Functional analysis of DNase-I hypersensitive sites at the mouse porphobilinogen deaminase gene locus. Different requirements for position-independent expression from its two promoters.
Porphobilinogen deaminase (EC 4.3.1.8; PBG-D) is the third enzyme of the heme biosynthetic pathway. In both human and mouse, the gene encoding PBG-D possesses two promoters, lying in close proximity. We have previously reported the mapping of six nuclear DNase-I hypersensitive sites at the PBG-D locus which could contribute to the regulation of the gene. In the present study, and in order to define all the elements necessary for a high level of expression and an integration site independence, we studied the pattern and the level of expression of a cloned PBG-D gene following integration into a host genome. The longest construct that we tested (12.5 kilobases) contained sufficient regulatory elements to promote expression levels similar to that of the endogenous gene, both in transgenic mice and in transfected cells. The overall contribution of individual DNase-I hypersensitive sites to the expression of the gene was then studied using a series of mutants that were stably transfected into mouse erythroleukemia cells. Two regions seem to play a critical role in the erythroid-specific expression of the PBG-D gene: the proximal promoter and a region situated at -1000 relative to the initiation site. Study of individual clones of mouse erythroleukemia cells revealed that the erythroid-specific expression of the gene was submitted to position effects in the absence of the upstream region, although the housekeeping transcription is not sensitive to such effects. The tandem arrangement of the housekeeping and tissue-specific promoters of the PBG-D gene raises some questions about the functioning of these two overlapping transcriptional units in erythroid cells. Previous data have suggested that in erythroid cells most of the transcripts initiated at the upstream promoter stop downstream of the first ubiquitous exon, between the two promoters. Here, we show that the deletion of a constitutive DNase-I hypersensitive site that is located in the region of the elongation block results in opposite effects on the steady state levels of housekeeping and tissue-specific RNA. This finding is consistent with the hypothesis that this region promotes premature termination of the housekeeping transcripts therefore preventing promoter interference.
Porcher
C
C
INSERM U409, Faculté de Médecine Xavier Bichat, Paris, France.
Picat
C
C
Daegelen
D
D
Beaumont
C
C
Grandchamp
B
B
D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D003850:Deoxyribonuclease I / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic; D011163:Hydroxymethylbilane Synthase / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D014162:Transfection
D012333:RNA, Messenger; D011163:Hydroxymethylbilane Synthase; D003850:Deoxyribonuclease I