Transcription factor IIA mutations show activator-specific defects and reveal a IIA function distinct from stimulation of TBP-DNA binding.

The general transcription factor IIA (TFIIA) binds to the TATA binding protein (TBP) and mediates transcriptional activation by distinct classes of activators. To elucidate the function of TFIIA in transcriptional activation, point mutants were created in the human TFIIA-gamma subunit at positions conserved with the yeast homologue. We have identified a class of TFIIA mutants that stimulate TBP-DNA binding (T-A complex) but fail to support transcriptional activation by several different activators, suggesting that these mutants are defective in their ability to facilitate an activation step subsequent to TBP promoter binding. Point mutations of the hydrophobic core of conserved residues from 65 to 74 resulted in various activation-defective phenotypes. These residues were found to be important for TFIIA gamma-gamma interactions, suggesting that gamma-gamma interactions are critical for TFIIA function as a coactivator. A subset of these TFIIA-gamma mutations disrupted transcriptional activation by all activators tested, except for the Epstein-Barr virus-encoded Zta protein. The gamma Y65F, gamma W72A, and gamma W72F mutants mediate Zta activation, but not GAL4-AH, AP-1, GAL4-CTF, or GAL4-VP16 activation. The gamma W72A mutant failed to stimulate TFIID-DNA binding (D-A complex) but was able to form a complex with TFIID and DNA in the presence of Zta (Z-D-A complex). Thus, the ability of Zta to activate transcription with gamma W72A appears to result from a unique ability to form the stable Z-D-A complex with this mutant. Our results show that different activators utilize the general factor TFIIA in unique ways and that TFIIA contributes transcription activation functions in addition to the facilitation of TBP-DNA binding.

Molecular cloning of the small (gamma) subunit of human TFIIA reveals functions critical for activated transcription.

TFIIA is thought to play an important role in transcriptional regulation in higher eukaryotes, but its precise function is unclear. A human cDNA encoding a protein with 45% identity to the small subunit of yeast TFIIA has been isolated. TFIIA activity could be reconstituted by the mixing of recombinant large (alpha beta) and small (gamma) subunits. TFIIA-depleted HeLa nuclear extracts were used to demonstrate that TFIIA is essential for basal and activated transcription by several distinct classes of activators. Recombinant TFIIA functioned in transcriptional activation whether expressed as a dimer (alpha beta+gamma) or as a trimer (alpha+beta+gamma), which closely resembles the native form. Yeast TFIIA also functioned in transcriptional activation, and the human gamma subunit was functionally interchangeable with TOA2, its yeast homolog. Recombinant TFIIA mediated the stimulation of TFIID binding to the TATA region and downstream promoter sequences by the Zta transcriptional activator. Significantly, we found that TFIIA bound directly to Zta in an activation domain-dependent manner. One consequence of the TFIIA-mediated interaction between Zta and TFIID was the formation of a promoter-bound complex resistant to TATA oligonucleotide competition. These results demonstrate that TFIIA is an evolutionarily conserved general factor critical for activator-regulated transcription.

Distinct promoters direct neuronal and nonneuronal expression of rat aromatic L-amino acid decarboxylase.

Aromatic L-amino acid decarboxylase (AADC, EC 4.1.1.28) catalyzes the decarboxylation of L-dopa to dopamine in catecholamine cells and 5-hydroxytryptophan to serotonin in serotonin-producing neurons. This enzyme is also expressed in relatively large quantities in nonneuronal tissues such as liver and kidney, where its function is unknown. Neuronal and nonneuronal tissues express AADC mRNAs with distinct 5' untranslated regions. To understand how this is accomplished at the genomic level, we have isolated rat genomic DNA encoding AADC. The organization of the AADC gene suggests that there are two separate promoters specific for the transcription of neuronal and nonneuronal forms of the AADC message. A small exon containing 68 bases of the neuronal-specific 5' end is located approximately 9.5 kilobases upstream of the translation start site, which is contained in the third exon. Approximately 7 kilobases upstream from the neuron-specific promoter is another small exon containing 71 bases of the 5' end of the nonneuronal AADC message. These data suggest that transcription initiating at distinct promoters, followed by alternative splicing, is responsible for the expression of the neuronal and nonneuronal forms of the AADC message.

Primary DNA sequence determines sites of maintenance and de novo methylation by mammalian DNA methyltransferases.

Analysis of the enzymatic methylation of oligodeoxynucleotides containing multiple C-G groups showed that hemimethylated sites in duplex oligomers are not significantly methylated by human or murine DNA methyltransferase unless those sites are capable of being methylated de novo in the single- or double-stranded oligomers. Thus, the primary sequence of the target strand, rather than the methylation pattern of the complementary strand, determines maintenance methylation. This suggests that de novo and maintenance methylation are the same process catalyzed by the same enzyme. In addition, the study revealed that complementary strands of oligodeoxynucleotides are methylated at different rates and in different patterns. Both primary DNA sequence and the spacing between C-G groups seem important since in one case studied, maximal methylation required a specific spacing of 13 to 17 nucleotides between C-G pairs.

DNA methylation: sequences flanking C-G pairs modulate the specificity of the human DNA methylase.

Synthetic single-stranded oligodeoxynucleotides of known sequence have been used as in vitro substrates for a partially purified HeLa cell DNA methylase. Although most oligonucleotides tested cannot be used by the HeLa DNA methylase in vitro, we have found a unique 27mer, containing 2 C-G pairs, that is an excellent substrate for the enzyme. Analysis of the methylation of the 27mer, its derivatives and other oligomer substrates reveal that the HeLa DNA methylase does not significantly methylate an oligomer which contains just one C-G pair. In addition, only one of the two C-G pairs in the 27mer is methylated and this methylation is abolished if the other C-G pair is converted to a C-A pair. Furthermore, the HeLa enzyme apparently cannot methylate C-G pairs located in compounds containing a high A + T content. The most efficient methylation occurs with multiple separated C-G pairs in a compound with a high G + C content (greater than 65%). The results suggest that clustering of C-G pairs in regions of the DNA high in G + C content may be the preferred site for DNA methylation in vivo.

The effect of flanking sequences on the de novo methylation of C-G pairs by the human DNA methylase.

The HeLa DNA methylase can methylate selected cytosine residues in oligodeoxynucleotides as small as 12-16 nucleotides in length in vitro. The maximum methylation rate seems to require oligomers having more than one C-G in the molecule even when only one of the C-G pairs is methylated. Compounds which contain a high G+C content also seem to be favored substrates. The use of defined synthetic oligodeoxynucleotides permits one to demonstrate that flanking DNA sequences can be critical in determining whether a C-G site can be methylated.