• 
  Jessen, M., Gertzmann, D., Liss, F., Zenk, F., B\"a}hner, L., Sch\"o}ffler, V., Schulte, C., Maric, H.M., Ade, C.P., von Eyss, B., & Gaubatz, S.Inhibition of the YAP-MMB interaction and targeting NEK2 as potential therapeutic strategies for YAP-driven cancersOncogene, 43(8): 578–593.
  • [ Abstract ]
  • [ DOI ]
  YAP activation in cancer is linked to poor outcomes, making it an attractive therapeutic target. Previous research focused on blocking the interaction of YAP with TEAD transcription factors. Here, we took a different approach by disrupting YAP's binding to the transcription factor B-MYB using MY-COMP, a fragment of B-MYB containing the YAP binding domain fused to a nuclear localization signal. MY-COMP induced cell cycle defects, nuclear abnormalities, and polyploidization. In an AKT and YAP-driven liver cancer model, MY-COMP significantly reduced liver tumorigenesis, highlighting the importance of the YAP-B-MYB interaction in tumor development. MY-COMP also perturbed the cell cycle progression of YAP-dependent uveal melanoma cells but not of YAP-independent cutaneous melanoma cell lines. It counteracted YAP-dependent expression of MMB-regulated cell cycle genes, explaining the observed effects. We also identified NIMA-related kinase (NEK2) as a downstream target of YAP and B-MYB, promoting YAP-driven transformation by facilitating centrosome clustering and inhibiting multipolar mitosis.

• 
  Fetiva, M.C., Liss, F., Gertzmann, D., Thomas, J., Gantert, B., Vogl, M., Sira, N., Weinstock, G., Kneitz, S., Ade, C.P., & Gaubatz, S.Oncogenic YAP mediates changes in chromatin accessibility and activity that drive cell cycle gene expression and cell migrationNucleic Acids Research. https://doi.org/10.1093/nar/gkad107.
  • [ DOI ]

• 
  Hanselmann, S., Gertzmann, D., Shin, W.J., Ade, C.P., & Gaubatz, S.Expression of the cytokinesis regulator PRC1 results in p53-pathway activation in A549 cells but does not directly regulate gene expression in the nucleusCell Cycle, 22(4): 419–432.
  • [ DOI ]

• 
  Leone, M., Cazorla-V\’a}}zquez, S., Ferrazzi, F., Wiederstein, J.L., Gründl, M., Weinstock, G., Vergarajauregui, S., Eckstein, M., Krüger, M., Gaubatz, S., & Engel, F.B.IQGAP3, a YAP Target, Is Required for Proper Cell-Cycle Progression and Genome StabilityMolecular Cancer Research, 19(10): 1712–1726.
  • [ DOI ]

• 
  Gründl, M., Walz, S., Hauf, L., Schwab, M., Werner, K.M., Spahr, S., Schulte, C., Maric, H.M., Ade, C.P., & Gaubatz, S.Interaction of YAP with the Myb-MuvB (MMB) complex defines a transcriptional program to promote the proliferation of cardiomyocytesPLOS Genetics, 16(5): 1–27.
  • [ Abstract ]
  • [ DOI ]
  Author summary YAP, the major downstream transducer of the Hippo pathway, is a potent inducer of proliferation. Here we show that the Myb-MuvB complex (MMB) mediates cardiomyocyte proliferation by YAP. We find that YAP and MMB regulate an overlapping set of pro-proliferative genes which involves binding of MMB to the promoters of these genes. We also identified a direct interaction between the B-MYB subunit of MMB and YAP. Based on the binding studies, we created a tool called MY-COMP that interferes with the association YAP to B-MYB and strongly inhibits proliferation of cardiomyocytes. Together, our data suggests that the YAP-MMB interaction is essential for division of cardiomyocytes, underscoring the functional relevance of the crosstalk between these two pathways for proper heart development.

