(Received for publication, May 9, 1995; and in revised form, June 21, 1995)
From the
The aldolase C gene product is a glycolytic isoenzyme specifically detected in brain. We have previously defined a short 115-base pair promoter fragment able to confer on a reporter chloramphenicol acetyltransferase (CAT) gene a specific expression in brain of transgenic mice. In this promoter fragment, two GC-rich regions (A/A` and B boxes) were detected by in vitro DNase1 footprinting experiments with brain, fibroblast, or liver nuclear extracts. Both A/A` and B boxes, sharing structural homology, are able to interact with Sp1, Krox20/Krox24 factors and with other proteins (Thomas, M., Makeh, I., Briand, P., Kahn, A., and Skala, H.(1993) Eur. J. Biochem. 218, 143-151). In this paper, we describe a new ubiquitous factor termed Ub able to bind the A/A` box. We also delimit a third element (box C) binding a hepatocyte-enriched protein displaced by a hepatocyte nuclear factor 3-specific oligonucleotide. The functional involvement of each binding site in brain-specific transcription of the aldolase C gene has been tested in transgenic mice carrying different mutant promoters cloned in front of the CAT gene. A promoter containing only box C was totally inactive, suggesting an essential role of the region containing A/A` and B boxes. However, mutations or deletions of either the A/A` or the B box have no significant effect on the CAT gene expression. We therefore hypothesize that the A/A` and B sites may be functionally redundant. Indeed, constructs harboring only one of these two boxes (A/A` or B) linked to the C box displayed a brain-specific CAT activity similar to that obtained with the wild-type promoter. Furthermore, a transgene with disruption of the C box, keeping intact the A/A` and B boxes, was totally inactive, suggesting a crucial role of the hepatocyte nuclear factor 3 binding site in activation of the aldolase C gene.
Genes specifically expressed in the brain can be divided in two
main groups according to the range of their specificity. A subset of
these genes is active in restricted and well defined types of neurons.
For instance, pcp2 gene is specifically expressed in retinal
bipolar cells and cerebellar Purkinje cells(1, 2) ,
while the homeodomain protein Pit-1 is detected in somatotroph,
lactotroph, and thyrotroph cells of the anterior pituitary(3) .
In contrast, other brain-specific genes do not exhibit any sharp
specificity and are expressed in many neurons, e.g. the genes
for synapsin I (review (4) ), amyloid precursor
protein(5) , neuron-specific enolase (6) , and
Thy-1(7) . The aldolase C gene belongs to this latter group;
indeed, in situ hybridization experiments allowed to detect
throughout the brain different levels of aldolase C mRNA(8) .
These ``nonspecific'' neuronal genes often contain
housekeeping-like promoters lacking TATA and CAAT boxes and covering a
GC-rich region(5, 9, 10, 11) . To
understand the mechanisms underlying the widespread cerebral expression
characterizing such genes, we studied the sequences involved in
brain-specific transcription of the rat aldolase C gene. In previous
works, we have defined a short 115-bp ()sequence as a
minimal efficient promoter of the aldolase C gene. This promoter is
indeed sufficient for driving a CAT reporter gene expression
specifically in cultured PC12 cells and in brain of transgenic
mice(12, 13) . Two GC-rich boxes A/A`
(-197/-166) and B (-161/-138) were defined by
footprinting experiments on this 115-bp sequence. Both were shown to be
able to interact in vitro with Sp1 and Krox20/Krox24
factors(12) . In addition, the B box contains a direct repeat
sequence located 5` to the GC-rich region and binding, in
vitro, unknown X and Y proteins(12) . We
report here another ubiquitous factor, termed Ub, binding the A/A` box
upstream of the Sp1/Krox2O/Krox24 binding site. We also describe on the
promoter, from bp -114 to -99 (Fig. 1), a third box,
termed box C, detected only with liver nuclear extracts and which seems
to interact with factors of the fork head/HNF3 family. In
transgenic mice harboring the chloramphenicol acetyltransferase (CAT)
gene controlled by various mutant versions of the aldolase C promoter,
we found that boxes A and B are redundant; indeed, the presence of only
one of them is sufficient to ensure a brain-specific expression. In
addition, an intact box C seems to be required for the expression of
the aldolase C transgenes.
