Chin-Hwa Hu

xUBF, an RNA polymerase I transcription factor, binds crossover DNA with low sequence specificity.

Xenopus UBF (xUBF) is a transcription factor for RNA polymerase I which contains multiple DNA-binding motifs. These include a short basic region adjacent to a dimer motif plus five high-mobility-group (HMG) boxes. All of these DNA-binding motifs exhibit low sequence specificity, whether assayed singly or together. In contrast, the HMG boxes recognize DNA structure that is formed when two double helices are crossed over each other. HMG box 1, in particular, requires association of two double helices before it will bind and, either by itself or in the context of the intact protein, will loop DNA and organize it into higher-order structures. We discuss how this mode of binding affects the function of xUBF as a transcription factor.

Detection of Ah receptor and Ah receptor nuclear translocator mRNAs in the oocytes and developing embryos of zebrafish (Danio rerio)

The heterodimeric complex of Ah receptor (AHR) – Ah receptor nuclear translocator (ARNT) is an ubiquitous transcription factor which mediates the expression of vertebrate xenobiotic-response genes, such as CYP1A1 and CYP1A2. AHR also performs key functions in murine tissue differentiation. Both AHR and ARNT factors share conserved function domains with PAS domain families, such as Drosophila CNS-developing modulator, SIM, and biological rhythm factor, PER. By using RT-PCR technique, we have obtained partial cDNA fragments of zebrafish AHR and ARNT from fish tissues. We found that both ahr and arnt genes are active during fish embryogenesis. The mRNAs of AHR and ARNT are also transcribed in oocytes as maternal mRNA. The deduced amino acid sequences derived from the amplified cDNA fragments share significant homology with the respective mammalian AHR and ARNT PAS domain sequences. The xenobiotic, 2,3,7,8-TCDD, strongly induces zebrafishCYP1A expression during embryogenesis. Apparently, both AHR and ARNT factors are present in fish embryos in forms that can accurately respond to the proper ligands to induce CYP1A. We speculate these factors play similar functional roles in fish development as they do during developemnt of murine embryos.

Influence of TCDD on Zebrafish CYP1B1 Transcription during Development

Cytochrome P450 1B1 (CYP1B1) is a heme-containing monooxygenase that metabolizes various polycyclic aromatic hydrocarbons and aryl amines, as well as retinoic acid and steroid hormones. Here we report the cloning of an ortholog of CYP1B1 from zebrafish and the demonstration that transcription of zebrafish CYP1B1 was modulated by two types of mechanisms during different developmental stage. First in late pharyngula stage before hatching, CYP1B1 was constitutively transcribed in retina, midbrain-hindbrain boundary and diencephalon regions through a close coordination between aryl hydrocarbon receptor 2 (AHR2)-dependent and AHR2-independent pathways. After hatching, the basal transcription was attenuated and it could not be elicited upon 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. In contrast, TCDD exposure induced de novo CYP1B1 transcription in larval branchial arches and heart tissues via an AHR2-dependent pathway. Blocking AHR2 translation completely eliminated the TCDD-mediated CYP1B1 transcription. However, we did not detect any types of CYP1B1 transcription in liver and kidney tissues through the developmental stage. It suggests that the constitutive and TCDD-inducible types of CYP1B1 transcriptions are modulated by distinct pathways with different tissue specificities. Finally, we investigated the role of CYP1B1 in TCDD-mediated embryonic toxicity. Because knockdown of CYP1B1 did not prevent TCDD-induced pericardial edema and cranial defects, it suggests that CYP1B1 is not involved in the developmental toxicity of dioxin.