• 
  Weinstock, G., & Gaubatz, S.One way to rule them all: G2/M gene regulation by oncogenic YAP1 and B-MYBMolecular \& Cellular Oncology, 6(6): e1648026.
  • [ DOI ]
• 
  Pattschull, G., Walz, S., Gründl, M., Schwab, M., Rühl, E., Baluapuri, A., Cindric-Vranesic, A., Kneitz, S., Wolf, E., Ade, C.P., Rosenwald, A., von Eyss, B., & Gaubatz, S.The Myb-MuvB Complex Is Required for YAP-Dependent Transcription of Mitotic GenesCell Reports, 27(12): 3533 – 3546.e7.
  • [ Abstract ]
  • [ DOI ]
  Summary YAP and TAZ, downstream effectors of the Hippo pathway, are important regulators of proliferation. Here, we show that the ability of YAP to activate mitotic gene expression is dependent on the Myb-MuvB (MMB) complex, a master regulator of genes expressed in the G2/M phase of the cell cycle. By carrying out genome-wide expression and binding analyses, we found that YAP promotes binding of the MMB subunit B-MYB to the promoters of mitotic target genes. YAP binds to B-MYB and stimulates B-MYB chromatin association through distal enhancer elements that interact with MMB-regulated promoters through chromatin looping. The cooperation between YAP and B-MYB is critical for YAP-mediated entry into mitosis. Furthermore, the expression of genes coactivated by YAP and B-MYB is associated with poor survival of cancer patients. Our findings provide a molecular mechanism by which YAP and MMB regulate mitotic gene expression and suggest a link between two cancer-relevant signaling pathways.

• 
  Hanselmann, S., Wolter, P., Malkmus, J., & Gaubatz, S.The microtubule-associated protein PRC1 is a potential therapeutic target for lung cancerOncotarget, 9(4): 4985–4997.
  • [ DOI ]
• 
  Gründl, M., Engel, F.B., & Gaubatz, S.GAS2L3: Coordinator of cardiomyocyte cytokinesis?Cell Cycle, 16(20): 1853–1854.
  • [ DOI ]
• 
  Wolter, P., Hanselmann, S., Pattschull, G., Schruf, E., & Gaubatz, S.Central spindle proteins and mitotic kinesins are direct transcriptional targets of MuvB, B-MYB and FOXM1 in breast cancer cell lines and are potential targets for therapyOncotarget, 8(7): 11160–11172.
  • [ DOI ]
• 
  Stopp, S., Gründl, M., Fackler, M., Malkmus, J., Leone, M., Naumann, R., Frantz, S., Wolf, E., von Eyss, B., Engel, F.B., & Gaubatz, S.Deletion of Gas2l3 in mice leads to specific defects in cardiomyocyte cytokinesis during developmentProceedings of the National Academy of Sciences, 114(30): 8029–8034.
  • [ Abstract ]
  • [ DOI ]
  Here, we demonstrate that mice lacking GAS2L3, a cytoskeleton-associated protein that interacts with actin filaments and tubulin, develop cardiomyopathy and heart failure after birth. During embryogenesis, cardiomyocytes rapidly divide. In the perinatal and neonatal period, cardiomyocytes withdraw from the cell cycle, binucleate, and the further increase in cardiac mass is achieved by hypertrophy. Germ-line deletion of Gas2l3 results in decreased cardiomyocyte proliferation and in cardiomyocyte hypertrophy. Embryonal cardiomyocytes from Gas2l3-deficient mice exhibit increased expression of the cell cycle inhibitor p21 and display premature binucleation of cardiomyocytes due to defects in cytokinetic abscission. Together these results suggest that GAS2L3 plays a central role in cardiomyocyte proliferation and cytokinesis during development. GAS2L3 is a recently identified cytoskeleton-associated protein that interacts with actin filaments and tubulin. The in vivo function of GAS2L3 in mammals remains unknown. Here, we show that mice deficient in GAS2L3 die shortly after birth because of heart failure. Mammalian cardiomyocytes lose the ability to proliferate shortly after birth, and further increase in cardiac mass is achieved by hypertrophy. The proliferation arrest of cardiomyocytes is accompanied by binucleation through incomplete cytokinesis. We observed that GAS2L3 deficiency leads to inhibition of cardiomyocyte proliferation and to cardiomyocyte hypertrophy during embryonic development. Cardiomyocyte-specific deletion of GAS2L3 confirmed that the phenotype results from the loss of GAS2L3 in cardiomyocytes. Cardiomyocytes from Gas2l3-deficient mice exhibit increased expression of a p53-transcriptional program including the cell cycle inhibitor p21. Furthermore, loss of GAS2L3 results in premature binucleation of cardiomyocytes accompanied by unresolved midbody structures. Together these results suggest that GAS2L3 plays a specific role in cardiomyocyte cytokinesis and proliferation during heart development.

