C. DEPARTMENT OF DEVELOPMENTAL GENETICS
C-b. Division of Gene Expression - Susumu Hirose Group

RESEARCH ACTIVITIES

(1) Role of histone modifications and chromatin remodeling in epigenetic gene expression

Takahiro Nakayama, Tsukasa Shimojima, Kazuma Hanai, Kenichi Nishioka, Koji Akasaka¹ and Susumu Hirose (¹Department of Biological Science, University of Tokyo, Tokyo, Japan)

--In multicellular organisms some patterns of gene expression are remembered in the chromatin structure and maintained through many rounds of cell cycle. These are termed epigenetic gene expression. Two types of epigenetic gene expression are known in Drosophila melanogaster: (1) maintenance of Hox gene expression governed by Polyomb (Pc) and trithorax (trx) group genes, and (2) position effect variegation (PEV). Trithorax-like (Trl) encoding GAGA factor is involved in the both types of regulation. Thus Trl is a member of trx group and Trl mutation is an enhancer of PEV. We have shown that GAGA factor recruits FACT, a heterodimer of dSPT16 and dSSRP1, facilitates chromatin remodeling around its binding site and contributes to the maintenance of Hox gene expression.
--When an actively transcribed white (w) gene is juxtaposed with heterochromatin by chromosome rearrangement such as w^m4, its expression is subject to variable but heritable silencing. This is PEV. We found that GAGA factor-FACT complex binds to a site just down stream of w, facilitates chromatin remodeling, and plays an important role in the maintenance of w expression against heterochromatin silencing.
--RSF (remodeling and spacing factor) has been first purified from human cells as a heterodimer of RSF1 and SNF2H (a human counterpart of Drosophila ISWI), which can assemble regularly spaced nucleosome arrays in vitro. To investigate in vivo role of RSF, we analyzed its Drosophila counterpart, a heterodimer of dRSF1 and ISWI. Genetic studies implicate Drosophila RSF in PEV through facilitating the spreading of silent chromatin.

(2) Chromatin transcription

Mikage Nakajima, Kenichi Nishioka, Tadashi Wada¹, Hiroshi Handa¹ and Susumu Hirose (¹Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuda, Yokohama, Japan)

--SPT6 is a transcription factor that is conserved across species from yeast to human. While biochemical studies on human SPT6 have revealed it to be a transcription elongation factor, genetic studies on yeast SPT6 have suggested its role in the recovery of nucleosome structure from deformation due to the passage of RNA polymerase II through a nucleosome. To study in vivo role of SPT6 in mulicellular organism, we started genetic study on Drosophila SPT6. We also continued studies on SPT6 and FACT using chromatin transcription in vitro in collaboration with Drs. T. Wada and H. Handa.

(3) Role of DNA topology in the formation of active chromatin

Kuniharu Matsumoto, Hirofumi Furuhashi, Youhei Ogasawara and Susumu Hirose

--Bulk DNA within the eukaryotic genome is torsionarily relaxed. However unconstrained negative supercoils of DNA have been detected in few local domains of the genome through preferential binding of psoralen. To make genome-wide survey for such domains, we introduced biotinylated psoralen into Drosophila salivary glands and visualized it on polytene chromosomes with fluorescent streptavidin. We observed bright psoralen signals on many transcriptionally active interbands and puffs. Upon heat shock, the signals appeared on heat-shock puffs. The signals were resistant to RNase treatment but disappeared or became faint by prior nicking of DNA or inhibition of transcription with α-amanitin. These data demonstrate that transcription-coupled, unconstrained negative supercoils of DNA exist in approximately 150 loci within the interphase genome.⁴⁾
--Supercoiling factor (SCF) is a protein capable of introducing negative supercoils into DNA in conjunction with topoisomerase II. Localization of SCF on puffs of polytene chromosomes has suggested its role in the formation of active chromatin. Knocking down of the SCF function in vivo by RNAi implicated SCF in twice activation of genes on male X chromosome. In good agreement with this, overexpression of SCF resulted in bloated appearance of the male X chromosome.

