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Alternative splicing removes an Ets interaction domain from Lozenge during Drosophila eye development

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Abstract

Physical and functional characteristics of the RUNX family of transcription factors are conserved between vertebrates and the Drosophila protein Lozenge. The runt-homology domain responsible for DNA binding and also the C-terminus are both nearly identical between the two proteins. The mammalian and fly proteins heterodimerize with a non-DNA binding partner protein to form a core binding factor essential for gene regulation during cell differentiation. The mammalian protein RUNX1 (AML1/PEBP2αB) interacts with the transcription factor Ets-1 to increase DNA binding and transactivation potential. Alternative splicing of the mammalian RUNX1 removes a domain required for this cooperative transactivation. In this work we determine the structure of the lozenge transcription unit and map 21 mutations. We show that the lozenge transcript is alternatively spliced during eye development to remove an Ets interaction domain. Emphasis is placed on Pointed the Drosophila homolog of the vertebrate Ets-1 protein; both Lozenge and Pointed proteins are needed for the activation of prospero expression. We use site-directed mutagenesis and yeast two-hybrid analysis to show that conserved amino acids within the alternate Lozenge exon are important for interaction with Pointed. Furthermore, the ectopic expression of Lozenge is sufficient to rescue Prospero expression in the presence of the Pointed competitor, YanACT. We show that both lozenge isoforms are expressed during eye development and that the relative ratio of the transcripts for the two isoforms is sensitive to changes in Ras activity. We suggest that during eye development, Lozenge isoforms function in divergent roles, either interacting with Pointed on downstream targets or by functioning independently to establish distinct cell fates.

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Acknowledgements

We thank Jana Van Patton and Claudia Almaguer for help with the Yeast Two-Hybrid system. We thank Ranga Rao and Joe Lepo for the use of their lab at UWF. We thank A. Javier Lopez, Richard Carthew and Andreas Nocker for discussion and advice. We thank Raghuram Selvaraju for critical review of the manuscript. We thank Sam Saba and Paul Keller for their help in optimizing PCR conditions, and James M. Burnette for assisting with MacTargSearch analysis. We thank Utpal Banerjee for flies and for the lozenge 3.5 clone, Gerald Rubin for the gift of flies, Richard Carthew for the PntP2 clone, and Chris Doe for the gift of Prospero antibody. Parts of this work were performed at Carnegie Mellon University and the University of West Florida. This work was supported in part by Duquesne University of the Holy Ghost and the following grants to J.A.P.: SURG/HHMI Awards to undergraduate researchers at Carnegie Mellon University; NIH grant EY09093; NSF/STC grant BIR-8920118; March of Dimes Birth Defects Foundation FY93-1010; Duquesne University Faculty Development Awards; Samuel and Emma Winters Foundation; and international collaborative support from the NSF INT-9605205 and the University of Melbourne.

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  1. Kristina Jackson Behan

    Present address: University of West Florida, Pensacola, FL, 32514, USA

  2. Fiona Cunningham

    Present address: Royal Children’s Hospital Parkville, Murdoch Children’s Research Institute, Victoria, 3052, Australia

  3. Charles D. Nichols

    Present address: Louisiana State University, New Orleans, LA, 70112, USA

Authors and Affiliations

  1. Department of Genetics, University of Melbourne, Parkville, Victoria, Australia

    Jason Fair, Michael Bogwitz, Trent Perry, Vladimir Grubor, Fiona Cunningham & Philip Batterham

  2. Department of Biological Sciences, Duquesne University, Pittsburgh, PA, 15282, USA

    Shalini Singh, Tara L. Cheung & John Archie Pollock

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  1. Kristina Jackson Behan
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Correspondence to John Archie Pollock.

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Communicated by C. Desplan

K.J. Behan and J. Fair contributed equally to the data presented

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Jackson Behan, K., Fair, J., Singh, S. et al. Alternative splicing removes an Ets interaction domain from Lozenge during Drosophila eye development. Dev Genes Evol 215, 423–435 (2005). https://doi.org/10.1007/s00427-005-0490-0

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