• 
  Iltzsche, F., Simon, K., Stopp, S., Pattschull, G., Francke, S., Wolter, P., Hauser, S., Murphy, D.J., Garcia, P., Rosenwald, A., & Gaubatz, S.An important role for Myb-MuvB and its target gene KIF23 in a mouse model of lung adenocarcinomaOncogene, 36(1): 110–121.
  • [ DOI ]

• 
  Kumari, G., Ulrich, T., Krause, M., Finkernagel, F., & Gaubatz, S.Induction of p21CIP1 Protein and Cell Cycle Arrest after Inhibition of Aurora B Kinase Is Attributed to Aneuploidy and Reactive Oxygen SpeciesJournal of Biological Chemistry, 289(23): 16072–16084.
  • [ DOI ]
• 
  Fackler, M., Wolter, P., & Gaubatz, S.The GAR domain of GAS2L3 mediates binding to the chromosomal passenger complex and is required for localization of GAS2L3 to the constriction zone during abscissionFEBS Journal, 281(9): 2123–2135.
  • [ DOI ]

• 
  Kumari, G., Ulrich, T., & Gaubatz, S.A role for p38 in transcriptional elongation of p21(CIP) in response to Aurora B inhibitionCell Cycle, 12(13): 2051–2060.
  • [ DOI ]
• 
  Esterlechner, J., Reichert, N., Iltzsche, F., Krause, M., Finkernagel, F., & Gaubatz, S.LIN9, a Subunit of the DREAM Complex, Regulates Mitotic Gene Expression and Proliferation of Embryonic Stem CellsPLOS ONE, 8(5): 1–14.
  • [ Abstract ]
  • [ DOI ]
  The DREAM complex plays an important role in regulation of gene expression during the cell cycle. We have previously shown that the DREAM subunit LIN9 is required for early embryonic development and for the maintenance of the inner cell mass in vitro. In this study we examined the effect of knocking down LIN9 on ESCs. We demonstrate that depletion of LIN9 alters the cell cycle distribution of ESCs and results in an accumulation of cells in G2 and M and in an increase of polyploid cells. Genome-wide expression studies showed that the depletion of LIN9 results in downregulation of mitotic genes and in upregulation of differentiation-specific genes. ChIP-on chip experiments showed that mitotic genes are direct targets of LIN9 while lineage specific markers are regulated indirectly. Importantly, depletion of LIN9 does not alter the expression of pluripotency markers SOX2, OCT4 and Nanog and LIN9 depleted ESCs retain alkaline phosphatase activity. We conclude that LIN9 is essential for proliferation and genome stability of ESCs by activating genes with important functions in mitosis and cytokinesis.

• 
  Wolter, P., Schmitt, K., Fackler, M., Kremling, H., Probst, L., Hauser, S., Gruss, O.J., & Gaubatz, S.GAS2L3, a novel target gene of the dream complex, is required for proper cytokinesis and genomic stabilityJournal of Cell Science. https://doi.org/10.1242/jcs.097253.
  • [ DOI ]

• 
  Hauser, S., Ulrich, T., Wurster, S., Schmitt, K., Reichert, N., & Gaubatz, S.Loss of LIN9, a member of the DREAM complex, cooperates with SV40 large T antigen to induce genomic instability and anchorage-independent growthOncogene, 31(14): 1859–1868.
  • [ DOI ]
• 
  Houben, R., Adam, C., Baeurle, A., Hesbacher, S., Grimm, J., Angermeyer, S., Henzel, K., Hauser, S., Elling, R., Bröcker, E.-B., Gaubatz, S., Becker, J.C., & Schrama, D.An intact retinoblastoma protein-binding site in Merkel cell polyomavirus large T antigen is required for promoting growth of Merkel cell carcinoma cellsInternational Journal of Cancer, 130(4): 847–856.
  • [ DOI ]

• 
  Reichert, N., Wurster, S., Ulrich, T., Schmitt, K., Hauser, S., Probst, L., Götz R., Ceteci, F., Moll, R., Rapp, U., & Gaubatz, S.Lin9, a Subunit of the Mammalian DREAM Complex, Is Essential for Embryonic Development, for Survival of Adult Mice, and for Tumor SuppressionMolecular and Cellular Biology, 30(12): 2896–2908.
  • [ DOI ]
• 
  Meierjohann, S., Hufnagel, A., Wende, E., Kleinschmidt, M.A., Wolf, K., Friedl, P., Gaubatz, S., & Schartl, M.MMP13 mediates cell cycle progression in melanocytes and melanoma cells: in vitro studies of migration and proliferationMolecular Cancer, 9(1): 201-.
  • [ Abstract ]
  • [ DOI ]
  Melanoma cells are usually characterized by a strong proliferative potential and efficient invasive migration. Among the multiple molecular changes that are recorded during progression of this disease, aberrant activation of receptor tyrosine kinases (RTK) is often observed. Activation of matrix metalloproteases goes along with RTK activation and usually enhances RTK-driven migration. The purpose of this study was to examine RTK-driven three-dimensional migration of melanocytes and the pro-tumorigenic role of matrix metalloproteases for melanocytes and melanoma cells.

