Department of Biological Regulation echanisms controlling cell Atan Gross Yehudit Zaltsman, Mlife and death decisions Natalie Yivgi-Ohana, Iris Kamer, Galia Oberkovitz, Liat Shachnai, Maria Maryanovich

Programmed cell death or apoptosis in the extraembryonic (ExEm) region of is essential for both the development E7.5 wild type embryos, and this region and maintenance of tissue homeostasis is largely impaired in the Mtch2/Mimp-/- in multicellular organisms. The BCL-2 embryos. Thus, Mtch2/Mimp might play family members are critical regulators a critical role in the formation of the 972 8 934 3656 of the apoptotic program, whereas ExEm region. To study the connection the caspase proteases are the major between Mtch2/Mimp and apoptosis FAX 972 8 934 4116 executioners of this program. Members we generated Mtch2/Mimp-/- stable [email protected] of the BCL-2 family include both anti- and embryonic stem (ES) cell lines carrying www.weizmann.ac.il/ pro-apoptotic proteins. The BH3-only either an empty vector or Mtch2/Mimp. Biological_Regulation/gross proteins (e.g., BID) are an important Using these lines we demonstrated that subset of the pro-apoptotic proteins the presence of Mtch2/Mimp sensitizes that act as sentinels of intercellular cells to tBID-induced MOMP. Thus, we damage. In our laboratory, we are discovered that Mtch2/Mimp is critical S phase and inhibition of apoptosis focused on elucidating the mechanisms for normal embryonic development, following DNA damage. This surprising that balance between cell life and and is an important positive regulator and new pro-survival function of BID death, and BID is one of our primary of tBID-induced apoptosis at the is regulated by its phosphorylation tools to study these mechanisms. mitochondria. Future studies using on serines 61 and 78 by the ataxia- a newly generated Mtch2/Mimp telangiectasia mutated (ATM) kinase, The role of mitochondrial conditional knockout mouse will enable a key guardian of genome integrity Mtch2/Mimp in apoptosis and us to further elucidate the role of Mtch2/ (Kamer et al., 2005; Zinkel et al., 2005; embryogenesis Mimp in apoptosis and embryogenesis. Gross, 2006; Zinkel et al., 2006; Zinkel In the extrinsic death pathway, et al., 2007)(see Figure). We also found apoptosis is initiated through activation Using blue-native gel electrophoresis that BID’s pro-survival activity involves of the TNF/Fas receptors. Activation of (BN-PAGE) we have found that its nucleo-cytoplasmic shuttling since these receptors results in the cleavage Mtch2/Mimp exists as part of a large DNA damage triggers the nuclear of pro-apoptotic BID into truncated mitochondrial complex that contains export of BID, and artificially trapping BID (tBID), which translocates to the tBID, BAX and additional unidentified BID in the nucleus inhibits its ability to mitochondria to induce oligomerization proteins. We have named this multi- induce cell cycle arrest at the S phase of pro-apoptotic BAX, followed by protein complex the “Mtch2/Mimp- (Oberkovitz et al., 2007). To assess the mitochondrial outer membrane eosome”. Two independent and relevance of our findings to the in vivo permeabilization (MOMP). This event complimentary approaches are being setting, we generated a BID knock-in i results in the release of apoptogenic taken to identify the components mouse, in which the endogenous BID factors such as cytochrome c. comprising the Mtch2/Mimp-eosome: gene has been replaced with a gene However, the mechanism by which tBID 1) Chemical cross-linking. We are using that drives the expression of a non- triggers MOMP is largely unknown. Mtch2/Mimp and several BCL-2 family phosphorylatable BID protein (BIDS61A/ Mitochondria carrier homolog 2/Met- members (e.g., BAX and BIM) as baits to S78A or BID-AA). Using cells from the induced mitochondrial protein (Mtch2/ identify interacting proteins comprising BID-AA mice we found that primary T Mimp) was identified in our lab as part this complex; 2) A siRNA screen to and B cells demonstrate a defect in the of a complex with tBID in cells signaled identify resident mitochondrial proteins intra S-phase DNA damage checkpoint, to die by TNF (Grinberg et al., 2005; involved in the tBID-Mtch2/Mimp increased chromosomal damage, and Gross, 2005; Schwarz et al., 2007) signaling pathway. For this screen we increased apoptosis in response to DNA (see Figure). Mtch2/Mimp is a novel are using a SMART pool library designed damage. Thus, BID’s S phase arrest and previously uncharacterized 33-kDa against all the known mitochondrial function seems to be critical for T and B

Weizmann Institute of ScienceWeizmann protein, which is related to the family of proteins. This library includes siRNAs cells to preserve genomic stability and ∙ mitochondrial carrier proteins. We have against 708 mitochondrial proteins, and to survive following. revealed that knocking out Mtch2/Mimp was designed by Dharmacon according in mice results in embryonic lethality, to our specifications. We are in the To determine how BID is physically ∙ 2008 and analysis of time pregnancies process of establishing a number of live executing its function(s) in the revealed that Mtch2/Mimp-/- embryos imaging systems to follow MOMP. DNA damage response, we took a are not viable beyond E7.5. Histological biochemical approach to purify proteins analysis of the Mtch2/Mimp+/+ and the The role of ATM mediated BID that associate with BID using the Mtch2/Mimp-/- embryos indicated that phosphorylation in the DNA formaldehyde cross-linker. We revealed the null embryos are morphologically damage response that BID is found as part of a 50-kDa abnormal and do not seem to undergo In 2005, we have reported that BID cross-linked complex in healthy cells complete gastrulation. Moreover, is also localized to the nucleus and is and in cells treated with DNA damage,