(4) Nucleosomal histone kinase-1

Hitoshi Aihara¹, Takeya Nakagawa¹, Kiyoshi Yasui¹, Tsutomu Ohta², Susumu Hirose, Naoshi Dhomae³, Koji Takio⁴, Mayumi Kaneko⁵, Yukio Takeshima⁵, Masami Muramatsu⁶ and Takashi Ito¹ (¹Department of Biochemistry, Nagasaki University School of Medicine, Nagasaki, Japan; ²Medical Genomic Center, National Cancer Center Research Institute, Tokyo, Japan; ³The Institute of Physical and Chemical Research, Wako-shi, Saitama, Japan; ⁴RIKEN Harima Institute, Mikazuki-cho, Hyogo, Japan; ⁵Second Department of Pathology, Hiroshima University School of Medicine, Hiroshima, Japan; ⁶Saitama Medical School Research Center for Genomic Medicine, Hidaka, Saitama, Japan)

--Posttranslational histone modifications are important for the regulation of many biological phenomena. Here, we show the purification and characterization of nucleosomal histone kinase-1 (NHK-1). NHK-1 has a high affinity for chromatin and phosphorylates a novel site, Thr 119, at the C terminus of H2A. Notably, NHK-1 specifically phosphorylates nucleosomal H2A, but not free H2A in solution. In Drosophila embryos, phosphorylated H2A Thr 119 is found in chromatin, but not in the soluble core histone pool. Immunostaining of NHK-1 revealed that it goes to chromatin during mitosis and is excluded from chromatin during S phase. Consistent with the shuttling of NHK-1 between chromatin and cytoplasm, H2A Thr 119 is phosphorylated during mitosis but not in S phase. These studies reveal that NHK-1-catalyzed phosphorylation of a conserved serine/threonine residue in H2A is a new component of the histone code that might be related to cell cycle progression.¹⁾

(5) Tips in analyzing antibodies directed against specific histone tail modifications

Kavitha Sarma¹, Kenichi Nishioka and Danny Reinberg¹ (¹Howard Hughes Medical Institute, Division of Nucleic Acids Enzymology, Department of Biochemistry, University of Medicine and Dentistry of New Jersay, Piscataway, NJ, USA)

--Histone methylation has been known to exist for over 40 years but the enzymes that catalyze this reaction have remained elusive until the discovery that Suv39H1 methylates histone H3 specifically at lysine 9. This discovery was followed by a bevy of papers describing other methyltransferases specific for different residues and their apparent function in vivo. Histones are methylated as lysine as well as arginine residues. Lysines can be mono-, di-, or tri-methylated in vivo.
--Studies on the effect of these modifications on gene regulation have been greatly facilitated by the production of antibodies “specific" for the modified state. The need to carefully characterize antibodies raised against methylated histone peptides stems from the observation by several laboratories that these antibodies can be promiscuous depending on several factors such as concentration, peptide context, substrate, etc. Due to this, several papers have been subject to scrutiny in recent months as the specificity of the antibodies used was questionable.
--In this article, we present several parameters to be taken into consideration and some useful hints for systematic characterization of antibodies raised against methylated histone peptides. Although we have focused on antibodies against methylated residues: H3-K4, H3-K27, and H4-K20, the methods and procedures described herein are applicable for any antibody directed against the histone tail modification, including arginine methylation and lysine acetylation, among other modifications.⁶⁾

(6) Functional analysis of transcriptional coactivator MBF1

Marek Jindra¹, Ivana Gaziova¹, Mirka Uhlirova¹, Masataka Okabe², Yasushi Hiromi², Kenichi Tsuda³, Toshiro Tsuji³, Ken-ichi Yamazaki³ and Susumu Hirose (¹Department of Molecular Biology, University of South Bohemia and Institute of Entomology ASCR, Ceske Budejovice, Czech Republic; ²Division of Developmental Genetics, National Institute of Genetics; ³Laobratory of Environmental Molecular Biology, Graduate School of Environmental Earch Science, Hokkaido University, Sapporo, Japan)