• 
  Mannefeld, M., Klassen, E., & Gaubatz, S.B-MYB Is Required for Recovery from the DNA Damage\textendashInduced G2 Checkpoint in p53 Mutant CellsCancer Research, 69(9): 4073–4080.
  • [ Abstract ]
  • [ DOI ]
  In response to DNA damage, several signaling pathways that arrest the cell cycle in G1 and G2 are activated. The down-regulation of mitotic genes contributes to the stable maintenance of the G2 arrest. The human LINC or DREAM complex, together with the B-MYB transcription factor, plays an essential role in the expression of G2-M genes. Here, we show that DNA damage results in the p53-dependent binding of p130 and E2F4 to LINC and the dissociation of B-MYB from LINC. We find that B-MYB fails to dissociate from LINC in p53 mutant cells, that this contributes to increased G2-M gene expression in response to DNA damage in these cells, and, importantly, that B-MYB is required for recovery from the G2 DNA damage checkpoint in p53-negative cells. Reanalysis of microarray expression data sets revealed that high levels of B-MYB correlate with a p53 mutant status and an advanced tumor stage in primary human breast cancer. Taken together, these data suggest that B-MYB/LINC plays an important role in the DNA damage response downstream of p53. [Cancer Res 2009;69(9):4073{\textendash}80]
• 
  Kleinschmidt, M.A., Wagner, T.U., Liedtke, D., Spahr, S., Samans, B., & Gaubatz, S.lin9 Is Required for Mitosis and Cell Survival during Early Zebrafish DevelopmentJournal of Biological Chemistry, 284(19): 13119–13127.
  • [ Abstract ]
  • [ DOI ]
  LIN9 has been described as a regulator of G1/S and G2/M progression of the cell cycle in invertebrates and human cell lines. To elucidate the in vivo function of LIN9 during vertebrate development, we took advantage of the teleost zebrafish (Danio rerio). By means of antisense morpholinos we show here that Lin9-depleted embryonic cells accumulate in mitosis. Flow cytometry and confocal microscopy data demonstrate that the delay in mitotic progression is followed by apoptosis, which strongly manifests in the developing central nervous system. In accordance with these findings, we identified a cohort of Lin9-regulated genes required for different mitotic processes, including mitotic entry, metaphase/anaphase transition, and cytokinesis. Our data establish LIN9 as an essential regulator of mitosis in vertebrate development.
• 
  Schmit, F., Cremer, S., & Gaubatz, S.LIN54 is an essential core subunit of the DREAM/LINC complex that binds to the cdc2 promoter in a sequence‐specific mannerThe FEBS Journal, 276(19): 5703–5716.
  • [ DOI ]

• 
  Kehoe, S.M., Oka, M., Hankowski, K.E., Reichert, N., Garcia, S., McCarrey, J.R., Gaubatz, S., & Terada, N.A Conserved E2F6-Binding Element in Murine Meiosis-Specific Gene PromotersBiology of Reproduction, 79(5): 921–930.
  • [ DOI ]

• 
  Schmit, F., Korenjak, M., Mannefeld, M., Schmitt, K., Franke, C., von Eyss, B., Gagrica, S., Hänel, F., Brehm, A., & Gaubatz, S.LINC, a human complex that is related to pRB-containing complexes in invertebrates regulates the expression of G2/M genesCell Cycle, 6(15): 1903–1913.
  • [ DOI ]

• 
  Osterloh, L., von Eyss, B., Schmit, F., Rein, L., Hübner, D., Samans, B., Hauser, S., & Gaubatz, S.The human synMuv-like protein LIN-9 is required for transcription of G2/M genes and for entry into mitosisThe EMBO Journal, 26(1): 144–157.
  • [ DOI ]

