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Volume 272, Number 42,
Issue of October 17, 1997
pp. 26585-26594
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Transcriptional Regulation of the Human Erythroid
5-Aminolevulinate Synthase Gene
IDENTIFICATION OF PROMOTER ELEMENTS AND ROLE OF REGULATORY
PROTEINS
(Received for publication, February 10, 1997, and in revised form, July 7, 1997)
Katharina H.
Surinya
,
Timothy C.
Cox
and
Brian K.
May
From the Department of Biochemistry, University of Adelaide,
South Australia, 5005 Australia and the Department of
Genetics, University of
Adelaide, South Australia, 5005 Australia
We have characterized the 5 -flanking region of
the human erythroid-specific 5-amino levulinate synthase (ALAS) gene
(the ALAS2 gene) and shown that the first 300 base pairs of promoter sequence gives maximal expression in erythroid cells. Transcription factor binding sites clustered within this promoter sequence include GATA motifs and CACCC boxes, critical regulatory sequences of many
erythroid cell-expressed genes. GATA sites at 126/ 121 (on the
noncoding strand) and 102/ 97 were each recognized by GATA-1 protein
in vitro using erythroid cell nuclear extracts. Promoter mutagenesis and transient expression assays in erythroid cells established that both GATA-1 binding sites were functional and exogenously expressed GATA-1 increased promoter activity through these
sites in transactivation experiments. A noncanonical TATA sequence at
the expected TATA box location ( 30/ 23) bound GATA-1- or
TATA-binding protein (TBP) in vitro. Conversion of this
sequence to a canonical TATA box reduced expression in erythroid cells, suggesting a specific role for GATA-1 at this site. However, expression was also markedly reduced when the 30/ 23 sequence was converted to
a consensus GATA-1 sequence (that did not bind TBP in
vitro), suggesting that a functional interaction of both factors
with this sequence is important. A sequence comprising two overlapping CACCC boxes at 59/ 48 (on the noncoding strand) was demonstrated by
mutagenesis to be functionally important. This CACCC sequence bound
Sp1, erythroid Krüppel-like factor, and basic Krüppel-like factor in vitro, while in transactivation experiments
erythroid Krüppel-like factor activated ALAS2 promoter expression
through this sequence. A sequence at 49/ 39 with a 9/11 match to the consensus for the erythroid specific factor NF-E2 was not functional. Promoter constructs with 5 -flanking sequence from 293 base pairs to
10.3 kilobase pairs expressed efficiently in COS-1 cells as well as in
erythroid cells, indicating that an enhancer sequence located elsewhere
or native chromatin structure may be required for the tissue-restricted
expression of the gene in vivo.

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Copyright © 1997 by the American Society for Biochemistry and Molecular Biology.
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