--Basic leucine zipper proteins Jun and Fos form the dimeric transcription factor AP-1, essential for cell differentiation and immune and antioxidant defenses. AP-1 activity is controlled, in part, by the redox state of critical cysteine residues within the basic regions of Jun and Fos. The redox control is necessary since replacement of these cysteines contributes to oncogenic potential of Jun and Fos. How cells maintain the redox-dependent AP-1 activity at favorable levels is not known. We show that the conserved coactivator MBF1 is a positive modulator of AP-1. Via a direct interaction with the basic region of Drosophila Jun (D-Jun), MBF1 prevents an oxidative modification (S-cystenyl cystenylation) of the critical cysteine and stimulates AP-1 binding to DNA. Cytoplasmic MBF1 translocates to the nucleus together with a transfected D-Jun protein, suggesting that MBF1 protects nascent D-Jun also in Drosophila cells. Consistent with the role of AP-1 in antioxidant defense, mbf1-null mutants exhibit shorter life than mbf1⁺ control in the presence of hydrogen peroxide (H₂O₂). An AP-1-dependent process of epithelial closure becomes sensitive to H₂O₂ in flies lacking mbf1. These results indicate that by preserving the redox-sensitive AP-1 activity, MBF1 provides an advantage during oxidative stress.³⁾
--Multiprotein bridging factor 1 (MBF1) is known to be a transcriptional co-activator that mediates transcriptional activation by bridging between an activator and a TATA-box binding protein (TBP). We demonstrated that expression of every three MBF1 from Arabidopsis partially rescues the yeast mbf1 mutant phenotype, indicating that all of them function as co-activators for GCN4-dependent transcriptional activation. We also report that each of their subtypes shows distinct tissue-specific expression patterns and responses to phytohormones. These observations suggest that even though they share a similar biochemical function, each MBF1 has distinct roles in various tissues and conditions.⁷⁾

(7) Differential functions of G protein and Baz-aPKC signaling pathways in Drosophila neuroblast asymmetric division

Yasushi Izumi¹, Nao Ohta¹, Asako Itoh-Furuya¹, Naoyuki Fuse and Fumio Matsuzaki¹ (¹Laboratory for Cell Asymmetry, Center for Developmental Biology, Institute of Physical and Chemical Research, and CREST, Japan Science and Technology Corporation, Kobe, Japan)

--Drosophila melanogaster neuroblasts (NBs) undergo asymmetric divisions during which cell-fate determinants localize asymmetrically, mitotic spindles orient along the apical-basal axis, and unequal-sized daughter cells appear. We identified here the first Drosophila mutant in the Gγ1 subunit of heterotrimeric G protein, which produces Gγ1 lacking its membrane anchor site and exhibits phenotypes identical to those of Gβ 13F, including abnormal spindle asymmetry and spindle orientation in NB divisions. This mutant fails to bind Gβ 13F to the membrane, indicating an essential role of cortical G1-Gβ13F signaling in asymmetric divisions. In Gγ1 and Gβ13F mutant NBs, Pins-Gαi, which normally localize in the apical cortex, no longer distribute asymmetrically. However, the other apical components, Bazooka-atypical PKC-Par6-Inscuteable, still remain polarized and responsible for asymmetric Miranda localization, suggesting their dominant role in localizing cell-fate determinants. Further analysis of Gβγ and other mutants indicates a predominant role of Partner of Inscuteable-Gαi in spindle orientation. We thus suggest that the two apical signaling pathways have overlapping but different roles in asymmetric NB division.²⁾

(8) Introduction of p16^INK4a inhibits telomerase activity through transcriptional suppression of human telomerase reverse transcriptase expression in human gliomas

Masahiro Saito¹, Kou Nakagawa¹, Katsuyuki Hamada², Susumu Hirose, Hironobu Harada¹, Shohei Kohno¹, Shigeyuki Nagato¹ and Takanori Ohnishi¹. (Department of ¹Neurosurgery and ²Obstetrics and Gynecology, Ehime University School of Medicine, Ehime Japan)

--The p16 and p53 tumor suppressor proteins, which are frequently altered in malignant gliomas, have been noted as regulators of telomerase activity. However, the link between telomerase regulation and these suppressor proteins has not been adequately clarified. In the present study, we demonstrated that p16, as well as p53, suppress telomerase activity through transcriptional regulation of human telomerase reverse transcriptase (hTERT) in malignant glioma. To examine the effect of p16 and p53 on telomerase activity, we utilized wild-type p16 or p53 expression plasmid and three human glioma cell lines differing in their p53 and p16 status. Restoring p16_significantly reduced the level of telomerase activity of glioma cells. Furthermore, cotransfection of the p16 gene with 5'-deletion constructs of the hTERT promoter carrying Sp1 binding sites, repressed the transcriptional activity of hTERT promoter in p16-deleted cells. In addition, electrophoretic mobility shift assay revealed that p16 expression inhibited the binding of Sp1 to the consensus Sp1 responsive element, indicating that the recruitment of Sp1 to the hTERT proximal core promoter is inhibited by p16 protein. These results were similar to those from a p53 transfection study in p53-mutated cells. These findings implicate p16 in the transcriptional regulation of telomerase activity by inhibiting the function of Sp1 in human malignant gliomas.⁵⁾

