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Chee-Gun Lee

Publications and source records attributed to Chee-Gun Lee.

10 recordsLinked to original sources
PubMed2004

MLE functions as a transcriptional regulator of the roX2 gene.

Dosage compensation is a process that equalizes transcription activity between the sexes. In Drosophila, two non-coding RNA, roX1 and roX2, and at least six protein regulators, MSL-1, MSL-2, MSL-3, MLE, MOF, and JIL-1, have been identified as essential for dosage compensation. Although there is accumulating evidence of the intricate functional and physical interactions between protein and RNA regulators, little is known about how roX RNA expression and function are modulated in coordination with protein regulators. In this report, we have found that a relatively short (about 350 bp) upstream genomic region of the roX2 gene, Prox2, harbors an activity that drives transcription of the downstream gene. Our study has shown that MLE can stimulate the transcription activity of Prox2 and that MLE associates with Prox2 through direct interaction with a newly identified 54-bp repeat, Prox. Our observations suggest a novel mechanism by which roX2 RNA is regulated at the transcriptional level.

Animals↗
PubMed2004

A functional interaction between RHA and Ubc9, an E2-like enzyme specific for Sumo-1.

RNA helicase A (RHA) is a member of the DEAH helicase family of proteins. Recent studies imply the role of RHA in the regulation of the topology of chromatin DNA, which could influence diverse nuclear processes such as transcription activity of the chromatin DNA and chromosome condensation. We previously reported that Ubc9, an E2-like enzyme specific for small ubiquitin-like modifier 1 (Sumo-1), is required for the interaction between RHA and topoisomerase IIalpha. Here, we describe that Ubc9 is a novel factor that functionally interacts with RHA and activates the transcription activity of RHA, measured in the CREB-mediated pathway. We demonstrate that the N-terminal domain of RHA, encompassing amino acid residues 1-137, is sufficient for its interaction with Ubc9. Our data also show that interaction with Ubc9 leads to the Sumo-1 conjugation of RHA both in vitro and in vivo. However, the catalytic activity of Ubc9 seems to be dispensable for the transcription activation activity of RHA. Our observation suggests multiple roles for Ubc9 in the regulation of the RHA function.

Amino Acid Sequence↗
PubMed2004

Identification of toposome, a novel multisubunit complex containing topoisomerase IIalpha.

Topoisomerase IIalpha plays essential roles in chromosome segregation. However, it is not well understood how topoisomerase IIalpha exerts its function during mitosis. In this report, we find that topoisomerase IIalpha forms a multisubunit complex, named toposome, containing two ATPase/helicase proteins (RNA helicase A and RHII/Gu), one serine/threonine protein kinase (SRPK1), one HMG protein (SSRP1), and two pre-mRNA splicing factors (PRP8 and hnRNP C). Toposome separates entangled circular chromatin DNA about fourfold more efficiently than topoisomerase IIalpha. Interestingly, this decatenation reaction yields knotted circles, which are not seen in reactions provided with monomeric circular DNA. Our results also show that interaction among toposome-associated proteins is highest in G2/M phase but drastically diminishes in G1/S phase. These results suggest that toposome is a dynamic complex whose assembly or activation is subject to cell cycle regulation.

Amino Acid Sequence↗
PubMed2003

Selective regulation of gene expression by nuclear factor 110, a member of the NF90 family of double-stranded RNA-binding proteins.

Members of the nuclear factor 90 (NF90) family of double-stranded RNA (dsRNA)-binding proteins have been implicated in several biological processes including the regulation of gene expression. cDNA sequences predict that the proteins have a functional nuclear localization signal and two dsRNA-binding motifs (dsRBMs), and are identical at their N termini. Isoforms are predicted to diverge at their C termini as well as by the insertion of four amino acid residues (NVKQ) between the two dsRBMs. In this study, we verified the expression of four of the isoforms by cDNA cloning and mass spectrometric analysis of proteins isolated from human cells. Cell fractionation studies showed that NF90 and its heteromeric partner, NF45, are predominantly nuclear and largely chromatin-associated. The C-terminally extended NF90 species, NF110, are almost exclusively chromatin-bound. Both NF110 isoforms are more active than NF90 isoforms in stimulating transcription from the proliferating cell nuclear antigen reporter in a transient expression system. NF110b, which carries the NVKQ insert, was identified as the strongest activator. It stimulated transcription of some, but not all, promoters in a fashion that suggested that it functions in concert with other transcription factors. Finally, we demonstrate that NF110b associates with the dsRBM-containing transcriptional co-activator, RNA helicase A, independently of RNA binding.

Amino Acid Sequence↗
PubMed2003

DEN1 is a dual function protease capable of processing the C terminus of Nedd8 and deconjugating hyper-neddylated CUL1.

Nedd8 activates ubiquitination by increasing the efficiency of polyubiquitin chain assembly through its covalent conjugation to cullin molecules. Here we report the isolation, cloning, and characterization of a novel human Nedd8-specific protease called DEN1. Human DEN1 is encoded by AAH31411.1, a previously uncharacterized protein of 212 amino acids that shares homology with the Ulp1 cysteinyl SUMO deconjugating enzyme family. Recombinant human DEN1, purified from bacteria, selectively binds to Nedd8 and hydrolyzes C-terminal derivatives of Nedd8. Interestingly, DEN1 deconjugates cullin 1 (CUL1)-Nedd8 in a concentration-dependent manner. At a low concentration, DEN1 processes hyper-neddylated CUL1 to yield a mononeddylated form, which presumably contains the Lys-720CUL1-Nedd8 linkage. At elevated concentrations, DEN1 is able to complete the removal of Nedd8 from CUL1. These activities distinguish DEN1 from the COP9 signalosome, which is capable of efficiently cleaving the Lys-720CUL1-Nedd8 conjugate, but lacks Nedd8 C-terminal hydrolytic activity and poorly processes hyperneddylated CUL1. These results suggest a unique role for DEN1 in regulating the modification of cullins by Nedd8.

