Abstract
In human, germ line mutations in the tumor suppressor retinoblastoma (Rb) predispose individuals to retinoblastoma, whereas somatic inactivation of Rb contributes to the progression of a large spectrum of cancers. In mice, Rb is highly expressed in restricted cell lineages including the neurogenic, myogenic, and hematopoietic systems, and disruption of the gene leads to specific embryonic defects in these tissues. The symmetry between Rb expression and the defects in mutant mice suggest that transcriptional control of Rb during embryogenesis is pivotal for normal development. We have initiated studies to dissect the mechanisms of transcriptional regulation of Rb during development by promoter lacZ transgenic analysis. DNA sequences up to 6 kilobase pairs upstream of the mouse Rb promoter, isolated from two different genomic libraries, directed transgene expression exclusively to the developing nervous system, excluding skeletal muscles and liver. Expression of the transgene in the central and peripheral nervous systems, including the retina, recapitulated the expression of endogenous Rb during embryogenesis. A promoter region spanning approximately 250 base pairs upstream of the transcriptional starting site was sufficient to confer expression in the central and peripheral nervous systems. To determine whether this expression pattern was conserved, we isolated the human Rb 5' genomic region and generated transgenic mice expressing lacZ under control of a 1.6-kilobase pair human Rb promoter. The human Rb promoter lacZ mice also expressed the transgene primarily in the nervous system in several independent lines. Thus, transgene expression directed by both the human and mouse Rb promoters is restricted to a subset of tissues in which Rb is normally expressed during embryogenesis. Our findings demonstrate that regulatory elements directing Rb expression to the nervous system are delineated within a well defined core promoter and are regionally separated from elements, yet to be identified, that are required for expression of Rb in the developing hematopoietic and skeletal muscle systems.
Highlights
The tumor suppressor Rb1 was originally identified as a gene that predisposes individuals to retinoblastoma in infants and osteosarcoma and other malignancies in later stages of life [1]
Our findings demonstrate that regulatory elements directing Rb expression to the nervous system are delineated within a well defined core promoter and are regionally separated from elements, yet to be identified, that are required for expression of Rb in the developing hematopoietic and skeletal muscle systems
A defect in terminal myogenesis was observed in Rb mutant embryos partially rescued to birth by a Rb minigene composed of the mouse Rb promoter and Rb cDNA [7, 18]
Summary
Retinoblastoma; X-gal, 5-bromo-4chloro-3-indolyl- -galactopyranoside; lacZ, -galactosidase; E, embryonic day; kb, kilobase pair(s); bp, base pair(s); 3Ј-UTR, 3Ј-untranslated region; 5Ј-CR, 5Ј conserved region; CP, core promoter; HS, hypersensitive site. Transgene expression under control of the human Rb promoter was detected primarily in the nervous system in most transgenic lines. These findings delineate the promoter region required for expression of Rb during neurogenesis and indicate that different mechanisms, yet to be defined, govern Rb expression during hematopoiesis and skeletal myogenesis
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