PUBLICATIONS

Papers
1. Aihara, H., Nakagawa. T., Yasui, K., Ohta, T., Hirose, S., Dhomae, N., Takio, K., Kaneko, M., Takeshima, Y., Muramatsu, M. and Ito, T. (2004). Nucleosomal histone kinase-1 phosphorylates H2A Thr 119 during mitosis in the early Drosophila embryo. Genes Dev., 18, 877-888.
2. Izumi, Y., Ohta, N., Itoh-Furuya, A., Fuse, N. and Matsuzaki, F. (2004). Differential functions of G protein and Baz-aPKC signaling pathways in Drosophila neuroblast asymmetric division. J. Cell Biol. 164, 729-738.
3. Jindra, M., Gaziova, I., Uhlirova, M., Okabe, M., Hiromi, Y., Tsuda, K., Tsuji, T., Yamazaki, K. and Hirose, S. (2004). Coactivator MBF1 preserves the redox-dependent AP-1 activity during oxidative stress in Drosophila. EMBO J., 23, 3538-3547.
4. Matsumoto, K. and Hirose, S. (2004). Visualization of unconstrained negative supercoils of DNA on polytene chromosomes of Drosophila. J. Cell Sci., 117, 3797-3805.
5. Saito, M., Nakagawa, K., Hamada, K., Hirose, S., Harada, H., Kohno, S., Nagato, S. and Ohnishi, T. (2004). Introduction of p16^INK4a inhibits telomerase activity through transcriptional suppression of human telomerase reverse transcriptase expression in human gliomas. Int. J. Oncol., 24, 1213-1220.
6. Sarma, K., Nishioka, K. and Reinberg, D. (2004). Tips in analyzing antibodies directed against specific histone tail modifications. Methods Enzymol. 376, 255-269.
7. Tsuda, K., Tsuji, T., Hirose, S. and Yamazaki, K. (2004). Three Arabidopsis MBF1 homologs with distinct expression profiles play roles as transcriptional co-activators. Plant Cell Physiol., 45, 225-231.

Reviews
8. 兼崎琢磨,西岡憲一（2004）「Hox遺伝子群におけるエピジェネティックス制御の分子機構」細胞工学,23, 1155-1161.
9. 西岡憲一（2004）「エピジェネティックス制御機構におけるメチル化ヒストンの役割」実験医学,22, 1361-1370.
10. 広瀬 進（2004）「ホメオティック遺伝子の発現制御」実験医学,22, 1371-1375.
11. 広瀬 進（2004）「MBF1と寿命」医学のあゆみ,211, 769.

Books
12. 西岡憲一（2004）「メチル化ヒストンによるクロマチン構造のエピジェネティクス制御機構」“エピジェネティクス".佐々木裕之編（シュプリンガー・フェアラーク東京）,pp31-50.
13. 広瀬 進（2004）「転写コファクター」“キーワードで理解する転写イラストマップ".田村隆明編（羊土社）,pp52-58.

