The RING Domain of Siaz, the Zebrafish Homologue of Drosophila seven in absentia, Is Essential for Cellular Growth Arrest

Myungchull Rhee,Hyunju Ro,장영주 한국분자세포생물학회 2004 Molecules and cells Vol.17 No.1

Siah is a mammalian homologue of Drosophila seven in absentia (sina) that is required for R7 photoreceptor development. Both the SINA and Siah family interact with ubiquitin-conjugating enzymes via an N-terminal RING domain and the C-terminal domain of SINA/ Siahs interacts with proteins targeted for degradation. Siah induces cell growth arrest by promoting -catenin degradation in a phosphorylation-independent manner as a result of indirect binding to -catenin. We previ-ously cloned a zebrafish homologue (Siaz) of Siah. Siaz shares high sequence homology with vertebrate Siah-2. We have now examined the role of Siaz in growth regulation using the trypan blue exclusion assay and flow cytometry and found that Siaz induces cellular growth arrest by inhibiting the G2/M transition. The C-terminal domain of Siaz that interacts with target proteins is not required for growth inhibition. We con-clude that the N-terminal RING and central domain of Siaz are sufficient to block the G2/M phase transition.

Characterization of Orphan Receptor DNA-Binding Enhancer

Rhee, Myungchull 충남대학교 생물공학연구소 1996 생물공학연구지 Vol.4 No.-

Apolipoprotein AI regulatory protein-1 (ARP-1) binds to a regulatory element which is composed of two direct repeat motifs (site A). TGAACCCT and TGACCCCT in the apolipoprotein AI (apo AI) gene as a dimer. Because the ligand of ARP-1 has not been identified, ARP-1 is classified as an orphan receptor. ARP-1 binds to various hormone response elements as a homodimer and/or a heterodimer. In most cases, they antagonize the transcriptional stimulation exerted by various nuclear receptors and their corresponding ligands. To better understand the molecular mechanism wherein ARP-1 functions as a transcriptional repressor, COS-1 cellular extract was analyzed for its effect on the DNA binding capacity of ARP-1. First, this report showed that a heat-stable protein (complex), designated orphan receptor DNA-binding enhancer (ORDE), enhanced orphan receptors’, such as ARP-1, chicken ovalbumin upstream promoter transcriptional factor (COUP-TF), and hepatocyte nuclear factor 4 (HNF4) binding to site A. Second, ORDE changed the DNA binding capacity, but neither the affinity nor the location of the band in EMSA, of ARP-1. Finally, ORDE interacted with the DNA binding domain of ARP-1. Taken together, these results suggest that a battery of orphan receptors may require ORDE to bind their cognate DNA sequences.

Characterization of Orphan Receptor DNA-Binding Enhacer

Rhee, Myungchull Korean Society of Molecular Biology 1994 Molecules and cells Vol.4 No.4

제브라피쉬 발생배에서 microRNA-206의 시공간적 발현양상

이충근,이명철 충남대학교 생물공학연구소 2012 생물공학연구지 Vol.18 No.-

Transcriptional Repression of Apolipoprotein AI Gene Expression by Orphan Receptor ARP-1

Ge, Ruowen,Rhee, Myungchull,Malik, Sohail,Karathanasis, Sotirios K. 충남대학교 생물공학연구소 1996 생물공학연구지 Vol.4 No.-

Expression of the apolipoprotein AI (apoAI) gene in the liver is controlled by a liver-specific enhancer. The function of this enhancer depends on synergistic interactions between transcription factors bound to at least three sites (designated A, B, and C) located within this enhancer. We have previously shown that an apoAI gene reporter construct containing the entire enhancer is expressed efficiently in a hepatoma cell line and that its activity is repressed by the orphan receptor ARP-1. Moreover, repression by ARP-1 is overcome by the retinoid X receptor RXRαin the presence of retinoic acid. In this study, we show that ARP-1 represses the apoAI promoter by binding to site A of the apoAI liver-specific enhancer, the repression being a promoter context-specific event. Mapping analysis of ARP-1 indicated that its DNA binding domain is essential but not sufficient for repression. Two separate repression domains located at the amino-and carboxyl-terminal halves of ARP-1 were found to individually complement the DNA binding domain for efficient repression. We also demonstrate the reversibility of ARP-1 repression by transcription factors C/EBP and Egr-1, which might also be involved in apoAI gene expression. Significantly, repression by ARP-1 was found to be a prerequisite for C/EBP-mediated transactivation. We interpret our results in terms of a model in which ARP-1 repression via its interaction with site A is an obligatory intermediate step in switching from one activated state of the apoAI gene to another.