• 
  Storre, J., Schäfer, A., Reichert, N., Barbero, J.L., Hauser, S., Eilers, M., & Gaubatz, S.Silencing of the Meiotic Genes SMC1β and STAG3 in Somatic Cells by E2F6Journal of Biological Chemistry, 280(50): 41380–41386.
  • [ Abstract ]
  • [ DOI ]
  E2F6, a member of the E2F-family of transcription factors, is a retinoblastoma protein-independent transcriptional repressor. E2F6 associates with polycomb group (Pc-G) multiprotein complexes that contain histone H3 methyltransferases, suggesting that E2F6 represses genes by covalent histone modification. However, genes that are repressed by E2F6 via a mechanism that involves histone H3 methylation have not been identified. Using cDNA microarray experiments comparing wild-type and E2f6-/- mouse embryonic fibroblasts, we now found that E2F6 is required to silence the meiosis-specific genes SMC1β and STAG3 in somatic cells. Re-expression of E2F6 in E2f6-/- cells was sufficient to restore their repression. E2F6 binds in vivo to the promoters of these genes through a conserved binding site. Transcriptional repression of SMC1β and STAG3 by E2F6 involves multiple mechanisms, including methylation of histone H3 on lysine 9 and lysine 27. Our findings suggest a molecular mechanism for the stable transcriptional silencing of meiotic genes in somatic cells by E2F6.

• 
  Gagrica, S., Hauser, S., Kolfschoten, I., Osterloh, L., Agami, R., & Gaubatz, S.Inhibition of oncogenic transformation by mammalian Lin-9, a pRB-associated proteinThe EMBO Journal, 23(23): 4627–4638.
  • [ DOI ]
• 
  Giangrande, P.H., Zhu, W., Schlisio, S., Sun, X., Mori, S., Gaubatz, S., & Nevins, J.R.A role for E2F6 in distinguishing G1/S- and G2/M-specific transcriptionGenes &Amp; Development, 18(23, /content/18/23.cover.gif): 2941–2951.
  • [ DOI ]

• 
  Ohtani, N., Brennan, P., Gaubatz, S., Sanij, E., Hertzog, P., Wolvetang, E., Ghysdael, J., Rowe, M., & Hara, E.Epstein-Barr virus LMP1 blocks p16INK4a\textendashRB pathway by promoting nuclear export of E2F4/5Journal of Cell Biology, 162(2): 173–183.
  • [ DOI ]

• 
  Storre, J., Elsässer, H.-P., Fuchs, M., Ullmann, D., Livingston, D., & Gaubatz, S.Homeotic transformations of the axial skeleton that accompany a targeted deletion of E2F6EMBO Reports, 3: 695–700.
• 
  Ogawa, H., Ishiguro, K., Gaubatz, S., Livingston, D.M., & Nakatani, Y.A complex with chromatin modifiers that occupies E2F- and Myc-responsive genes in G0 cellsScience, 296(5570): 1132–1136.
  • [ Abstract ]
  • [ DOI ]
  E2F-6 contributes to gene silencing in a manner independent of retinoblastoma protein family members. To better elucidate the molecular mechanism of repression by E2F-6, we have purified the factor from cultured cells. E2F-6 is found in a multimeric protein complex that contains Mga and Max, and thus the complex can bind not only to the E2F-binding site but also to Myc- and Brachyury-binding sites. Moreover, the complex contains chromatin modifiers such as a novel histone methyltransferase that modifies lysine 9 of histone H3, HP1gamma, and Polycomb group (PcG) proteins. The E2F-6 complex preferentially occupies target promoters in G0 cells rather than in G1 cells. These data suggest that these chromatin modifiers contribute to silencing of E2F- and Myc-responsive genes in quiescent cells.
• 
  Dahme, T., Wood, J., Livingston, D.M., & Gaubatz, S.Two different E2F6 proteins generated by alternative splicing and internal translation initiationEur J Biochem, 269(20): 5030–5036.
  • [ Abstract ]
  • [ DOI ]
  E2F transcription factors play an important role in the regulation of cell cycle progression. E2F6, the most recently identified member of the E2F family, is a retinoblastoma-protein-independent transcriptional repressor that is required for developmental patterning of the axial skeleton. It has recently been shown that the E2f6 locus produces two different mRNAs, E2F6 and E2F6b. The E2F6b mRNA contains an additional exon that is inserted by alternative splicing. This exon contains an in-frame stop-codon and an in-frame translation initiation codon. However, whether a protein is translated from the E2F6b mRNA has not yet been addressed. We now show that internal translation initiation gives rise to E2F6b, an amino-terminal truncated E2F6 protein. We also show that E2F6 and E2F6b mRNAs are ubiquitously expressed in primary mouse tissues. During the cell cycle, the highest expression of both forms is found at the G1 to S transition. The 5' untranslated regions of E2F6 and E2F6b are unusually long, and they contain several upstream AUG codons followed by short reading frames. Our results suggest that translation of E2F6b is initiated by internal ribosome entry. We propose that regulated translation initiation can produce distinct E2F6 isoforms under different physiological conditions.