Overexpression of a Zebrafish ARNT2-like Factor Represses CYP1A Transcription in ZLE Cells

Abstract: Aryl hydrocarbon receptor nuclear translocator (ARNT) factors belong to a novel basic-helix-loop-helix–PAS (bHLH-PAS) transcription factor family that controls a variety of physiological and developmental processes. In a previous study, we obtained a partial complementary DNA fragment of an ARNT2-like factor from zebrafish embryo, liver, and other tissues by reverse transcription–polymerase chain reaction. In an effort to characterize the function of this factor, we screened an embryonic complementary DNA library and obtained a complete cDNA of this ARNT2-like factor, zARNT2A. The deduced protein sequence of zARNT2A encompasses the basic-helix-loop-helix and PAS-A/B motifs and shares highest sequence similarity with the amino-terminal half of mouse ARNT2 factor. However, it lacks a carboxy-terminal transactivation motif following the PAS-A/B motifs. Transient expression of zARNT2A in cultured cells resulted in repression of TCDD-dependent CYP1A transcription. Whole-mount in situ hybridization revealed that zARNT2A is expressed in brain and pronephros at prime-5 stages. In adult fish, zARNT2A messenger RNA is transcribed in a wide range of tissues, which indicates that zARNT2A and its corresponding signal transduction mechanisms have important roles in fish development and other physiological aspects.

Hypoxia-Inducible Factor 2 Alpha Is Essential for Hepatic Outgrowth and Functions via the Regulation of leg1 Transcription in the Zebrafish Embryo

The liver plays a vital role in metabolism, detoxification, digestion, and the maintenance of homeostasis. During development, the vertebrate embryonic liver undergoes a series of morphogenic processes known as hepatogenesis. Hepatogenesis can be separated into three interrelated processes: endoderm specification, hepatoblast differentiation, and hepatic outgrowth. Throughout this process, signaling molecules and transcription factors initiate and regulate the coordination of cell proliferation, apoptosis, differentiation, intercellular adhesion, and cell migration. Hifs are already recognized to be essential in embryonic development, but their role in hepatogenesis remains unknown. Using the zebrafish embryo as a model organism, we report that the lack of Hif2-alpha but not Hif1-alpha blocks hepatic outgrowth. While Hif2-alpha is not involved in hepatoblast specification, this transcription factor regulates hepatocyte cell proliferation during hepatic outgrowth. Furthermore, we demonstrated that the lack of Hif2-alpha can reduce the expression of liver-enriched gene 1 (leg1), which encodes a secretory protein essential for hepatic outgrowth. Additionally, exogenous mRNA expression of leg1 can rescue the small liver phenotype of hif2-alpha morphants. We also showed that Hif2-alpha directly binds to the promoter region of leg1 to control leg1 expression. Interestingly, we discovered overrepresented, high-density Hif-binding sites in the potential upstream regulatory sequences of leg1 in teleosts but not in terrestrial mammals. We concluded that hif2-alpha is a key factor required for hepatic outgrowth and regulates leg1 expression in zebrafish embryos. We also proposed that the hif2-alpha-leg1 axis in liver development may have resulted from the adaptation of teleosts to their environment.

Estrogen modulates osteogenic activity and estrogen receptor mRNA in mesenchymal stem cells of women

ABSTRACT Objective To determine whether estrogen regulates mesenchymal stem cell (MSC) activity in bone marrow from osteoporotic postmenopausal women. Methods MSCs were collected from bone marrows which were aspirated simultaneously during iliac bone graft procedures in spine fusion surgery in osteoporotic postmenopausal women. We investigated proliferation, differentiation, osteogenic activity, and estrogen receptor (ER) α and β mRNA expression of primary culture MSCs isolated from four osteoporotic postmenopausal women, treated in vitro with or without 17β-estradiol. The expression of alkaline phosphatase (ALP), osteocalcin, interleukin-6, ERα and ERβ mRNA was evaluated. Results The expression of ALP and osteocalcin mRNA was detected during the cultures of MSCs and was observed to increase up to day 20. As compared with MSCs not treated with estradiol, a significant increase in DNA content, ERα mRNA, and ALP mRNA expression was observed in cultures with estradiol. The mRNA expression of osteocalcin and interleukin-6 was significantly lower in MSCs treated with estradiol than those without estradiol. There was no significant difference in the mRNA expression of ERβ between MSCs cultured with and without estradiol. Conclusions In the proper environment, MSCs from osteoporotic women can differentiate into osteoblasts and estrogen enhances the osteogenic activity possibly via ERα activity.

