Sequences and Factors Required for the F9 Embryonal Carcinoma Stem Cell E1a-Like Activity

Abstract

F9 embryonal carcinoma (EC) stem cells contain an E1a-like activity that is absent from differentiated derivatives. We have previously characterized proteins present in F9 EC cell extracts that bind to the E1a-dependent E2A promoter and have shown that two of them, TF68 and DRTF1, are required for efficient transcription in vitro (N. B. La Thangue, B. Thimmapaya, and P. W. J. Rigby, Nucleic Acids Res. 18:2929-2938, 1990). We now show that the E1a-like activity is detectable in transient transfection assays. Deletion mutations show that a distal sequence element, which includes the ATF/CREB consensus, is required for expression in both cell types, although it does not mediate the down-regulation of promoter activity that accompanies differentiation. A series of point mutations generated by in vitro mutagenesis confirm this and show that sequences around -60 are necessary for efficient expression in stem cells but not in differentiated derivatives. These sequences bind DRTF1, the activity of which is strongly down-regulated during differentiation. Surprisingly, mutations in a previously uncharacterized region of the promoter restore activity to a promoter carrying the -60 mutation and lead to the formation of a new DNA-protein complex.

UTF1, a novel transcriptional coactivator expressed in pluripotent embryonic stem cells and extra-embryonic cells

“…This E1A‐LA had originally been postulated to explain the specific activation of the adenovirus early E2A gene in the absence of viral E1A in EC cells (Imperiale et al ., 1984). This increase in transcriptional activation occurs via the E2F–DP‐1 complex(es) which interact at the E2F sites of the E2A promoter (Bandara and La Thangue, 1991; Murray et al ., 1991). In contrast to the E1A‐LA, UTF1 does not exert its stimulatory effect through this complex but rather functions through the upstream ATF‐2 site.…”