Figure 1: Structure of the aldolase C gene promoter. The footprinted A/A` (-197/-166), B(-161/-138), and C(-114/-99) boxes detected in the 0.115-bp aldolase C promoter are indicated. For each box, recognition sites for factors are underlined, and nucleotides modified in each mutation are indicated by arrows.
The coding strand of the 0.115/CAT plasmid was end-labeled at the XbaI site by filling in with
[-
P]dNTP using Klenow fragment of DNA
polymerase I. Then, the 0.115 probe was excised by SacI
digestion and purified. After incubation with 50 µg of liver
extract proteins and 250 ng of poly(dI-dC), 0.1 ng of probe was
partially digested by DNase1 (Sigma). 50 ng of specific competitor were
or were not added. In control experiments, probe was incubated with 2.5
µg of sonicated salmon sperm DNA prior to the DNase1 treatment.
Samples were ethanol-precipitated and loaded in 8% (w/v)
polyacrylamide, 8 M urea gel.
Probes were end-labeled with
[-
P]ATP using T4 polynucleotide kinase. Gel
shift reaction mixtures included 300 ng of poly(dI-dC) (Pharmacia
Biotech Inc.) as a nonspecific competitor, 0.05-0.1 ng of
radiolabeled probe, and 6 µg of nuclear extract proteins in a
classical binding buffer(21) . Various amounts of specific
competitor were or were not added. After a 5-min incubation at 0
°C, samples were loaded on a non-denaturing 6% (w/v) polyacrylamide
gel.
For methylation interference assays(22, 23) , partially methylated 5`-labeled or 3`-labeled A/A`mut probe was used. Bound and free DNA fractions were separated by gel shift electrophoresis and then eluted from the gel, cleaved at modified guanine residues with 10% (v/v) piperidine, and loaded on a 15% (w/v) polyacrylamide, 8 M urea gel.
Figure 2: Detection of a new binding activity upon A/A`mut oligonucleotide. A, radiolabeled A/A`mut oligonucleotide was incubated with 6 µg of nuclear proteins prepared from 3T6 fibroblasts, or from whole organs (liver or brain). Homologous or heterologous competitors (20 ng) were or were not added. The complex named Ub is indicated by the arrow. Free, probe incubated without nuclear extracts. B and C, A/A` mut or Sp1cons oligonucleotide was used as radiolabeled probe and incubated with 6 µg of brain nuclear proteins. As indicated, 20 ng or increasing amounts (5, 10, and 20 ng) of homologous or heterologous competitors were or were not added to the reaction mixture. Free, probe incubated without nuclear extracts. Ub and Sp1 complexes are indicated by arrows. The fast migrating N.S complex obtained with A/A`mut probe appeared nonspecific since it was unaffected by addition of unlabeled homologous competitor.
We further investigated which nucleotides were contacted by the Ub factor by in vitro methylation interference assays using radiolabeled A/A`mut oligonucleotide and nuclear proteins extracted from adult brain, in which the gene is active, and from adult liver, in which the gene is inactive. In both extracts, methylation at guanines -195 and -194 of the non-coding strand interfered with Ub complex formation strongly and weakly, respectively (Fig. 3). On the coding strand, three residues were mainly contacted by the Ub factor: nucleotides -192, -187, and -182, while three minor interferences were detected at positions -186, -185, and -184 (Fig. 3). These results indicate that the 5`-region of the A/A` box, which contains a 15-bp sequence conserved between rat and human, is involved in the binding of the Ub complex, both in the brain and the liver.
Figure 3: Protein/DNA contact points of brain- and liver-Ub complexes. Binding reactions were performed with brain or liver nuclear extracts and with partially methylated A/A`mut oligonucleotide end-labeled on the coding or non-coding strand. G, Maxam-Gilbert sequence reaction. Residues at which methylation interfered with protein binding are indicated by circles and summarized in the bottom of the figure. Solidcircles represent a greater degree of interference than opencircles. Nucleotides conserved between rat and human sequences are indicated.
