The protein encoded by the retinoblastoma susceptibility gene (Rb) functions as a tumour suppressor and negative growth regulator. As actively growing cells require the ongoing synthesis of ribosomal RNA, we considered that Rb might interact with the ribosomal DNA transcription apparatus. Here we report that (1) there is an accumulation of Rb protein in the nucleoli of differentiated U937 cells which correlates with inhibition of rDNA transcription; (2) addition of Rb to an in vitro transcription system inhibits transcription by RNA polymerase I; (3) this inhibition requires a functional Rb pocket; and (4) Rb specifically inhibits the activity of the RNA polymerase I transcription factor UBF (upstream binding factor) in vitro. This last observation was confirmed by affinity chromatography and immunoprecipitation, which demonstrated an interaction between Rb and UBF. These results indicate that there is an additional mechanism by which Rb suppresses cell growth, namely that Rb directly represses transcription of the rRNA genes.

Gene targeting studies have shown that T cell receptor (TCR)-β gene expression and recombination are inhibited after deletion of an enhancer (Eβ) located at the 3′ end of the ∼500-kb TCR-β locus. Using knockout mouse models, we have measured, at different regions throughout the TCR-β locus, the effects of Eβ deletion on molecular parameters believed to reflect epigenetic changes associated with the control of gene activation, including restriction endonuclease access to chromosomal DNA, germline transcription, DNA methylation, and histone H3 acetylation. Our results demonstrate that, in early developing thymocytes, Eβ contributes to major chromatin remodeling directed to an ∼25-kb upstream domain comprised of the Dβ-Jβ locus regions. Accordingly, treatment of Eβ-deleted thymocytes with the histone deacetylase inhibitor trichostatin A relieved the block in TCR-β gene expression and promoted recombination within the Dβ-Jβ loci. Unexpectedly, however, epigenetic processes at distal Vβ genes on the 5′ side of the locus and at the 3′ proximal Vβ14 gene appear to be less dependent on Eβ, suggesting that Eβ activity is confined to a discrete region of the TCR-β locus. These findings have implications with respect to the developmental control of TCR-β gene recombination, and the process of allelic exclusion at this locus.

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Deletion of the TCR␤ transcriptional enhancer (E␤) results in nearly complete inhibition of V(D)Jrecombination at the TCR␤ locus and a block in ␣␤ T cell development. This result, along with previous work from many laboratories, has led to the hypothesis that transcriptional enhancers affect V(D)J recombination by regulating the accessibility of the locus to the recombinase. Here we test this hypothesis by performing a detailed analysis of the recombination defect in E␤-deleted (E␤ −/− ) mice using assays that detect various reaction intermediates and products. We found double-strand DNA breaks at recombination signal sequences flanking D␤ and J␤ gene segments in E␤ −/− thymuses at about one-third to one-thirtieth the level found in thymuses with an unaltered TCR␤ locus. These sites are also subject to in vitro cleavage by the V(D)J recombinase in both E␤ −/− and E␤ +/+ thymocyte nuclei. However, the corresponding D␤-to-J␤ coding joints are further reduced (by 100-to 300-fold) in E␤ −/− thymuses. Formation of extrachromosomal D␤-to-J␤ signal joints appears to be intermediately affected and nonstandard D␤-to-D␤ joining occurs at the E␤-deleted alleles. These data indicate that, unexpectedly, loss of accessibility alone cannot explain the loss of TCR␤ recombination in the absence of the E␤ element and suggest an additional function for E␤ in the process of DNA repair at specific TCR␤ sites during the late phase of the recombination reaction.[Key Words: V(D)J recombination; TCR␤ enhancer; RSS accessibility; double-strand break repair] Received January 27, 1998; revised version accepted June 3, 1998. V(D)J recombination, the only site-specific DNA rearrangement process known to occur in vertebrates, is required for the assembly of immunoglobulin and T cell receptor (TCR) genes and normal lymphocyte differentiation (for review, see Alt et al. 1992;Schatz et al. 1992;Lewis 1994;Bogue and Roth 1996). This process utilizes an enzyme complex, called the V(D)J recombinase, which targets conserved recombination signal sequences (RSSs) associated with all rearranging immunoglobulin and TCR V, D, and J gene segments. RSSs consist of conserved 7-and 9-nucleotide sequences (the heptamer and nonamer) separated by a 12-or 23-nucleotide spacer of nonconserved sequence. In vitro studies have demonstrated that two lymphoid-specific components of the recombinase, RAG1 and RAG2, are sufficient for recognition and double-strand cleavage of pairs of RSSs (for review, see Gellert 1997; Schatz 1997). Subsequent processing and joining of the cleaved intermediates require the additional activities of several factors involved in general DNA double-stranded break (DSB) repair (for review, see Weaver 1995;Lieber et al. 1997). In vivo, V(D)J recombination at endogenous immunoglobulin and TCR loci is tightly regulated with respect to cell lineage, stage of cell differentiation and, at particular gene segments and/or loci, allele usage (for review, see Willerford et al. 1996;Papavasiliou et al. 1997). Given that a common recombinase mediates all...