Processes that regulate gene transcription are directly under the influence of the genome organization. The epigenome contains additional information that is not brought by DNA sequence, and generates spatial and functional constraints that complement genetic instructions. DNA methylation on CpGs constitutes an epigenetic mark generally correlated with transcriptionally silent condensed chromatin. Replication of methylation patterns by DNA methyltransferases maintains genome stability through cell division. Here we present evidence of an unanticipated dynamic role for DNA methylation in gene regulation in human cells. Periodic, strand-specific methylation/demethylation occurs during transcriptional cycling of the pS2/TFF1 gene promoter on activation by oestrogens. DNA methyltransferases exhibit dual actions during these cycles, being involved in CpG methylation and active demethylation of 5mCpGs through deamination. Inhibition of this process precludes demethylation of the pS2 gene promoter and its subsequent transcriptional activation. Cyclical changes in the methylation status of promoter CpGs may thus represent a critical event in transcriptional achievement.
Expression of the adenine nucleotide translocator 2 (ANT2) gene is growth regulated. We report a feature of the ANT2 promoter that involves a novel regulatory function for the Sp1 transfactor. We show that expression from the ANT2 proximal promoter is modulated through three Sp1 elements, two of which activate and one of which partially inhibits transcription. The inhibitor site, box C, is juxtaposed to transcription start (nucleotides ؊7 to ؊2). Sp1 bound to box C decreases transcription initiation. This was demonstrated by introducing mutations in box C which (a) increased chloramphenicol acetyltransferase expression in the transient transfection assay and (b) inhibited binding of both purified Sp1 and Sp1 in crude nuclear extracts. The activating elements (A and B boxes) are located at adjacent sites in the distal region of the proximal promoter. Mutation of either box inhibits transfection by 90%, indicating that they act in a synergistic manner. Supershift experiments with crude nuclear extracts showed that only Sp1 was bound to the three GC boxes. The finding that Sp1 acts as an activator/inhibitor within the same promoter region was verified in NIH3T3, HeLa, JEG3, and COS-1, indicating that this dual effect of Sp1 is widely preserved. These data suggest a unique role for Sp1 and raise the possibility that growth activation of the ANT2 gene is regulated by the interaction of Sp1 on the A, B, and C boxes. ANT2 is one of three genes that encodes mammalian adenine nucleotide translocator (ANT, ATP/ADP translocase) proteins (1-4). The three genes are differentially expressed in a tissuespecific manner (5-7), and during cellular differentiation (6 -9). The ANT2 isoform is unique since it is expressed in a growthdependent fashion. ANT2 cDNA clones were first isolated as differentially expressed transcripts from growth-activated (10) and serum-stimulated (8) cells. Subsequently, growth-related expression of ANT2 was demonstrated in several cell lines (6,8) and in activated human peripheral lymphocytes (11). Induction of ANT2 transcripts is rapid (8, 12); this, together with the conditions under which the original clones were isolated (10), suggests that it might belong to the group of early-immediate genes. This conclusion is supported by the finding that ANT2 transcripts are induced by mitogens such as platelet-derived growth factor and epidermal growth factor and that induction is not inhibited by cycloheximide (8).To understand the growth-dependent control of ANT2 expression, we characterized the promoter region of the human gene. We report a unique role for the transactivating Sp1 protein. Sp1 is a ubiquitously expressed factor that is required for expression of a large number of constitutive and regulated genes. Sp1 has been described only as a transcription activator. However, Sp1 activation can be suppressed by a variety of mechanisms. For example, Sp3, a member of the Sp1 family (13, 14), competes for Sp1 binding sites (15) but is not able to activate transcription (15). Activation by Sp1 can also be supp...
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