• 
  Gaubatz, S., Lees, J.A., Lindeman, G.J., & Livingston, D.M.E2F4 is exported from the nucleus in a CRM1-dependent mannerMol Cell Biol, 21(4): 1384–1392.
  • [ Abstract ]
  • [ DOI ]
  E2F is a family of transcription factors required for normal cell cycle control and for cell cycle arrest in G1. E2F4 is the most abundant E2F protein in many cell types. In quiescent cells, it is localized to the nucleus, where it is bound to the retinoblastoma-related protein p130. During entry into the cell cycle, the protein disappears from the nucleus and appears in the cytoplasm. The mechanism by which this change occurs has, in the past, been unclear. We have found that E2F4 is actively exported from the nucleus and that leptomycin B, a specific inhibitor of nuclear export, inhibits this process. E2F4 export is mediated by two hydrophobic export sequences, mutations in either of which result in export failure. Individual export mutants of E2F4, but not a mutant with inactivation of both export signals, can be efficiently excluded from the nucleus by forced coexpression of the nuclear export receptor CRM1. Similarly, CRM1 overexpression can prevent cell cycle arrest induced by the cyclin kinase inhibitor p16(INK4a), an E2F4-dependent process. Taken together, these data suggest that nuclear export contributes to the regulation of E2F4 function, including its ability to regulate exit from G1 in association with a suitable pocket protein.

• 
  Gaubatz, S., Lindeman, G.J., Ishida, S., Jakoi, L., Nevins, J.R., Livingston, D.M., & Rempel, R.E.E2F4 and E2F5 play an essential role in pocket protein-mediated G1 controlMol Cell, 6(3): 729–735.
  • [ Abstract ]
  • [ DOI ]
  E2F transcription factors are major regulators of cell proliferation. The diversity of the E2F family suggests that individual members perform distinct functions in cell cycle control. E2F4 and E2F5 constitute a defined subset of the family. Until now, there has been little understanding of their individual biochemical and biological functions. Here, we report that simultaneous inactivation of E2F4 and E2F5 in mice results in neonatal lethality, suggesting that they perform overlapping functions during mouse development. Embryonic fibroblasts isolated from these mice proliferated normally and reentered from Go with normal kinetics compared to wild-type cells. However, they failed to arrest in G1 in response to p16INK4a. Thus, E2F4 and E2F5 are dispensable for cell cycle progression but necessary for pocket protein-mediated G1 arrest of cycling cells.

• 
  Elliott, K., Sakamuro, D., Basu, A., Du, W., Wunner, W., Staller, P., Gaubatz, S., Zhang, H., Prochownik, E., Eilers, M., & Prendergast, G.C.Bin1 functionally interacts with Myc and inhibits cell proliferation via multiple mechanismsOncogene, 18(24): 3564–3573.
  • [ Abstract ]
  • [ DOI ]
  The tumor suppressor Bin1 was identified through its interaction with the N-terminal region of Myc which harbors its transcriptional activation domain. Here we show that Bin1 and Myc physically and functionally associate in cells and that Bin1 inhibits cell proliferation through both Myc-dependent and Myc-independent mechanisms. Bin1 specifically inhibited transactivation by Myc as assayed from artificial promoters or from the Myc target genes ornithine decarboxylase (ODC) and alpha prothymosin (pT). Inhibition of ODC but not pT required the presence of the Myc binding domain (MBD) of Bin1 suggesting two mechanisms of action. Consistent with this possibility, a non-MBD region of Bin1 was sufficient to recruit a repression function to DNA that was unrelated to histone deacetylase. Regions outside the MBD required for growth inhibition were mapped in Ras cotransformation or HepG2 hepatoma cell growth assays. Bin1 required the N-terminal BAR domain to suppress focus formation by Myc whereas the C-terminal U1 and SH3 domains were required to inhibit adenovirus E1A or mutant p53, respectively. All three domains contributed to Bin1 suppression of tumor cell growth but BAR-C was most crucial. These findings supported functional interaction between Myc and Bin1 in cells and indicated that Bin1 could inhibit malignant cell growth through multiple mechanisms.

