Samson T. Jacob

Control of gene expression by lipophilic hormones.

Regulation of gene expression in response to steroids, thyroid hormone, and retinoids is mediated by an impressive array of intracellular receptors. Sequence analysis showed that the hormone receptors comprise a large superfamily of ligand‐responsive transcription factors. Upon binding to hormones, the receptors interact with specific hormone response elements located in the promoters of numerous genes. Promoter‐bound receptors communicate with distinct receptors and/or additional members of the transcriptional machinery, frequently evoking protein‐protein interactions. Ultimately, this results in the induction of complex gene systems that control hormone‐induced processes such as differentiation, cell growth, and homeostasis. In addition to the genes transcribed by RNA polymerase II, the lipophilic hormones, particularly glucocorticoids, can also modulate RNA polymerase I‐directed transcription of the ribosomal gene. For both transcription systems, activation and repression of genes in response to hormones have been reported. Finally, the involvement of hormone receptors in tumorigenesis has been discussed. It is likely that receptor studies will have major implications in the diagnosis and therapy of diseases such as leukemia.— Reichel, R. R., Jacob, S. T. Control of gene expression by lipophilic hormones. FASEB J. 7: 42 7‐436; 1993.

Characterization of the 130-bp repeat enhancer element of the rat ribosomal gene: functional interaction with transcription factor E1BF

The 130-bp repetitive element (RE) of the rat rDNA (ribosomal RNA-encoding gene) intergenic spacer stimulated the synthesis of rRNA four- to sixfold, in comparison with that of the promoter alone, both in vivo and in vitro, when ligated to the rat rDNA promoter. The addition of increasing amounts of highly purified E1BF (enhancer-1 binding factor), which binds to the rat rDNA promoter and an upstream nonrepetitive enhancer element [Zhang and Jacob, Mol. Cell. Biol. 10 (1990) 5177-5186], to an in vitro transcription system resulted in enhancement of rDNA transcription from the recombinant plasmids containing the promoter or promoter-RE. However, E1BF-mediated stimulation of transcription under the influence of the RE continued at higher concentrations of E1BF than did the control transcription from the promoter alone. The binding affinity of E1BF for the RE was comparable to its affinity for the nonrepetitive far upstream enhancer element previously characterized in our laboratory. The sequences protected by E1BF in the RE differed from those protected by UBF (upstream control element-binding factor), a well characterized pol I transcription factor. These data suggest that E1BF belongs to a class of transcription factors which interact with the promoter and spacer cis-acting RE to modulate rDNA transcription.

Multiple functional enhancer motifs of rat ribosomal gene

Previous studies from this laboratory have characterized a 174 bp enhancer element which is located 2 kb upstream of the initiation site. Half of the enhancer action is controlled by a 37 bp element at the 3’ end of the 174 bp region. We now report that a 43 bp adjacent domain which is located upstream of the 37 bp element constitutes an additional motif of the rDNA enhancer. When the plasmid consisting of the 43 bp DNA upstream of the rDNA core promoter was transcribed in a fractionated rat tumor cell extract (fraction DE-B), transcription of rDNA was augmented 4 fold. Electrophoretic mobility shift and DNAase I footprinting analyses showed that the purified 37 bp enhancer (E1-binding protein, (E1BF) not only interacted with the enhancer motif E1 but also interacted with the neighbouring 43 bp enhancer domain E2. The specificity of the binding was demonstrated by competition with unlabeled 37 bp and 43 bp fragment and lack of competition with nonspecific DNAs in the mobility shift assay. These studies have shown that a single pol I transcription factor can bind to multiple enhancer domains with no significant sequence homologies and such multiple interactions may result in maximal transcription of ribosomal gene from the core promoter.

Heat shock inhibits pre‐rRNA processing at the primary site in vitro and alters the activity of some rRNA binding proteins

The effect of heat shock on pre‐rRNA processing at the primary site within external transcribed spacer region 1 (ETS1) was studied in S‐100 extract derived from mouse lymphosarcoma cells. In vivo labeling with [32P]orthophosphate showed that the synthesis of the rRNA precursor and its processing to 28S and 18S rRNAs were inhibited significantly due to heat shock. The processing activity was reduced by 50% at 1 h and was completely blocked following 2‐h exposure of cells at 42°C. Mixing S‐100 extracts from the control and heat‐treated cells did not affect the processing activity in the control extract, which proves the absence of a nuclease or other inhibitor(s) of processing in the extract from the heat‐shocked cells. Heat shock did not affect interaction between pre‐rRNA and U3 snoRNA, a prerequisite for the processing at the primary site, but significantly altered RNA‐protein interaction. Three polypeptides of 200, 110, 52 kDa that specifically cross‐link to pre‐rRNA spanning the primary processing site were inactivated after heat shock. Hyperthermia did not alter 3′ end processing of SV40L pre‐mRNA.