Life Science Open Day Mtch2/Mimp mRNA is highly expressed required for cell cycle arrest at the suggesting that BID may execute its BID-AA BID-AA and wild-type mice with Eμ breeding that found we Interestingly, as well as for genomic instability instability genomic for syndromes. as well as lineage, lymphoid the in development tumor for implications important have in lymphoid tissues then our studies will a pivotal in such role cell fate decisions playing indeed is BID if that anticipate between linkBID and the DNA damage response. We the strengthen further studies and the studies described above These lymphomagenesis. Myc-induced is playing a in that role BID phosphorylation suggesting background, BID-AA the in lymphomas cell B Myc-induced of onset accelerated in resulted mice was Eµ- the in Ser61 on BID phosphorylated be to Finally, shown recently protein. with another interaction stable a by function Fig.1 to generate tBID, which translocates to the mitochondria to interact with the Mtch2/Mimp- the with interact to mitochondria the to translocates which tBID, generate to eosome and BAX/BAK to induce MOMP. Right: Following DNA damage, ATM and ATR are are ATR and ATM damage, DNA Following Right: MOMP. induce to BAX/BAK and eosome activated and can lead to either survival or apoptosis. BID is an ATM target, which is is which target, ATM an is BID apoptosis. or survival either to lead can and activated important for cell cycle arrest, DNA repair and survival. It still remains unknown how BID BID how unknown remains still It survival. and repair DNA arrest, cycle cell for important connects between the DNA damage pathway in the nucleus and the apoptosis pathway at at pathway apoptosis the and nucleus the in pathway damage DNA the between connects the mitochondria. Both pathways that involve BID are eventually critical for inhibition of of inhibition for critical eventually are BID involve that pathways Both mitochondria. the tumorigenesis. BID as a double agent. Left: In the TNF/Fas death-receptor pathway, BID is cleaved cleaved is BID pathway, death-receptor TNF/Fas the In Left: agent. double a as BID myc mouse model. mouse - myc myc Zinkel, S.S., Hurov, K.E., Ong, Kamer, Y., I., R., Zaltsman, Sarig, Carrier (2005) A. Mitochondrial Gross, M., M., Zatsman, Schwarz, Grinberg, publications Selected response. Cell, 122, Cell, 579-591.response. damage BID DNA in the apoptotic Korsmeyer, pro- (2005) S.J. for Arole F.M.,C., Abtahi, A., and Gross, 122, 593-603. Cell, response. damage DNA in the BID ATM an is effector Pro-apoptotic (2005) A. R.C., Gross, and Marcellus, Y., G.,L., Haimovich, Lerenthal, Niv, G., Regev, H., Oberkovitz, 37, Biomembranes, and 113-119. BCL-2 riddle? family J. Bioenergetics the 2: cracking to Aclue homolog Biol., 25, 4579-4590. by die TNF. to signaled Cell. Mol. tBID of in cells atarget 2is homolog carrier (2005) A. Mitochondrial Gross, Y., Eini, T., S., and Pietrokovski, Zinkel, S., Hurov, A. K., Gross, and M., M., Gross, Andrade, and Schwarz, G., Regev, A. Oberkovitz, L., Gross, and Yacobi, K., Tsafriri, A. Gross, A., and A.,Zinkel, &Yang, S., Gross, (2006) E. (2006) agent A. BID adouble as Gross, Joint Research Program Research Joint award, McGill-Weizmann ICR research Center, MDM Research Woman Health support INTERNAL Association. Cancer Israel the and Foundation, Research Cancer (DKFZ-MOST), Israel the Research in Cancer Program Research German-Israeli Joint the Foundation, Israeli German The Foundation, Science Binational USA-Israel the Foundation, (A-TCP),Foundation Science Israel the Ataxia-Telangiectasia by the supported is work Our research. cancer of Chair Development Family Career Armour the of incumbent the is Gross Atan Acknowledgements damage response. Cell, 130, Cell, 9-10. response. damage DNA (2007) in the playsarole Bid 12, 869-876. Apoptosis, program? apoptotic mitochondrial the of Key regulators pores: and carriers (2007)A. Mitochondrial 1628-1634. &Diff., Death 14, Cell response. DNA-damage in the activities its BIDof in regulating involved is shuttling (2007) Nucleo-cytoplasmic 148,Endocrinology, 1717-1726. steroidogenesis. mitochondrial to coupled is follicles preovulatory in rat activation caspase (2007) LH-induced 1351-1359. &Diff., Death 13, Cell control. cycle family, cell and damage, BCL2 DNA 582-584. 5, Cycle, Cell death. and life in cell

Life Science Open Day ∙ 2008 ∙ Weizmann Institute of Science ii