insects. The tropical carnivorous plant Nepenthes produces trapping organs called pitchers at the tips of tendrils elongated from leaf ends. Acidic fluid is secreted at the bottoms of the pitchers. The pitcher fluid includes several hydrolytic enzymes, and some, such as aspartic proteinase, are thought to be involved in nitrogen acquisition from insect proteins. To understand the nitrogen-acquisition process, it is essential to identify the protein-degradation products in the pitcher fluid. To gain insight into protein degradation in pitcher fluid, we used the oxidized B-chain of bovine insulin as a model substrate, and its degradation by the pitcher fluid of N. alata was investigated using liquid chromatography-mass spectrometry (LC-MS). LC-MS analysis of the degradation products revealed that the oxidized B-chain of bovine insulin was initially cleaved at aromatic amino acids such as phenylalanine and tyrosine. These cleavage sites are similar to those of aspartic proteinases from other plants and animals. The presence of a series of peptide fragments as degradation products suggests that exopeptidase(s) is also present in the pitcher fluid. Amino acid analysis and peptide fragment analysis of the degradation products demonstrated that three amino acids plus small peptides were released from the oxidized B-chain of bovine insulin, suggesting that insect proteins are readily degraded to small peptides and amino acids in the pitcher fluid of N. alata.

Aspartic proteinases are expressed in pitchers of the carnivorous plant Nepenthes alata Blanco.

Carnivorous plants acquire significant amounts of nitrogen from insects. The tropical carnivorous plant Nepenthes accumulates acidic fluid containing aspartic proteinase (AP) in its trapping organs (pitchers), suggesting that the plant utilizes insect protein as a nitrogen source. Aspartic proteinases have been purified and characterized from sterile pitcher fluid of several species of Nepenthes; however, there is, as of yet, no information about sequence and expression of Nepenthes AP genes. To identify the pitcher AP, we cloned plant AP homologs from N. alata and examined their expressions. Five AP homologs ( NaAP1-NaAP5) were obtained by reverse transcription-polymerase chain reaction with degenerate primers designed for the conserved sequences of plant APs. Alignment of deduced amino acid sequences with other plant APs demonstrated that NaAP1-NaAP4 contained a plant-specific insert (PSI), a unique sequence of plant AP. However, NaAP5 did not possess the insert, and had a shorter sequence (by >100 amino acids) than the other APs. Northern analysis using a part of the coding region of NaAP1 as a probe showed that bands of approx. 1.8 kb corresponding to the sizes of NaAP1-NaAP4 mRNA were present in roots, stems, leaves, tendrils, and lower part of the pitchers, but a band of approx. 1.3 kb corresponding to the size of NaAP5 mRNA was not observed in any organs. In pitchers, highest expressions of NaAP1-NaAP4 were seen in the lower part of open pitchers containing natural prey, suggesting that the expressions of NaAP1-NaAP4 are coupled with prey capture. Transcripts of NaAP2 and NaAP4 were detected in the digestive glands, where AP secretion may occur. This result suggests that NaAP2 and NaAP4 are the possible APs secreted into the pitcher of N. alata.