Ethanol inhibits retinal and CNS differentiation due to failure of cell cycle exit via an apoptosis-independent pathway

Alcohol exposure during embryogenesis results in a variety of developmental disorders. Here, we demonstrate that continuous exposure to 1.5% ethanol causes substantial apoptosis and abrogated retinal and CNS development in zebrafish embryos. Chronic exposure to ethanol for 24h before hatching also induces apoptosis and retinal disorder. After the 2-day post-fertilization (dpf) stage, chronic exposure to ethanol continued to induce apoptosis, but did not block retinal differentiation. Although continuous ethanol exposure induces substantial accumulation of reactive oxygen species (ROS) and increases p53 expression, depletion of p53 did not eliminate ethanol-induced apoptosis. On the other hand, sequestering ROS with the antioxidant reagent N-acetylcysteine (NAC) successfully inhibited ethanol-associated apoptosis, suggesting that the ethanol-induced cell death primarily results from ROS accumulation. Continuous ethanol treatment of embryos reduced expression of the mature neural and photoreceptor markers elavl3/huC, rho, and crx; in addition, expression of the neural and retinal progenitor markers ascl1b and pax6b was maintained at the undifferentiated stage, indicating that retinal and CNS neural progenitor cells failed to undergo further differentiation. Moreover, ethanol treatment enhanced BrdU incorporation, histone H3 phosphorylation, and pcna expression in neural progenitor cells, thereby maintaining a high rate of proliferation. Ethanol treatment also resulted in sustained transcription of ccnd1/cyclin D1 and ccne/cyclin E throughout development in neural progenitor cells, without an appropriate increase of cdkn1b/p27 and cdkn1c/p57 expression, suggesting that these cells failed to exit from the cell cycle. Although NAC was able to mitigate ethanol-mediated apoptosis, it was unable to ameliorate the defects in visual and CNS neural differentiation, suggesting that abrogated neural development in ethanol-exposed embryos is unlikely to arise from excessive apoptosis. In conclusion, we demonstrate that the pathological effect of ethanol on zebrafish embryos is partially attributable to cell death and inhibition of visual and CNS neuron differentiation. Excessive apoptosis largely results from the accumulation of ROS, whereas abrogated neural development is caused by failure of cell cycle arrest, which in turn prevents a successful transition from proliferation to differentiation.

Expression of Estrogen Receptors Alfa and Beta mRNA and Alkaline Phosphatase in the Differentiation of Osteoblasts from Elderly Postmenopausal Women: Comparison with Osteoblasts from Osteosarcoma Cell Lines

OBJECTIVE To evaluate the expression of estrogen receptors (ER) alpha and beta, and activity of alkaline phosphatase during differentiation of primary osteoblast cells (hOB) from aged postmenopausal women and human osteosarcoma cell lines (HOS, MG63). MATERIALS AND METHODS Osteoblast cultures were prepared from the upper femur of postmenopausal patients (age, 60-74 years) and HOS. At the indicated times (days 5, 10, 15, 20, and 25), alkaline phosphatase activity and expression of ERalpha and ERbeta mRNA were evaluated. RESULTS In both cultures of primary hOB and HOS, alkaline phosphatase activity decreased at the osteoblast proliferation stage, whereas it subsequently increased at the matrix maturation stage. ER beta mRNA was strongly expressed in HOS on day 15 and remained at high levels of transcription through to day 25 (matrix maturation phase), whereas ERalpha mRNA was barely detectable during osteoblast differentiation. In hOB, transcription of ERalpha mRNA was much stronger than that of ERbeta mRNA. CONCLUSION The presence of ERalpha and ERbeta mRNA in osteoblasts supports the involvement of estrogen in human bone formation. The developmental expression of alkaline phosphatase was not correlated to ER mRNA expression during osteoblast differentiation. ER isoforms may have different functions or interact with each other during osteoblast differentiation. Since the expression of ER isoforms is different between postmenopausal women and osteosarcoma cell lines, characteristics of osteosarcoma cell lines may not be suitable as a model for the evaluation of estrogen effects on postmenopausal osteoporosis.

