Regulation of RB Transcription In Vivo by RB Family Members

Abstract

In cancer cells, the retinoblastoma tumor suppressor RB is directly inactivated by mutation in the RB gene or functionally inhibited by abnormal activation of cyclin-dependent kinase activity. While variations in RB levels may also provide an important means of controlling RB function in both normal and cancer cells, little is known about the mechanisms regulating RB transcription. Here we show that members of the RB and E2F families bind directly to the RB promoter. To investigate how the RB/E2F pathway may regulate Rb transcription, we generated reporter mice carrying an eGFP transgene inserted into a bacterial artificial chromosome containing most of the Rb gene. Expression of eGFP largely parallels that of Rb in transgenic embryos and adult mice. Using these reporter mice and mutant alleles for Rb, p107, and p130, we found that RB family members modulate Rb transcription in specific cell populations in vivo and in culture. Interestingly, while Rb is a target of the RB/E2F pathway in mouse and human cells, Rb expression does not strictly correlate with the cell cycle status of these cells. These experiments identify novel regulatory feedback mechanisms within the RB pathway in mammalian cells.The RB tumor suppressor gene was first identified through its direct mutation or deletion in human retinoblastoma. The RB protein is thought to function largely as a transcriptional cofactor that can repress or potentiate the functions of numerous transcription factors, affecting the expression of a broad number of target genes. Since its initial discovery, RB function has been demonstrated to be inactivated in virtually all human cancers through a variety of mechanisms. In particular, in addition to direct mutation events in the RB gene, the RB protein is often functionally inactivated by phosphorylation in tumor cells with constitutive activation of cyclin/Cdk complexes (44,55). RB inactivation through reduced transcription may also participate in the development of cancer (4, 41); surprisingly, however, little is known about the mechanisms regulating RB transcription in normal and tumor cells.Interestingly, the RB promoter contains a conserved binding site for the E2F transcription factors, some of which are direct partners of RB and key downstream mediators of RB (9). Increasing evidence suggests that this site may contribute to the regulation of RB transcription. For instance, overexpression of E2F1 can activate RB transcription, which may contribute to the variations in RB mRNA levels during cell cycle progression that are observed in some contexts (30, 43) but not in others (5,13,20). In addition, methylation of the E2F site in the RB promoter is sufficient to recruit repressor complexes to inhibit RB transcription (15). Accordingly, reporter assays show that mutation of the E2F site in the RB promoter leads to an absence of RB repression (33). Furthermore, mutation of the E2F (and Sp1) sites in the mouse Rb promoter in transgenic mice has revealed the importance of these sites for Rb expression in vivo (1).Togeth...

The Retinoblastoma Tumor Suppressor Modulates DNA Repair and Radioresponsiveness

“…Tumor volumes were measured weekly with calipers, serum PSA levels were determined, and PSA doubling times were calculated. In shRB xenografts, maintenance of RB knockdown in vivo was verified by quantitative PCR (qRT-PCR) analysis of the human RB1 (shRB) transcript was performed as described in (8, 18). RB transcript levels as measured by qRT-PCR ranged from 10% to 90%.…”

Overlapping and distinct pRb pathways in the mammalian auditory and vestibular organs

“…28,29 It has been shown that, in the absence of pRb, negative cell growth genes, including other Rb family members, may be upregulated to compensate for pRb function. [30][31][32] Correlating with this, microarray analysis showed upregulation of Rbl1 (p107), Cdkn1a (p21 Cip1 ) and Cdkn2d (p19 Ink4d ) in the pRb -/-inner ear.…”

Characterization of the cell of origin for small cell lung cancer

“…Tamoxifen (Tam) resuspended in corn oil was injected intraperitoneally daily for five consecutive days at a dose of 2 mg per mouse. 49 All mouse brief, a CreER cDNA 50 was cloned downstream of a 3.7 kb promoter fragment 24 and transferred into an adenoviral vector backbone (pAd/PL-DEST TM Gateway ® Vector Kit, Invitrogen) before amplification. Viral infections were performed as described in reference 51.…”