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CRTF is a novel transcription factor that regulates multiple stages of Dictyostelium development

Xiuqian Mu^*, Seth A. Spanos, Joseph Shiloach and Alan Kimmel

Laboratory of Cellular and Developmental Biology, NIDDK (MMDS; Bldg 50/3351), National Institutes of Health, Bethesda, MD 20892-8028, USA
^* Present address: Department of Biochemistry and Molecular Biology, 1515 Holcombe, M. D. Anderson Cancer Center, University of Texas, Houston, TX 77030, USA
Present address: School of Medicine, 50 North Medical Drive, University of Utah, Salt Lake City, UT 84132, USA

Author for correspondence (e-mail: ark1{at}helix.nih.gov)

Accepted April 20, 2001

During aggregation, Dictyostelium establish nanomolar oscillation waves of extracellular cAMP, but as development progresses, cells become responsive to higher, non-fluctuating concentrations of cAMP. The regulation of the promoter responsible for expression of cAMP receptor subtype 1, CAR1, during aggregation reflects these signaling variations. Transcription of CAR1 from the early, aggregation promoter is activated by cAMP pulsing, but is repressed by continuous exposure to micromolar concentrations of cAMP. Deletion and mutation analyses of this promoter had defined an element essential for cAMP-regulated expression, and mobility shift assay, DNA crosslinking and DNase I footprinting experiments had identified a nuclear protein (CRTF) with zinc-dependent sequence binding specificity. In our study, CRTF was purified to homogeneity, peptides were sequenced and full-length cDNAs were obtained. The deduced CRTF protein is 100 kDa with a C-terminal, zinc finger-like motif required for DNA binding; CRTF purified from cells, however, represents only a 40 kDa C-terminal fragment that retains DNA-binding activity.

As might have been predicted if CRTF were essential for the regulation of CAR1, crtf-null strains fail to develop under standard conditions or to exhibit induced expression of CAR1 or other cAMP-regulated genes. Furthermore, crtf-nulls also fail to sporulate, even under conditions that bypass the dependence on early cAMP signaling pathways. In addition, early developmental events of crtf-null strains could be rescued with exogenous cAMP treatment, constitutive expression of CAR1 or co-development with wild-type cells; however, these treatments were insufficient to promote sporulation. This suggests a cell-autonomous role for CRTF during late development that is separate from its capacity to control CAR1 expression. Finally, ablation of CRTF promotes a precocious induction of certain cAMP-dependent gene expression pathways. We suggest that CRTF may function to help insulate distinct pathways from simultaneous and universal activation by cAMP. CRTF, thus, exhibits multiple complex and independent regulatory functions during Dictyostelium development.

Key words: Transcription factor, Development, Gene regulation, Dictyostelium

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