Analysis of core promoter sequences located downstream from the TATA element in the hsp70 promoter from Drosophila melanogaster.

TFIID recognizes multiple sequence elements in the hsp70 promoter of Drosophila. Here, we investigate the function of sequences downstream from the TATA element. A mutation in the initiator was identified that caused an eightfold reduction in binding of TFIID and a fourfold reduction in transcription in vitro. Another mutation in the +24 to +29 region was somewhat less inhibitory, but a mutation in the +14 to +19 region had essentially no effect. The normal promoter and the mutants in the initiator and the +24 to +29 region were transformed into flies by P element-mediated transformation. The initiator mutation reduced expression an average of twofold in adult flies, whereas the mutation in the +24 to +29 region had essentially no effect. In contrast, a promoter combining the two mutations was expressed an average of sixfold less than the wild type. The results suggest that the initiator and the +24 to +29 region could serve overlapping functions in vivo. Protein-DNA cross-linking was used to identify which subunits of TFIID contact the +24 to +29 region and the initiator. No specific subunits were found to cross-link to the +24 to +29 region. In contrast, the initiator cross-linked exclusively to dTAF230. Remarkably, dTAF230 cross-links approximately 10 times more efficiently to the nontranscribed strand than to the transcribed strand at the initiator.

DNA-PK, the DNA-activated protein kinase, is differentially expressed in normal and malignant human tissues.

DNA-PK is a nuclear, serine/threonine protein kinase required for repairing DNA double-strand breaks and for V(D)J recombination. To determine the distribution of DNA-PK in human tissues, we assayed paraffin-embedded sections of normal and cancerous tissues for DNA-PKcs and Ku80 by immunohistochemistry. We also assayed for Brca2, a human tumor suppressor gene that is implicated in the repair of DNA strand-breaks. Brca2 was strongly expressed in epithelial cells of the breast, endometrium, and thymus, in tingible body macrophages of follicular germinal centers of lymphoid tissue, and in reticuloendothelial cells in the spleen. DNA-PKcs and Ku80 expression was usually parallel, but both were expressed in a highly cell- and tissue-specific manner. The highest levels were observed in spermatogenic cells (but not in spermatozoa), and in neurons and glial cells of the central and autonomic nervous system. Neither protein was consistently expressed in liver nor in resting mammary epithelium, but lactating breast epithelium was strongly positive for DNA-PKcs and Ku80. In contrast to established human cell cultures, expression between cells in the same tissue was highly selective in the epidermis, exocrine pancreas, renal glomeruli, the red pulp of the spleen, and within cellular compartments of tonsils, lymph nodes, and thymus. Most cancerous tissues were consistently positive for DNA-PKcs and Ku80, except invasive carcinoma of the breast. DNA-PKcs, Ku80, and Ku70 mRNAs were expressed in all normal tissues with relatively little variation in levels. Our results suggest that the apparent absence of DNA-PKcs and Ku80 from some cells or tissues is a consequence of post-transcriptional mechanisms that regulate protein levels.

Protein/DNA crosslinking of a TFIID complex reveals novel interactions downstream of the transcription start.

A protein--DNA complex containing TFIID has been analyzed by crosslinking. The TBP subunit of TFIID crosslinked to the TATA element but not to any of the regions further downstream which were tested. A 150 kd polypeptide, which corresponds in size to one of the TBP-associated factors (TAFs), crosslinked to a region between +10 and +15 and a second region between +35 and +47. Another polypeptide of greater than 205 kd (also a potential TAF) crosslinked preferentially to the region between +35 and +42. The +10 to +15 region has been recently implicated in hsp70 promoter recognition by TFIID, and the most downstream contacts overlap with the region where RNA polymerase II pauses on the hsp70 promoter in noninduced cells. Crosslinking revealed that as the salt concentration was increased, the TBP interaction was largely unaffected whereas the protein/DNA interactions downstream of the TATA element were disrupted. We propose that during the formation of a transcription complex, TATA-dependent interactions could be disrupted in the vicinity of the start site and the region immediately downstream. A protein contact downstream of +35 might function in pausing polymerase.