Thyroid Hormone Receptor Monomer, Homodimer, and Heterodimer(with Retinoid-X Receptor) Contact Different Nucleotide Sequences in Thyroid Hormone Response Elements

IKEDA, MASATO,RHEE, MYUNGCHULL,CHIN, WILLIAM W. 충남대학교 생물공학연구소 1996 생물공학연구지 Vol.4 No.-

Thyroid hormone receptors (TR_s) bind as monomers, homodimers, and heterodimers with nuclear proteins such as retinoid-X receptors (RXR_s) to thyroid hormone response elements (TRE_s). However, it is not known which nucleotides each TR complex contacts in a particular TRE. To identify the precise contact sites on a synthetic DR4 (TRE half-sites arranged as a direct repeat with a four-nucleotide spacer) and a chick lysoenzyme TRE, F2 (half-sites arranged as an inverted palindrome with a six-nucleotide spacer), for various TR complexes, mobility shift assays, and dimethylsulfate and KMnO_4 DNA modification interference assays were employed. First, TRαmonomer bound to the downstream half-site of these TREs, whereas TRαhomodimer bound to both half-sites. TRα/RXRαheterodimer also bound to both half-sites, but "preferrde" to contact the down-stream half-site. Second, the specific flanking and spacing sequences of DR4 influenced the contact sites and the binding of TRαmonomer and homodimer, but not TRα/RXRαheterodimer. Finally, cotransfection studies, using reporter plasmids containing DR4 or F2 in both orientations with respect to the basal promoter, provide evidence that preferential contact with the down-stream half-site by TR/RXR heterodimer may be important for maximal transcriptional activation. (Endocrinology 135:1628-1638, 1994)

Transcriptomic networks of gba3 governing specification of the dopaminergic neurons in zebrafish embryos

Kumar Ajeet,Rhee Myungchull 한국유전학회 2022 Genes & Genomics Vol.44 No.12

Background: Molecular networks associated with dopaminergic (DA) neurogenesis remain undefined within mammalian models. To address this issue, the transient zebrafish model lmx1al: EGFP was generated, which expresses GFP in the DA precursor cells as well as in the DA neurons of the ventral diencephalon (VD). We found that the novel pseudogene gba3 has not been well studied in zebrafish neurogenesis. Objective: Crucial networks associated with gba3 transcripts were investigated because the biological functions of these networks have not been reported in DA neurogenesis in zebrafish. Methods: RNA isolation and sequencing were employed with GFP-expressing cells from 20-, 22-, and 24 h post-fertilization (hpf), while subsequent transcriptomic analysis generated differentially expressed genes with DA neurogenesis (DEG-DA) list. Hierarchical cluster analysis provided absolute guidance for the collection of gba3, an essential transcript that is strictly spatiotemporally expressed during DA neurogenesis, which was proven with whole-mount in situ hybridization (WISH) and knockdown and rescue of the gba3 transcripts in zebrafish embryos. Results: The gba3 transcripts were restricted to the midbrain at 24 hpf and the midbrain and pectoral fins at 30 hpf in zebrafish embryos. Functional studies with knockdown of gba3 found a diminished state in the midbrain and midbrain-hindbrain boundary (MHB) and an underdeveloped condition in the anteroposterior and dorsolateral axis relative to the wild type (WT) at 24 hpf. However, it was recovered after forced expression of gba3 transcripts at 24 hpf. Molecular markers for the DA precursors and mature neurons lmx1al, nurr1, th, and pitx3 were analyzed in the gba3 MOs. The levels of transcripts lmx1al, nurr1, and th were significantly reduced in the midbrain ventral diencephalon (VD) and hindbrain of gba3 morphants compared to the WT at 24 hpf, while expression patterns of pitx3 transcripts showed no differences in the identical regions between gba3 MOs and the controls. Conclusions: We discuss transcriptional networks in which transcripts of gba3 plausibly govern the specification of dopaminergic neurogenesis in zebrafish embryos.

