Functional analysis of XXXX hypersensitive sites at the @entity19 porphobilinogen deaminase gene locus. Different requirements for position-independent expression from its two promoters.

@entity1 @entity19 @entity10473 @entity6007 @entity64598 @entity9828 @entity1446
@entity10473 deaminase (EC 4.3.1.8; @entity64598 ) is the third enzyme of the @entity1446 biosynthetic pathway. In both @entity1 and @entity19 , the gene encoding @entity64598 possesses two promoters, lying in close proximity. We have previously reported the mapping of six nuclear @entity9828 hypersensitive sites at the @entity64598 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 @entity64598 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 @entity19 and in transfected cells. The overall contribution of individual @entity9828 hypersensitive sites to the expression of the gene was then studied using a series of mutants that were stably transfected into @entity19 @entity6007 cells. Two regions seem to play a critical role in the erythroid-specific expression of the @entity64598 gene: the proximal promoter and a region situated at -1000 relative to the initiation site. Study of individual clones of @entity19 @entity6007 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 @entity64598 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 @entity9828 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.