Amino Acid Sequence↗
PubMed2003

RNA helicase A interacts with dsDNA and topoisomerase IIalpha.

RNA helicase A (RHA) is a multifunctional protein involved in various nuclear processes such as transcription and RNA export. It is believed that the interacting factors play important roles in determining the functional specificity of RHA. Here we show that RHA directly interacts with double-stranded (ds) nucleic acids (NAs) and assembles complexes with topoisomerase IIalpha. First, electrophoresis mobility shift assays demonstrate that RHA interacts with dsDNAs of different lengths ranging from 15 to 104 bp. Secondly, the binding of RHA to closed circular dsDNA stimulates the relaxation reaction catalyzed by either calf thymus topoisomerase I or HeLa topoisomerase IIalpha. Thirdly, immunoprecipitation, coupled with western blot analysis using anti-RHA and anti-topoisomerase IIalpha antibodies, shows that RHA and topoisomerase IIalpha assemble a complex in the presence of as yet unknown RNA molecules and additional protein factors such as Ubc9. Our observation suggests physical and functional interaction between RHA and topoisomerase IIalpha, which, perhaps, play important roles in regulating chromatin structure. The putative role of RHA-topoisomerase IIalpha complex in RNA polymerase II-mediated transcription is discussed.

Adenosine Triphosphatases↗
PubMed2003

The growth factor granulin interacts with cyclin T1 and modulates P-TEFb-dependent transcription.

Cyclin T1, together with the kinase CDK9, is a component of the transcription elongation factor P-TEFb which binds the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. P-TEFb facilitates transcription by phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase II. Cyclin T1 is an exceptionally large cyclin and is therefore a candidate for interactions with regulatory proteins. We identified granulin as a cyclin T1-interacting protein that represses expression from the HIV-1 promoter in transfected cells. The granulins, mitogenic growth factors containing repeats of a cysteine-rich motif, were reported previously to interact with Tat. We show that granulin formed stable complexes in vivo and in vitro with cyclin T1 and Tat. Granulin bound to the histidine-rich domain of cyclin T1, which was recently found to bind to the CTD, but not to cyclin T2. Binding of granulin to P-TEFb inhibited the phosphorylation of a CTD peptide. Granulin expression inhibited Tat transactivation, and tethering experiments showed that this effect was due, at least in part, to a direct action on cyclin T1 in the absence of Tat. In addition, granulin was a substrate for CDK9 but not for the other transcription-related kinases CDK7 and CDK8. Thus, granulin is a cellular protein that interacts with cyclin T1 to inhibit transcription.

3T3 Cells↗
PubMed2002

RH70, a bidirectional RNA helicase, co-purifies with U1snRNP.

In this report, I describe the co-purification of a novel 70-kDa RNA helicase (RH70) and U1snRNP through six column steps. Peptide sequence analysis by mass spectrometry and Edman degradation revealed that RH70 is the previously reported DDX17. Biochemical characterization of RH70, obtained by partial separation from U1snRNP, yielded the following results. (a) RH70 mediates the unwinding of duplex RNA but not DNA in an ATP-dependent manner. (b) Both the RNA-dependent ATPase and RNA helicase activities of RH70 are highly specific for ATP, exhibiting an apparent K(m) of 0.5 mm. (c) RH70 catalyzes the unwinding of duplex RNA containing single-stranded regions at either the 5'- or 3'-end. Its association with U1snRNP and ATP specificity suggest a role for RH70 in pre-mRNA splicing, in particular, at the early stages of the splicing reaction involving U1snRNP.

Adenosine Triphosphatases↗
PubMed2002

Effect of an exogenous trigger on the pathogenesis of lupus in (NZB x NZW)F1 mice.

OBJECTIVE: This study examined the interactions between exogenous and endogenous factors shaping the phenotype of lupus in autoimmune (NZB x NZW)F(1) mice exposed to pristane, a model environmental trigger. METHODS: Frequencies of various autoantibodies in untreated NZB/NZW mice were determined by various means (immunoprecipitation, enzyme-linked immunosorbent assay [ELISA], Crithidia luciliae kinetoplast staining). Pristane or saline was administered intraperitoneally to 9-12-week-old NZB/NZW mice, followed by serial studies of autoantibodies, total Ig levels (ELISA), and proteinuria (dipstick). RESULTS: Besides antichromatin/DNA responses, NZB/NZW mice spontaneously produced novel autoantibodies against the double-stranded RNA binding protein RNA helicase A (RHA). In contrast, NZB/NZW mice (n = 70) did not produce autoantibodies against the nuclear RNP (nRNP), Sm, Ro, or La antigens. Pristane exposure synergistically activated the production of antichromatin/DNA antibodies and dramatically accelerated renal disease. Production of anti-nRNP/Sm and Su autoantibodies also was induced, indicating that the unresponsiveness of NZB/NZW mice to these antigens can be overcome. Curiously, pristane treatment did not enhance the production of anti-RHA, suggesting that these autoantibodies are regulated differently than anti-DNA/chromatin and Sm. In contrast to previous reports that suggest a critical role of deficient interleukin-12 (IL-12) production in the pathogenesis of lupus, there was overproduction of IL-12 in the peritoneal cavity of pristane-treated NZB/NZW mice, and their spleen cells also produced large amounts of IL-12. CONCLUSION: These data lead us to propose that environmental influences exacerbate autoimmune manifestations in genetically lupus-susceptible mice through their stimulatory effects on proinflammatory cytokines, such as IL-12.

Animals↗