Transcription factor IIIA gene expression in Xenopus oocytes utilizes a ...

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Apr 26, 1989 - ROBERT K. HALL AND WILLIAM L. TAYLOR*. Department of Molecular ...... Weintraub, H., P. F. Cheng, and K. Conrad. 1986. Expression of.
Vol. 9, No. 11

MOLECULAR AND CELLULAR BIOLOGY, Nov. 1989, p. 5003-5011 0270-7306/89/115003-09$02.00/0 Copyright C 1989, American Society for Microbiology

Transcription Factor IIIA Gene Expression in Xenopus Oocytes Utilizes a Transcription Factor Similar to the Major Late Transcription Factor ROBERT K. HALL AND WILLIAM L. TAYLOR* Department of Molecular Physiology and Biophysics, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232 Received 26 April 1989/Accepted 20 July 1989

Xenopus transcription factor MA (TFIIIA) gene expression is stringently regulated during development. The steady-state level of TFIIIA mRNA in a somatic cell is approximately 106 times less than in an immature oocyte. We have undertaken studies designed to identify differences in how the TFiIA gene is transcribed in oocytes and somatic cells. In this regard, we have localized an upstream transcriptional control element in the TFIIIA promoter that stimulates transcription from the TFIIIA promoter approximately threefold in microinjected oocytes. The upstream element, in cis, does not stimulate transcription from the TFHA promoter in somatic cells. Thus, the element appears to be oocyte specific in the context of the TFIA promoter. However, both oocytes and somatic cells contain a protein (or a related protein) that binds the upstream element. We have termed this protein from oocytes the TFIA distal element factor. The sequence of the upstream element is similar to the sequence of the upstream element found in the adenovirus major late promoter that is a binding site for the majior late transcription factor. By gel shift analysis, chemical footprinting, methylation intereference, and point mutation analysis, we demonstrate that the TFIA distal element factor and major late transcription factor have similar DNA-binding properties.

transcriptional control elements that are required for expression of the TFIIIA gene. The 425 base pairs (bp) upstream of the TFIIIA mRNA start site appear to be sufficient for expression of the TFIIIA gene in both oocytes and somatic cells (25). Within this 425-bp region are sequences typical of genes transcribed by RNA polymerase II, such as a TATA box centered at -30 and a CAAT sequence motif centered at -90 relative to the mRNA start site (25, 45). Matsumoto and Korn (25) have recently identified, by linker substitution analysis, regions between bases -283 and -238 and bases -167 and -122 that appear to be important for efficient transcription of the TFIIIA gene in oocytes. We have also found the distal region (from -325 to -235) to be important for TFIIIA gene expression in oocytes and have identified a protein-binding site within this region. We demonstrate in this report that a protein (referred to as the TFIIIA distal element factor [TDEF]) binds to the distal element and that this binding is apparently required for stimulation of TFIIIA gene expression in oocytes. Although Xenopus somatic cells also contain a protein that binds to the same region, the binding site does not appear to play a significant role in the transcription of the TFIIIA gene in somatic cells. Interestingly, the TDEF-binding site contains a 6-bp palindromic sequence that is also found in the binding site of the major late transcription factor, MLTF/USF (5, 35). Using mobility shift assays and in vitro competition experiments with defined double-stranded oligonucleotides, we demonstrate that TDEF can bind to the MLTF-binding site in the adenovirus type 2 (Ad2) major late promoter (MLP) and that MLTF (from HeLa nuclear extracts) can bind to the TDEF-binding site. TDEF and MLTF bind to each binding site with the same affinity. The footprints as determined by chemical methods and methylation interference patterns of the two proteins on their respective binding sites are also very similar. In addition, point mutations that abolish binding of TDEF to its binding site also abolish

TFIIIA is a transcription factor required for RNA polymerase III-mediated transcription of SS rRNA genes (12, 33). Its concentration in the cell is critical for the developmental regulation of oocyte-type and somatic-type 5S rRNA genes (1, 4, 47). The Xenopus oocyte produces and stores TFIIIA such that it accounts for about 10% of the total cytoplasmic protein (31, 42). This high level of TFIIIA is accumulated in oocytes to ensure that enough 5S rRNA is transcribed for the synthesis of ribosomes, which occurs at a rate approximately 1,000 times greater in oocytes than in somatic cells (24). In Xenopus laevis, TFIIIA gene expression decreases dramatically during early embryogenesis. Measurements of the steady-state levels of TFIIIA mRNA range from 2 x 107 (43) to 5 x 106 (13) in a stage 1 (immature) oocyte and from 6 in a cultured somatic cell (43) to