Human Heme Oxygenase-1 Promoter Activity Is Mediated by Z-DNA Formation

Cigarette smoke, containing reactive oxygen species, is the most important risk factor for chronic pulmonary emphysema (CPE). Heme oxygenase-1 (HO-1) plays a protective role as an antioxidant in the lung. A (GT)n dinucleotide repeat in the 5'-flanking region of human HO-1 gene shows length polymorphism and could modulate the level of gene transcription. To investigate the correlation between the length of the (GT)n repeat and susceptibility to the development of CPE, we screened the frequencies of alleles with varying numbers of (GT)n repeats in the HO-1 gene in 101 smokers with CPE and in 100 smokers without CPE. Polymorphisms of the (GT)n repeat were grouped into three classes: class S alleles ( /=30 repeats). The proportion of allele frequencies in class L, as well as the proportion of genotypic frequencies in the group with class L alleles (L/L, L/M, and L/S), was significantly higher in the smokers with CPE than in smokers without CPE. Moreover, we analyzed the promoter activities of the HO-1 gene carrying different (GT)n repeats (n=16, 20, 29, and 38), by transient-transfection assay in cultured cell lines. H2O2 exposure up-regulated the transcriptional activity of the HO-1 promoter/luciferase fusion genes with (GT)16 or (GT)20 but did not do so with (GT)29 or (GT)38. These findings suggest that the large size of a (GT)n repeat in the HO-1 gene promoter may reduce HO-1 inducibility by reactive oxygen species in cigarette smoke, thereby resulting in the development of CPE.

Editing of RNA changes the read-out of information from DNA by altering the nucleotide sequence of a transcript. One type of RNA editing found in all metazoans uses double-stranded RNA (dsRNA) as a substrate and results in the deamination of adenosine to give inosine, which is translated as guanosine. Editing thus allows variant proteins to be produced from a single pre-mRNA. A mechanism by which dsRNA substrates form is through pairing of intronic and exonic sequences before the removal of noncoding sequences by splicing. Here we report that the RNA editing enzyme, human dsRNA adenosine deaminase (DRADA1, or ADAR1) contains a domain (Zalpha) that binds specifically to the left-handed Z-DNA conformation with high affinity (KD = 4 nM). As formation of Z-DNA in vivo occurs 5' to, or behind, a moving RNA polymerase during transcription, recognition of Z-DNA by DRADA1 provides a plausible mechanism by which DRADA1 can be targeted to a nascent RNA so that editing occurs before splicing. Analysis of sequences related to Zalpha has allowed identification of motifs common to this class of nucleic acid binding domain.

The mammalian BAF complex regulates gene expression by modifying chromatin structure. In this report, we identify 80 genes activated and 2 genes repressed by the BAF complex in SW-13 cells. We find that prior binding of NFI/CTF to the NFI/CTF binding site in CSF1 promoter is required for the recruitment of the BAF complex and the BAF-dependent activation of the promoter. Furthermore, the activation of the CSF1 promoter requires Z-DNA-forming sequences that are converted to Z-DNA structure upon activation by the BAF complex. The BAF complex facilitates Z-DNA formation in a nucleosomal template in vitro. We propose a model in which the BAF complex promotes Z-DNA formation which, in turn, stabilizes the open chromatin structure at the CSF1 promoter.