• 
  Lindeman, G.J., Dagnino, L., Gaubatz, S., Xu, Y., Bronson, R.T., Warren, H.B., & Livingston, D.M.A specific, nonproliferative role for E2F-5 in choroid plexus function revealed by gene targetingGenes Dev, 12(8): 1092–1098.
  • [ Abstract ]
  • [ DOI ]
  Homozygous E2F-5 knockout embryos and mice have been generated. Although embryonic development appeared normal, newborn mice developed nonobstructive hydrocephalus, suggesting excessive cerebrospinal fluid (CSF) production. Although the CSF-producing choroid plexus displayed normal cellular organization, it contained abundant electron-lucent epithelial cells, consistent with excessive CSF secretory activity. Moreover, E2F-5 CNS expression in normal animals was largely confined to the choroid plexus. Cell cycle kinetics were not perturbed in homozygous knockout embryo fibroblasts. Thus, E2F-5 is not essential for cell proliferation. Rather, it affects the secretory behavior of a differentiated neural tissue.
• 
  Gaubatz, S., Wood, J.G., & Livingston, D.M.Unusual proliferation arrest and transcriptional control properties of a newly discovered E2F family member, E2F-6Proc Natl Acad Sci USA, 95(16): 9190–9195.
  • [ Abstract ]
  • [ DOI ]
  E2F transcription factors play an important role in the regulation of cell cycle progression. We report here the cloning and characterization of an additional member of this family, E2F-6. E2F-6 lacks pocket protein binding and transactivation domains, and it is a potent transcriptional repressor that contains a modular repression domain at its carboxyl terminus. Overproduction of E2F-6 had no specific effect on cell cycle progression in asynchronously growing Saos2 and NIH 3T3 cells, but it inhibited entry into S phase of NIH 3T3 cells stimulated to exit G0. Taken together, these data suggest that E2F-6 can regulate a subset of E2F-dependent genes whose products are required for entry into the cell cycle but not for normal cell cycle progression.

• 
  Lindeman, G.J., Gaubatz, S., Livingston, D.M., & Ginsberg, D.The subcellular localization of E2F-4 is cell-cycle dependentProc Natl Acad Sci U S A, 94(10): 5095–5100.
  • [ Abstract ]
  • [ DOI ]
  The E2F family of transcription factors plays a crucial role in cell cycle progression. E2F activity is tightly regulated by a number of mechanisms, which include the timely synthesis and degradation of E2F, interaction with retinoblastoma protein family members ("pocket proteins"), association with DP heterodimeric partner proteins, and phosphorylation of the E2F/DP complex. Here we report that another mechanism, subcellular localization, is important for the regulation of E2F activity. Unlike E2F-1, -2, or -3, which are constitutively nuclear, ectopic E2F-4 and -5 were predominantly cytoplasmic. Cotransfection of expression vectors encoding p107, p130, or DP-2, but not DP-1, resulted in the nuclear localization of E2F-4 and -5. Moreover, the transcriptional activity of E2F-4 was markedly enhanced when it was invariably nuclear. Conversely, it was reduced when the protein was excluded from the nucleus, implying that E2F-4 transcription function depends upon its cytological location. In keeping with this, the nuclear/cytoplasmic ratios of endogenous E2F-4 changed as cells exited G0, with high ratios in G0 and early G1 and a progressive increase in cytoplasmic E2F-4 as cells approached S phase. Thus, the subcellular location of E2F-4 is regulated in a cell cycle-dependent manner, providing another potential mechanism for its functional regulation.

• 
  Desbarats, L., Gaubatz, S., & Eilers, M.Discrimination between different E-box-binding proteins at an endogenous target gene of c-mycGenes Dev, 10(4): 447–460.
  • [ Abstract ]
  • [ DOI ]
  c-myc plans a key role in regulating mammalian cell proliferation and apoptosis. The gene codes for a transcription factor, Myc, that belongs to the helix-loop-helix/leucine zipper (HLH/LZ) family of proteins. Myc heterodimerizes with a partner protein termed Max; the heterodimeric complex binds to CAC(G/A)TG (E-box) sequences and activates transcription from these sites. However, several other HLH/LZ proteins, including USF and TFE-3, bind to and trans-activate from the same element, yet have no documented effect on cell proliferation or apoptosis. Therefore, it is likely that mechanisms exist that discriminate between these proteins for activation of natural target genes of Myc. We now show that trans-activation from the E-box in the rat prothymosin-alpha intron enhancer is indeed specific for Myc, and identify both the distance from the start site of transcription and a second E-box element adjacent to that recognized by Myc as critical determinants of specificity. Surprisingly, transcription activation domains required for Myc to activate from this distal enhancer position differ from previously mapped domains and closely correlate with those domains essential for transformation. As observed in transformation assays, Myc and Max strongly synergize in activation from a distal enhancer position. Our data suggest that trans-activation from the prothymosin intron enhancer is a faithful reflection of the transforming properties of the Myc protein.

