Cis‐acting elements in the promoter region of the human aldolase C gene

Abstract

We i~vesti~f~d the c&acting sequences involve m the expression of the human Adolph C gene by transient tmnsf~t~ons into b~man neuroblastoma cells (SKNBE). We demonstrate that 420 bp of the Y-flanking DNA dimct at high efficiency the transcription of the CAT reporter gene. A deletion between -420 bp and -164 bp causes a 60% decrease ofCAT activity. Gel shift and DNase I footprmtmg analyses revealed four protected elements: A, B, C and D. Competition analyses indicate that Spl or factors sharing a similar sequence specificity bind to elements A and B, but not to elements C and D. Sequence analysts shows a half palindromic ERE motrf(GGTCA), in elements B and D. Region D binds a transactivatmg factor which appears also essential to stabtlize the initiation complex.Aldolase C; Gene expression; Neuroblastoma cell (SKNBE), &-acting elements; Spl transacting factor Three distinct types of mammalian fructose-1 ,6-bisphosphate aldolase have been characterized by a variety of structural, biochemical and immunological methods [l]. These closely related isoenzymes, designated aldolase A, B and C, are expressed in a tissue-specific fashion and are regulated during ontogenesis and cancerogenesis [2,3]. Three genes, A, B and C, locaiized on chromosomes 16% 9 and 17, respectively, encode for the three ~so~~z~mes (ih,S,@ We and others have eIoned and sequenced the human genes and cDNAs for the three aldolases [7--l 7].Aldolase C is eharaGteristic of brain, where it is mainly localized in neurons and astroghal cells fl8.19]. The protein sequence of aldolase C has been deduced from the nucleotide sequence of mouse and rat complementary cDNA clones [20,21] and from human and rat genomic clones [13,14,17,22]. The 5' non-coding region has been analyzed in rat and man. Multiple start sites of transcription have been reported in rat f22], and one major start site has been described in man ]17].In this paper we report the analysis of the ~~ornot~r region performed by ~~a~s~~~~* of fusion constructs with a reporter gene into human neuronaf (SKNBE) cefl line and the identification of the bus-elements involved in the regulation of the human aldoias~ C gene expression MATERIALS AND METHODS I.. Phwtid constructionThe aldolase C promoter region was excused from the gene as an XbaIlPsfI fragment. spanning from -1190 bp to +20 relative to the major transc~pt~onaI start site fI7], and cloned into the Ml3 rap19 vector. The deletions were made by cuttmg the Y-end with X&rI and subsequently digesting with ExQIII m&ease (Boehringer Ma~n~e~m) @3]. The digested fragments were flush-ended by Klenow poiymerase and hgated into the Mi3 mp19 vector. The deleted fragments were checked by sequencing wtth the chain te~~nat~on method 124], excised from the vector with the appropriate res~~~ct~on enzyme, and then cloned m SmaIlPsrI sites upstream from the CAT reporter gene in the pEMBL-&CAT expression vector [25]. Chrmeric aldolase C-CAT plasmids were purified on a cesium chloride gradient. Cell culture, truxrrent trunsfections and CAT assaysHuman neuroblastoma ...

Genomic sequences of aldolase C (Zebrin II) direct lacZ expression exclusively in non-neuronal cells of transgenic mice

“…A normal TATA box is missing; instead a GC-rich region can be found just upstream of the transcription start site (20). A CAAT box is present, but it is located far upstream from its expected position (21). Northern analysis using a probe specific for aldolase C mRNA demonstrates a 1.6-kb band; there is no evidence of alternative splicing.…”

Diverse human aldolase C gene promoter regions are required to direct specific LacZ expression in the hippocampus and Purkinje cells of transgenic mice

“…3, black bar). It also reinforces in vitro [14,15] and in vivo [10][11][12][13] results obtained with human and rat promoter sequences.…”

“…The black rectangle in the distal region (heavy black bar) is element D [15]; the transcription factor-binding sites in the distal region (GC-rich, AT-rich and CAAT motif) are circled. The hatched rectangles represent the proximal promoter region that is conserved (more than 75% homology) among the mouse, rat and human genes [11,14,15] Copy numbers ranged from 5 to 24. There was no correlation between copy number and b-gal activity.…”

“…The upstream region binds Pax-6 transcriptional factor and is involved in the activation of rat aldolase C gene expression in the brain of transgenic mice [13]. We previously showed, in human-derived neuronal cells (SKNBE), that two regions (proximal and distal) are required for the complete transcriptional expression of the CAT-reporter gene of chimeric aldolase C [14]. We also characterized in the distal region a functional binding-site (element D) for the transcriptional activator NGFI-B.…”

In vivoregulation of single copy and amplified N-myc in human neuroblastoma cells

“…Primer set A was used for footprinting the promoter of the human aldolase C gene to control for the quality of nicked DNA. It covers a region of the aldolase C gene starting from position+27 relative to the transcription start site to 7150 that was previously shown by in vitrofootprinting experiments to span three closely spaced regulatory elements (called footprints A, B, and C; Buono et al, 1993). All DNA preparations used for LM ± PCR with N-myc-speci®c primer sets showed at least 50% protection of the aldolase C promoter region containing footprint B when tested in LM ± PCR with primer set A (data not shown).…”