Figure 4: Detection of a liver-specific footprint on the 115-bp promoter. The 115-bp fragment used as probe was excised from the 0.115/CAT plasmid by XbaI and SacI digestion. Prior to the attack with DnaseI, this fragment was incubated with 50 µg of liver nuclear extract proteins and, as indicated, with 30 ng of unlabeled oligonucleotides representing the binding sites for HNF1 (box L1 of the pyruvate kinase gene promoter(17) , HNF3 (sequence derived from the transthyretin gene promoter)(16) , and C/EBP (site D of the albumin gene promoter)(18, 19) . G+A, Maxam-Gilbert sequence reaction; Free, digestion of the probe without extracts. Borders of the footprinted sequences are indicated.
Figure 5:
Distribution of CAT activity in transgenic
mice carrying wild-type or mutated transgene. A, scheme of the
different transgenes injected. B, CAT activities, expressed in
cpmmin
(µg
protein)
, were measured on 200 µg of proteins
from brain and other tissues (liver, heart, and lung), previously
heated 10 min at 65 °C to eliminate endogenous deacetylase
activity. For each line, the transgene copy number was estimated by
Southern blot hybridization of transgenic mouse DNAs and of standard
amounts of the injected transgene. CAT activities obtained with the
0.115/CAT fragment were more extensively described in (12) and (13) ). F indicates that founders were sacrificed
before mating. For line 39, carrying the A/A`/C/CAT transgene, an
ectopic CAT expression was also observed with heart
extracts.
To test the in vivo involvement of the B region in brain-specific expression of the aldolase C gene, the DRmut/CAT and Sp1Bmut/CAT constructs previously described (12) were used to generate several independent transgenic lines. The DRmut mutation prevents the binding of the X and Y proteins(12) , while the Sp1Bmut mutation suppresses the binding of Sp1 (12) and Krox20/Krox24 (not shown) (Fig. 1). Fig. 5shows that a comparable brain-specific expression was observed with either wild-type or mutant transgenes.
To test the role of A/A` and B boxes in the promoter activity, both regions were deleted, yielding the construct Del.A/A`/B/CAT (Fig. 5). In six independent lines carrying the Del.A/A`/B/CAT transgene, no significant CAT activity was detected (Fig. 5), suggesting an essential involvement of the region containing the A/A` and B boxes in the expression of the transgene. As independent mutations of binding sites in either A or B box had no effect, we then supposed that these two sequences could have a redundant role in transcriptional activity. To further test this hypothesis, each box (A/A` or B) was independently cloned in front of the C box, resulting in A/A`/C/CAT and B/C/CAT plasmids. CAT activities obtained in transgenic mice were comparable to that obtained with wild-type construct (Fig. 5). The presence of only one box (A/A` or B) in front of the C region was therefore able to restore a specific activity of the aldolase C promoter.
In lines 70 (Del.Ub/CAT), 1 and 80 (Sp1Bmut/CAT), and 20, 32, 29, and 73 (A/A`/C/CAT), the CAT transgene was totally inactive, probably owing to a position effect. We already reported that for the 115/CAT transgene the CAT expression was dependent on the integration site and not correlated with the transgene copy number(13) .
The involvement of the C box in transcription was tested by replacing the genuine box by an HNFmut sequence mutated at 5 nucleotides, -111, -109, -107, -104, and -103 (Fig. 1), resulting in the HNFmut/CAT transgene. In the 6 lines obtained, the transgene was inactive in all tissues tested (Fig. 5).
We can therefore assume that an integral C box associated with at least one of the two A/A` or B boxes is necessary to ensure a brain-specific activity of the aldolase C promoter.
We had previously shown that a CAT construct harboring a consensus Sp1 binding site in place of the whole A/A` box (Sp1cons/CAT) appears very weakly and ubiquitously expressed in transgenic mice(12) . The A/A` box was then assumed to be essential to achieve a brain-specific expression (12) . Present results show, in fact, that the B box is also able to ensure a brain-specific activity in the absence of the A/A` box. Considering this result, we can now assume that, in the Sp1cons/CAT construct, the presence of a high affinity binding site for the ubiquitous Sp1 protein may have induced a new array of interactions upon this promoter region, responsible for the widespread activity of the Sp1cons/CAT transgene.
