Suppression of cell cycle progression by Jun dimerization protein (JDP2) involves down-regulation of cyclin A2

We report here a novel role for Jun dimerization protein-2 (JDP2) as a regulator of the progression of normal cells through the cell cycle. To determine the role of JDP2 in vivo, we generated Jdp2 knock-out (Jdp2KO) mice by targeting exon 1 to disrupt the site of initiation of transcription. The healing of wounded skin of Jdp2KO mice proceeded more rapidly than that of control mice and more proliferating cells were found at wound margins. Fibroblasts derived from embryos of Jdp2KO mice proliferated more rapidly and formed more colonies than wild-type fibroblasts. JDP2 was recruited to the promoter of the gene for cyclin A2 (ccna2) at a previously unidentified AP-1 site. Cells lacking Jdp2 had elevated levels of cyclin A2 mRNA. Moreover, reintroduction of JDP2 resulted in repression of transcription of ccna2 and of cell cycle progression. Thus, transcription of the gene for cyclin A2 appears to be a direct target of JDP2 in the suppression of cell proliferation

Identification of mouse Jun dimerization protein 2 as a novel repressor of ATF-211The nucleotide sequence reported herein has been deposited in the DDBJ, EMBL and GenBank databanks under the accession number AB034697.

AbstractA mouse cDNA that encodes a DNA-binding protein was identified by yeast two-hybrid screening, using activating transcription factor-2 (ATF-2) as the bait. The protein contained a bZIP (basic amino acid-leucine zipper region) domain and its amino acid sequence was almost identical to that of rat Jun dimerization protein 2 (JDP2). Mouse JDP2 interacted with ATF-2 both in vitro and in vivo via its bZIP domain. It was encoded by a single gene and various transcripts were expressed in all tested tissues of adult mice, as well as in embryos, albeit at different levels in various tissues. Furthermore, mouse JDP2 bound to the cAMP-response element (CRE) as a homodimer or as a heterodimer with ATF-2, and repressed CRE-dependent transcription that was mediated by ATF-2. JDP2 was identified as a novel repressor protein that affects ATF-2-mediated transcription

Efficacy and safety of cold forceps polypectomy utilizing the jumbo cup: a prospective study

Background/Aims There are few prospective studies on cold forceps polypectomy (CFP) using jumbo cup forceps. Therefore, we examined patients with diminutive polyps (5 mm or smaller) treated with CFP using jumbo cup forceps to achieve an adenoma-free colon and also assessed the safety of the procedure and the recurrence rate of missed or residual polyp after CFP by performing follow-up colonoscopy 1 year later. Methods We included patients with up to 5 adenomas removed at initial colonoscopy and analyzed data from a total of 361 patients with 573 adenomas. One-year follow-up colonoscopy was performed in 165 patients, at which 251 lesions were confirmed. Results The one-bite resection rate with CFP was highest for lesions 3 mm or smaller and decreased significantly with increasing lesion size. Post-procedural hemorrhage was observed in 1 of 573 lesions (0.17%). No perforation was noted. The definite recurrence rate was 0.8% (2/251 lesions). The probable recurrence rate, which was defined as recurrence in the same colorectal segment, was 17%. Adenoma-free colon was achieved in 55% of patients at initial resection. Multivariate analysis revealed that achievement of an adenoma-free colon was significantly associated with number of adenomas and years of endoscopic experience. Conclusions CFP using jumbo biopsy forceps was safe and showed a high one-bite resection rate for diminutive lesions of 3 mm or smaller. The low definite recurrence rate confirms the reliability of CFP using jumbo biopsy forceps. Number of adenomas and years of endoscopic experience were key factors in achieving an adenoma-free colon

