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Molecular and Cellular Biology, June 2006, p. 4448-4461, Vol. 26, No. 12
0270-7306/06/$08.00+0     doi:10.1128/MCB.02101-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Visualizing Dynamic E2F-Mediated Repression In Vivo{dagger}

Monica Agromayor,{ddagger} Elzbieta Wloga, Benedetta Naglieri, John Abrashkin, Kapil Verma, and Lili Yamasaki*

Department of Biological Sciences, Columbia University, New York, New York 10027

Received 28 October 2005/ Returned for modification 30 November 2005/ Accepted 12 March 2006

Although many E2F target genes have been identified recently, very little is known about how any single E2F site controls the expression of an E2F target gene in vivo. To test the requirement for a single E2F site in vivo and to learn how E2F-mediated repression is regulated during development and tumorigenesis, we have constructed a novel series of wild-type and mutant Rb promoter-LacZ transgenic reporter lines that allow us to visualize the activity of a crucial E2F target in vivo, the retinoblastoma tumor suppressor gene (Rb). Two mutant Rb promoter-LacZ constructs were used to evaluate the importance of a single E2F site or a nearby activator (Sp1/Ets) site that is found mutated in low-penetrance retinoblastomas. The activity of the wild-type Rb promoter is dynamic, varying spatially and temporally within the developing nervous system. While loss of the activator site silences the Rb promoter, loss of the E2F site stimulates its activity in the neocortex, retina, and trigeminal ganglion. Surprisingly, E2F-mediated repression of Rb does not act globally or in a static manner but, instead, is a highly dynamic process in vivo. Using neocortical extracts, we detected GA-binding protein {alpha} (GABP{alpha}, an Ets family member) bound to the activator site and both E2F1 and E2F4 bound to the repressor site of the Rb promoter in vitro. Additionally, we detected binding of both E2F1 and E2F4 to the Rb promoter in vivo using chromatin immunoprecipitation analysis on embryonic day 13.5 brain. Unexpectedly, we detect no evidence for Rb promoter autoregulation in neuroendocrine tumors from Rb+/–; RbP-LacZ mice that undergo loss of heterozygosity at the Rb locus, in contrast to the situation in human retinoblastomas where high RB mRNA levels are found. In summary, this study provides the first demonstration that loss of an E2F site is critical for target gene repression in vivo and underscores the complexity of the Rb and E2F family network in vivo.

* Corresponding author. Mailing address: Department of Biological Sciences, Columbia University, 1212 Amsterdam Avenue, 1102 Fairchild Building, Mail Code 2428, New York, NY 10027. Phone: (212) 8544384. Fax: (212) 854-5662. E-mail: ly63{at}columbia.edu.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

{ddagger} Present address: Department of Infectious Diseases, King's College, London SE1 9RT, United Kingdom.

Molecular and Cellular Biology, June 2006, p. 4448-4461, Vol. 26, No. 12
0270-7306/06/$08.00+0     doi:10.1128/MCB.02101-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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