The existence of specific transcriptional factors able to bind both A/A` and B boxes and responsible for brain-specific expression of the aldolase C gene remains to be established. Two types of hypotheses can be proposed. 1) The brain-specific activity of the aldolase C promoter could result from interaction between ubiquitous transcriptional factors (like Sp1) and brain-restricted factors not yet identified. Such an implication of Sp1 in tissue-restricted transcription has been already described(41, 42, 43) . Mutations in the Sp1 site in A/A`/C/CAT or in B/C/CAT construct should help to test this hypothesis. 2) The brain-specific activity of the aldolase C promoter should result from direct interaction of a brain-specific factor with A/A` and B boxes.
Fig. 6shows that a GGGAG sequence is shared by A/A` and B boxes. Interestingly, a similar element is located in the Pcp2 gene promoter involved in the widespread expression of a reporter gene in almost all neurons of transgenic mice(44) . The aldolase C sequence is also similar to an element of the neuron-specific enolase gene promoter (6) and with a hemipalindrome described in the heavy neurofilament gene promoter (NF-H) (45) (Fig. 6). The palindromic sequence of this latter promoter was described to be important in brain-specific enhancement of in vitro transcription. We are currently investigating whether the role of the A/A` and B boxes in the brain-specific transcription of the aldolase C gene can indeed be ascribed to this conserved G-rich sequence.
Figure 6: Similarities between A/A` and B boxes of the aldolase C gene promoter and sequences present in other brain-specific promoters. G-rich and GGGA core elements found in A/A` and B boxes, and shared by brain-specific promoters of several genes, are underlined.
It should be observed that the results obtained in vivo in transgenic mice are significantly different from those obtained ex vivo in cultured PC12 cells; in these cells, a single mutation in the Sp1/Krox20/Krox24 binding site of the A/A` box was sufficient to inactivate the gene, and mutations of the B box also decreased expression(12) . In fact, it appears that the redundance between boxes A/A` and B observed in vivo was not operative ex vivo, where integrity of both boxes was required. This could be explained by a lower level of differentiation in PC12 cells than in the brain in vivo, such that a strict cooperation between the functionally redundant boxes A/A` and B would be required ex vivo and dispensable in vivo. A similar observation was reported for the cell-specific elastase I enhancer where three distinct enhancer domains are all required in transfected cells but have a redundant function in transgenic mice (46) . Other examples point out the differences between activity of cis-acting sequences tested ex vivo, in transient transfection assays, and in vivo, in the normal developmental and chromosomal context of transgenic animals(47, 48) .
Mutations performed in the aldolase C gene promoter HNF3 binding site led to the complete inactivity of this promoter in transgenic mice (Fig. 5). To explain this unexpected result, two hypotheses could be proposed. 1) The C box is a binding site for a positive brain-specific activator related to the HNF3/fork head family. Such a protein, essential in developing brain, has been described as the brain factor-1 (BF-1) and the brain factor-2 (BF-2)(49, 50) . However, by footprint experiments (12) as well as by gel shift assays (data not shown), we failed to detect any binding activity on the C box with adult brain nuclear extracts. Further experiments (e.g.in vivo footprinting experiments) are in progress to study in more details protein interaction on the C box in fetal and adult brains. 2) The binding of an HNF3-like factor leads to the formation of an active aldolase C promoter by a chromatin structure modification at an early stage of development. In line with this hypothesis, HNF3 factors have been suggested to be essential for the establishment of the phased nucleosome arrangement in the albumin gene enhancer, necessary for gene activation(51) . Since HNF3 proteins are synthesized early during development, especially in derivatives of the neural crest, we hypothesize that such a protein can contribute to make the aldolase C promoter accessible to other factors potentially interacting with the A/A` and/or B boxes and involved in brain-specific expression. Although further investigations are required to test this model, the present paper is the first report suggesting that a fixation of an HNF3-related protein could be involved in the activity of a brain-specific gene promoter.
In conclusion, activity and brain specificity of the aldolase C gene promoter require the presence of an AT-rich element, box C, that seems to be able to bind factors of the HNF3 family and of at least one of two composite GC-rich elements, boxes A/A` and B.