ショウジョウバエの転写因子、ＦＴＺ-Ｆ１強制発現の発生への影響

　FTZ-F1は､ショウジョウバエfushi tarazu(ftz)遺伝子の発現調節領域に塩基配列特異的に結合する因子であり､ステロイドホルモンレセプターと類似のアミノ酸配列を持つ｡FTZ-F1の生体内での機能を明らかにするためにFTZ-F1の発現パターンおよびトランスジェニックフライでのFTZ-F1の強制発現による影響を調べた｡　FTZ-F1には少なくとも二つのisoformが存在することがゲルシフトアッセイの結果から明らかになっている｡胚では産卵後4時間までにαFTZ-F1が､また､産卵後12時間以降にβFTZ-F1がそれぞれ存在するが､発現部位は不明であった｡そこで抗FTZ-F1抗血清を用いた組織染色をおこなった結果､初期胚ではembryonic stage 8(産卵後3.75-4.5時間)まで極細胞を除く胚全体で発現しその後消失すること後期胚ではembryonic stage 13(産卵後10.5-11.5時間)からふたたび胚全体で発現することがわかった｡　1齢幼虫から3齢幼虫でのFTZ-F1の発現をウエスタンプロッティングによって調べたところ､産卵後42-51時間および69-75時間にβFTZ-F1の発現を観察した｡この時期はそれぞれ1-2齢幼虫脱皮および2-3齢幼虫脱皮の時期にあたる｡ワンダリング幼虫および囲蛹殻形成後0から20時間の前蛹および蛹での発現をウエスタンブロッティングによって調べたところ､囲蛹殻形成後6時間から12時間にβFTZ-F1の発現が観察された｡　以上の結果から､FTZ-F1は時期特異的に多くの組織で発現すること､いづれの組織の発現でも核に局在することがわかった｡また､FTZ-F1は転写因子であることから､FTZ-F1の時期特異的な発現によって標的遺伝子の発現時期を調節していることが考えられた｡また､幼虫および蛹期の発現から､ショウジョウバエの脱皮や変態にかかわると予想された｡　ところで､これまでにFTZ-F1遺伝子の突然変異体は得られていない｡そこでFTZ-F1の生体内での機能を明らかにするために､熱ショックでFTZ-F1を発現できるトランスジェニックフライ(hsFTZ-F1系統)を作成した｡ショウジョウバエhsp70遺伝子プロモータ一領域をもつFTZ-F1融合遺伝子を作成し､P-elementによりwhite系統に導入することによってhsFTZ-F1系統を得た｡　まず熱ショックによってFTZ-F1が誘導されるかをウエスタンブロッティングで調べた｡hsα227系統とhsα338系統では38℃60分の熱ショックによって誘導されるFTZ-F1タンパクのレベルは熱ショック終了後2時間で最高に達し､内在性FTZ-F1のレベルに比べ2から3倍であった｡その後､約1時間の半減期をもって減少し､4時間後には検出できなくなった｡hsα332系統では38℃60分の熱ショックによって10倍以上の発現があり､32℃では､内在性のレベルに比べ約2倍であった｡　つぎに､FTZ-F1の時期特異的な強制発現による影響を調べるために､バランサー染色体とheterozygousにしたhsFTZ-F1系統をwhite系統とかけあわせ､F1世代のショウジョウバエに熱ショックを与え､FTZ-F1を強制的に発現させた｡強制発現の影響はバランスされた(コントロール)成虫の出現数に対するバランスされていない(hsFTZ-F1)成虫の出現数の比で求めた｡　3時間ごとにagingした産卵後24-108時間の幼虫に対して熱ショック(38℃60分)を与えたところ､産卵後36-42時間の1齢幼虫および､57-69時間の2齢幼虫でhsFTZ-F1系統の出現数が最低0.0にまで減った｡この時期にはウエスタンプロッティング法で調べた限りFTZ-F1は検出されず､本来FTZ-F1が発現する直前にあたる｡この実験から､FTZ-F1の強制発現の影響をうける時期に特異性があることがわかった｡その時期はFTZ-F1が本来発現していない時期であることから､FTZ-F1は､時期特異的に発現することが重要であると考えられた｡また､FTZ-F1が本来発現していない1齢幼虫初期(産卵後24-33時間)､2齢幼虫初期(産卵後51-54時間)､3齢幼虫初期(産卵後75-108時間)ではFTZ-F1の強制発現の影響が少ないことがわかった｡FTZ-F1が本来発現していないにも関わらずFTZ-F1の強制発現の影響が少なかったことに対して､FTZ-F1が転写因子として機能できなかった可能性と､FTZ-F1は転写因子として機能したが､標的遺伝子産物が発生に影響を及ぼさなかった可能性が考えられた｡　FTZ-F1強制発現による致死がどのように引き起こされるかを調べるために､産卵後57-60時間の2齢幼虫に38℃60分の熱ショックを与え､その後､25℃での発生を観察した｡white系統ではほとんどが3齢幼虫を経て蛹まで発生を続けたのに対し､hsFTZ-F1系統の多くが3齢幼虫特有のanterior spiracleの構造を示さないまま死んだ｡熱ショック後48時間の幼虫の形態をくわしく観察すると､white系統では3齢幼虫の形態を示していたが､hsFTZ-F1系統はmouth hookおよびspiracleを二組もっており､その形態は2齢および3齢幼虫のそれぞれの特徴をもっていた｡さらにtracheaは二重化していた｡この形態から考えて､hsFTZ-F1系統は3齢幼虫への脱皮の準備はできているが､脱皮はしていないと考えられた｡脱皮ができないために致死になるかどうかは不明であるが､この実験結果からFTZ-F1がショウジョウバエの脱皮にかかわっていることが示唆され､正確な脱皮のためにはecdysteroidsによって誘導される一群の遺伝子の発現順序が重要であることが考えられた

Genes encoded within 8q24 on the amplicon of a large extrachromosomal element are selectively repressed during the terminal differentiation of HL-60 cells