Molecular Evolution of Multiple-Level Control of Heme Biosynthesis Pathway in Animal Kingdom

Adaptation of enzymes in a metabolic pathway can occur not only through changes in amino acid sequences but also through variations in transcriptional activation, mRNA splicing and mRNA translation. The heme biosynthesis pathway, a linear pathway comprised of eight consecutive enzymes in animals, provides researchers with ample information for multiple types of evolutionary analyses performed with respect to the position of each enzyme in the pathway. Through bioinformatics analysis, we found that the protein-coding sequences of all enzymes in this pathway are under strong purifying selection, from cnidarians to mammals. However, loose evolutionary constraints are observed for enzymes in which self-catalysis occurs. Through comparative genomics, we found that in animals, the first intron of the enzyme-encoding genes has been co-opted for transcriptional activation of the genes in this pathway. Organisms sense the cellular content of iron, and through iron-responsive elements in the 5′ untranslated regions of mRNAs and the intron-exon boundary regions of pathway genes, translational inhibition and exon choice in enzymes may be enabled, respectively. Pathway product (heme)-mediated negative feedback control can affect the transport of pathway enzymes into the mitochondria as well as the ubiquitin-mediated stability of enzymes. Remarkably, the positions of these controls on pathway activity are not ubiquitous but are biased towards the enzymes in the upstream portion of the pathway. We revealed that multiple-level controls on the activity of the heme biosynthesis pathway depend on the linear depth of the enzymes in the pathway, indicating a new strategy for discovering the molecular constraints that shape the evolution of a metabolic pathway.

Functional enrichment analysis based on long noncoding RNA associations.

BackgroundDifferential gene expression analysis using RNA-seq data is a popular approach for discovering specific regulation mechanisms under certain environmental settings. Both gene ontology (GO) and KEGG pathway enrichment analysis are major processes for investigating gene groups that participate in common biological responses or possess related functions. However, traditional approaches based on differentially expressed genes only detect a few significant GO terms and pathways, which are frequently insufficient to explain all-inclusive gene regulation mechanisms.MethodsTranscriptomes of survivin (birc5) gene knock-down experimental and wild-type control zebrafish embryos were sequenced and assembled, and a differential expression (DE) gene list was obtained for traditional functional enrichment analysis. In addition to including DE genes with significant fold-change levels, we considered additional associated genes near or overlapped with differentially expressed long noncoding RNAs (DE lncRNAs), which may directly or indirectly activate or inhibit target genes and play important roles in regulation networks. Both the original DE gene list and the additional DE lncRNA-associated genes were combined to perform a comprehensive overrepresentation analysis.ResultsIn this study, a total of 638 DE genes and 616 DE lncRNA-associated genes (lncGenes) were leveraged simultaneously in searching for significant GO terms and KEGG pathways. Compared to the traditional approach of only using a differential expression gene list, the proposed method of employing DE lncRNA-associated genes identified several additional important GO terms and KEGG pathways. In GO enrichment analysis, 60% more GO terms were obtained, and several neuron development functional terms were retrieved as complete annotations. We also observed that additional important pathways such as the FoxO and MAPK signaling pathways were retrieved, which were shown in previous reports to play important roles in apoptosis and neuron development functions regulated by the survivin gene.ConclusionsWe demonstrated that incorporating genes near or overlapped with DE lncRNAs into the DE gene list outperformed the traditional enrichment analysis method for effective biological functional interpretations. These hidden interactions between lncRNAs and target genes could facilitate more comprehensive analyses.