• 
  Gaubatz, S., Imhof, A., Dosch, R., Werner, O., Mitchell, P., Buettner, R., & Eilers, M.Transcriptional activation by Myc is under negative control by the transcription factor AP-2EMBO J, 14(7): 1508–1519.
  • [ Abstract ]
  The Myc protein binds to and transactivates the expression of genes via E-box elements containing a central CAC(G/A)TG sequence. The transcriptional activation function of Myc is required for its ability to induce cell cycle progression, cellular transformation and apoptosis. Here we show that transactivation by Myc is under negative control by the transcription factor AP-2. AP-2 inhibits transactivation by Myc via two distinct mechanisms. First, high affinity binding sites for AP-2 overlap Myc-response elements in two bona fide target genes of Myc, prothymosin-alpha and ornithine decarboxylase. On these sites, AP-2 competes for binding of either Myc/Max heterodimers or Max/Max homodimers. The second mechanism involves a specific interaction between C-terminal domains of AP-2 and the BR/HLH/LZ domain of Myc, but not Max or Mad. Binding of AP-2 to Myc does not preclude association of Myc with Max, but impairs DNA binding of the Myc/Max complex and inhibits transactivation by Myc even in the absence of an overlapping AP-2 binding site. Taken together, our data suggest that AP-2 acts as a negative regulator of transactivation by Myc.

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  Gaubatz, S., Meichle, A., & Eilers, M.An E-box element localized in the first intron mediates regulation of the prothymosin alpha gene by c-mycMol Cell Biol, 14(6): 3853–3862.
  • [ Abstract ]
  • [ DOI ]
  In RAT1A fibroblasts, expression of the prothymosin alpha gene is under the transcriptional control of the c-myc proto-oncogene. We have now cloned the rat gene encoding prothymosin alpha and show that the cloned gene is regulated by c-myc in vivo. We find that regulation by c-myc is mediated by sequences downstream of the transcriptional start site, whereas the promoter is constitutive and not regulated by c-myc. We have identified an enhancer element within the first intron that is sufficient to mediate a response to Myc and Max in transient transfection assays and to activation of estrogen receptor-Myc chimeras in vivo. We find that this element contains a consensus Myc-binding site (CACGTG). Disruption of this site abolishes the response to Myc and Max in both transient and stable assays. Mutants of either Myc or Max that are deficient for heterodimerization fail to regulate the prothymosin alpha gene, suggesting that a heterodimer between Myc and Max activates the prothymosin alpha gene. Our data define the prothymosin alpha gene as a bona fide target gene for c-myc.

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  K\"u}es, U., Richardson, W.V., Tymon, A.M., Mutasa, E.S., G\"o}ttgens, B., Gaubatz, S., Gregoriades, A., & Casselton, L.A.The combination of dissimilar alleles of the A alpha and A beta gene complexes, whose proteins contain homeo domain motifs, determines sexual development in the mushroom Coprinus cinereusGenes Dev, 6(4): 568–577.
  • [ Abstract ]
  • [ DOI ]
  The A mating-type factor is one of two gene complexes that allows mating cells of the mushroom Coprinus cinereus to recognize self from nonself and to regulate a pathway of sexual development that leads to meiosis and sporulation. We have identified seven A genes separated into two subcomplexes corresponding to the classical A alpha and A beta loci. Four genes, one alpha and three beta, all coding for proteins with a homeo domain-related motif, determine A-factor specificity; their allelic forms are so different in sequence that they do not cross-hybridize. It requires only one of these four genes to be heteroallelic in a cell to trigger A-regulated sexual development, and it is the different combinations of their alleles that generate the multiple A factors found in nature. The other three genes cause no change in cell morphology and may regulate the activity of the four specificity genes.