Human acute myeloblastic leukemia HL-60 cells become resistant to differentiation during long­term cultivation. After 150 passages, double minute chromosomes (dmins) found in early­-passaged cells are replaced by large extrachromosomal elements (LEEs). In a DNA library derived from a purified fraction of LEEs, 12.6% (23/183) of clones were assigned to 8q24 and 9.2% (17/183) were assigned to 14q11 in the human genome. Fluorescence in situ hybridization (FISH) revealed a small aberrant chromosome, which had not been found in early­-passaged cells, in addition to the purified LEEs. We determined that each LEE consisted of six discontinuous segments in a region that extended for 4.4 Mb over the 8q24 locus. Five genes, namely, Myc (a proto­-oncogene), NSMCE2 (for a SUMO ligase), CCDC26 (for a retinoic acid­-dependent modulator of myeloid differentiation), TRIB1 (for a regulator of MAPK kinase) and LOC389637 (for a protein of unknown function), were encoded by the amplicon. Breaks in the chromosomal DNA within the amplicon were found in the NSMCE2 and CCDC26 genes. The discontinuous structure of the amplicon unit of the LEEs was identical with that of dmins in HL­60 early­-passaged cells. The difference between them seemed, predominantly, to be the number (10 to 15 copies per LEE versus2 or 3 copies per dmin) of constituent units. Expression of the Myc, NSMCE2, CCDC26 and LOC389637 and TRIB1genes was constitutive in all lines of HL­-60 cells and that of the first four genes was repressed during the terminal differentiation of early­passaged HL­-60 cells. We also detected abnormal transcripts of CCDC26. Our results suggest that these genes were selected during the development of amplicons. They might be amplified and, sometimes, truncated to contribute to the maintenance of HL­-60 cells in an undifferentiated state

Creation of haemoglobin A1c direct oxidase from fructosyl peptide oxidase by combined structure-based site specific mutagenesis and random mutagenesis

糖尿病マーカー、ヘモグロビンA1cを直接酸化できる酵素の創製 --シンプルかつ短時間測定が可能な新規測定試薬開発に大きく前進--. 京都大学プレスリリース. 2019-01-31.The currently available haemoglobin A1c (HbA1c) enzymatic assay consists of two specific steps: proteolysis of HbA1c and oxidation of the liberated fructosyl peptide by fructosyl peptide oxidase (FPOX). To develop a more convenient and high throughput assay, we devised novel protease-free assay system employing modified FPOX with HbA1c oxidation activity, namely HbA1c direct oxidase (HbA1cOX). AnFPOX-15, a modified FPOX from Aspergillus nidulans, was selected for conversion to HbA1cOX. As deduced from the crystal structure of AnFPOX-15, R61 was expected to obstruct the entrance of bulky substrates. An R61G mutant was thus constructed to open the gate at the active site. The prepared mutant exhibited significant reactivity for fructosyl hexapeptide (F-6P, N-terminal amino acids of HbA1c), and its crystal structure revealed a wider gate observed for AnFPOX-15. To improve the reactivity for F-6P, several mutagenesis approaches were performed. The ultimately generated AnFPOX-47 exhibited the highest F-6P reactivity and possessed HbA1c oxidation activity. HbA1c levels in blood samples as measured using the direct assay system using AnFPOX-47 were highly correlated with the levels measured using the conventional HPLC method. In this study, FPOX was successfully converted to HbA1cOX, which could represent a novel in vitro diagnostic modality for diabetes mellitus

JDP2, a Repressor of AP-1, Recruits a Histone Deacetylase 3 Complex To Inhibit the Retinoic Acid-Induced Differentiation of F9 Cells

Up-regulation of the c-jun gene is a critical event in the retinoic acid (RA)-mediated differentiation of embryonal carcinoma F9 cells. Activating transcription factor 2 (ATF-2) and p300 cooperate in the activation of transcription of the c-jun gene during the differentiation of F9 cells. We show here that the overexpression of Jun dimerization protein 2 (JDP2), a repressor of AP-1, inhibits the transactivation of the c-jun gene by ATF-2 and p300 by recruitment of the histone deacetylase 3 (HDAC3) complex, thereby repressing the RA-induced transcription of the c-jun gene and inhibiting the RA-mediated differentiation of F9 cells. Moreover, chromatin immunoprecipitation assays showed that the JDP2/HDAC3 complex, which binds to the differentiation response element within the c-jun promoter in undifferentiated F9 cells, was replaced by the p300 complex in response to RA, with an accompanying change in the histone acetylation status of the chromatin, the initiation of transcription of the c-jun gene, and the subsequent differentiation of F9 cells. These results suggest that JDP2 may be a key factor that controls the commitment of F9 cells to differentiation and shed new light on the mechanism by which an AP-1 repressor functions