ORAL PRESENTATIONS

1. Hirose, S. Chromatin remodeling directed by GAGA factor-FACT complex serves as a boundary against epigenetic silencing. The 2nd Annual CDB Symposium on Developmental Remodeling, Kobe, March, 2004.
2. Aihara, H., Nakagawa, T., Yasui, K., Ohta, T., Hirose, S., Muramatsu, M., Ito, T. Nucleosomal histone kinase-1 phosphorylates H2A Thr 119 during mitosis in the early Drosophila embryo. The 27th Annual Meeting of the Molecular Biology Society of Japan, Workshop, Kobe, December, 2004.
3. 広瀬 進「酸化ストレスにおけるコアクチベーターMBF1の役割」第3回転写研究会、筑波、2004年1月.
4. 広瀬 進「サイレントクロマチンの侵攻をたち切るクロマチンリモデリング」第21回染色体ワークショップ、湯河原、2004年1月.
5. 広瀬 進「コアアクチベーターMBFと寿命」ショウジョウバエ先端研究シンポジウム、京都、2004年3月.
6. 西岡憲一「クロマチン機能のエピジェネティクス制御機構」大阪大学蛋白質研究所セミナー、大阪、2004年6月.
7. 古橋寛史、広瀬 進「スーパーコイル化因子はX染色体量的補正に関与する」国立遺伝学研究所研究会、三島、2004年9月.
8. 広瀬 進「転写に伴なうDNA超らせんの可視化」東京工業大学生命工学フロンティアシンポジウム、長津田、2004年10月.
9. 西岡憲一、広瀬 進「エピジェネティクス制御の分子機構」第27回日本分子生物学会年会ワークショップ「クロマチンと遺伝子発現の核内クロストーク」神戸、2004年12月.
10. 津中康央、笠井信幸、楯 真一、梶村直子、的場京子、広瀬 進、森川耿右「FACT-HMGドメインとヌクレオソームの相互作用に関する構造解析」第27回日本分子生物学会年会ワークショップ「ゲノムとクロマチンの分子解剖:隠された情報と暗号を探る」、神戸、2004年12月.
11. 花井一馬、古橋寛史、霜鳥大信、西岡憲一、山本 卓、赤坂甲治、広瀬 進「クロマチンリモデリング複合体RSFの機能解析」第27回日本分子生物学会年会ワークショップ「ゲノムとクロマチンの分子解剖:隠された情報と暗号を探る」、神戸、2004年12月.
12. 上田 均、阿川泰夫、高井将圭、首藤一平、広瀬 進「昆虫の脱皮変態の時間的制御機構」第27回に本分子生物学会年会ワークショップ「昆虫特異的機能の分子基盤」、神戸、2004年12月.

POSTER PRESENTATONS
1. Furuhashi, H. and Hirose, S. DNA supercoiling factor is involved in Drosophila dosage compensation. 45th Annual Drosophila Research Conference, Washington DC, USA, March, 2004.
2. Agawa, Y., Hirose, S. and Ueda, H. p170, a novel Drosophila transcription factor that regulates expression of the FTZ-F1 gene during high ecdysteroid periods. 45th Annual Drosophila Research Conference, Washington DC, USA, March, 2004.
3. Hirose, S. Drosophila GAGA factor-FACT complex builds up a barrier against epigenetic silencing. Gordon Research Conference on Chromatin Structure and Function, Tilton, USA, July, 2004.
4. Liu, Q.-X., Ikeo, K., Hirose, S. and Gojobori, T. Coevolution of MBF1 and TBP across the species from Archea to human. The 27th Annual Meeting of the Molecular Biology Society of Japan, Kobe, December, 2004.
5. 小笠原洋平、古橋寛史、広瀬 進「ショウジョウバエDNA超らせん化因子SCFによる熱ショップ遺伝子およびホメオティック遺伝子の発現調節」第27回日本分子生物学会年会、神戸、2004年12月.
6. 中山貴博、霜島 司、西岡憲一、広瀬 進「GAGA因子ﾑdFACT複合体によるヘテロクロマチンサイレンシングの遮断」第27回日本分子生物学会年会、神戸、2004年12月.
7. 霜島 司、中山貴博、広瀬 進「GAGA-dFACT複合体はクロマチンリモデリング複合体と相互作用する」第27回日本分子生物学会年会、神戸、2004年12月.
8. 布施直之、広瀬 進「原腸陥入における細胞運動の協調性のメカニズム」第27回日本分子生物学会年会、神戸、2004年12月.

EDUCATION

1. Dr. S. Hirose was invited to give a seminar on “Chromatin remodeling as a barrier against the epigenetic silencing" at National Institute for Basic Biology, Okazaki, July, 2004 (in Japanese).
2. Dr. S. Hirose was invited to give a seminar on “GAGA factor-FACT complex as a boundary against the spreading of silent chromatin" at Tokyo Institute of Technology, Nagatsuda, August, 2004 (in Japanese).

SOCIAL CONTRIBUTION AND OTHERS

評価委員:生物資源研究所

特許
1. 出願番号:2004-315398,発明の名称:負の超らせんDNAの検出法,発明者:広瀬 進・松本国治,出願人:大学共同利用機関法人情報・システム研究機構