Kapd Is Essential for Specification of the Dopaminergic Neurogenesis in Zebrafish Embryos

Jung, Jangham,Kim, Eunhee,Rhee, Myungchull Korean Society for Molecular and Cellular Biology 2021 Molecules and cells Vol.44 No.4

To define novel networks of Parkinson's disease (PD) pathogenesis, the substantia nigra pars compacta of A53T mice, where a death-promoting protein, FAS-associated factor 1 was ectopically expressed for 2 weeks in the 2-, 4-, 6-, and 8-month-old mice, and was subjected to transcriptomic analysis. Compendia of expression profiles and a hierarchical clustering heat map of differentially expressed genes associated with PD were bioinformatically generated. Transcripts level of a particular gene was fluctuated by 20, 60, and 0.75 fold in the 4-, 6-, and 8-month-old mice compared to the 2 months old. Because the gene contained Kelch domain, it was named as Kapd (Kelch-containing protein associated with PD). Biological functions of Kapd were systematically investigated in the zebrafish embryos. First, transcripts of a zebrafish homologue of Kapd, kapd were found in the floor plate of the neural tube at 10 h post fertilization (hpf), and restricted to the tegmentum, hypothalamus, and cerebellum at 24 hpf. Second, knockdown of kapd caused developmental defects of DA progenitors in the midbrain neural keel and midbrain-hindbrain boundary at 10 hpf. Third, overexpression of kapd increased transcripts level of the dopaminergic immature neuron marker, shha in the prethalamus at 16.5 hpf. Finally, developmental consequences of kapd knockdown reduced transcripts level of the markers for the immature and mature DA neurons, nkx2.2, olig2, otx2b, and th in the ventral diencephalon of the midbrain at 18 hpf. It is thus most probable that Kapd play a key role in the specification of the DA neuronal precursors in zebrafish embryos.

Siah Ubiquitin Ligases Modulate Nodal Signaling during Zebrafish Embryonic Development

Kang, Nami,Won, Minho,Rhee, Myungchull,Ro, Hyunju Korean Society for Molecular and Cellular Biology 2014 Molecules and cells Vol.37 No.5

Siah acts as an E3 ubiquitin ligase that binds proteins destined for degradation. Extensive homology between siah and Drosophila Siah homologue (sina) suggests their important physiological roles during embryonic development. However, detailed functional studies of Siah in vertebrate development have not been carried out. Here we report that Siah2 specifically augments nodal related gene expression in marginal blastomeres at late blastula through early gastrula stages of zebrafish embryos. Siah2 dependent Nodal signaling augmentation is confirmed by cell-based reporter gene assays using 293T cells and 3TP-luciferase reporter plasmid. We also established a molecular hierarchy of Siah as a upstream regulator of FoxH1/Fast1 transcriptional factor in Nodal signaling. Elevated expression of nodal related genes by overexpression of Siah2 was enough to override the inhibitory effects of atv and lft2 on the Nodal signaling. In particular, E3 ubiquitin ligase activity of Siah2 is critical to limit the duration and/or magnitude of Nodal signaling. Additionally, since the embryos injected with Siah morpholinos mimicked the atv overexpression phenotype at least in part, our data support a model in which Siah is involved in mesendoderm patterning via modulating Nodal signaling.

Ubiquitin conjugation system for body axes specification in vertebrates

Ro, Hyunju,Hur, Tae-Lin,Rhee, Myungchull Taylor Francis 2015 Animal cells and systems Vol.19 No.2