Rat Aldolase Research Articles

An Intronic Enhancer Essential for Tissue-specific Expression of the Aldolase B Transgenes

Expression in mice of transgenes directed by regulatory regions of the rat aldolase B gene requires the presence of a B element located in the first intron, while constructs devoid of this intronic enhancer are silent. Histo- and immunochemical staining of transgenic tissue sections showed that the longer transgene was expressed in the proximal tubular cells of the kidney, enterocytes located in small intestine villi and liver parenchymal cells. In the liver, a maximal expression was observed in perivenous hepatocytes, while the transgene was weakly active in periportal hepatocytes, which reproduced the pattern of functional zonation already reported for other glycolytic and gluconeogenic genes in the liver. We also established that the transgene retained the necessary elements for a correct chronological expression during development but was lacking elements necessary for activation by high carbohydrate diet. Instead, transgene expression was paradoxically stimulated in fasted animals, suggesting that the endogenous gene, which must be active under both glycolytic and gluconeogenic conditions, could possess distinct elements activating it in fasted as well as in carbohydrate-fed animals; the former element might be conserved in the transgene and the latter one might be lost.

Copy-Dependent and Position-Independent Expression of Rat Aldolase A Gene1

In order to understand the molecular mechanisms of the temporal and spatial differences of gene expression in higher organisms, rat aldolase A gene carrying two distinct promoters was introduced into fertilized eggs and the resulting transgenic mice were analyzed. The transgene expression is tissue-specific and is developmentally regulated. In addition, the expression is regulated in a copy-dependent manner irrespective of where the transgene is integrated, suggesting that a mechanism excluding the effect of the integration site exists within the transgene itself. To explore the conformational change of this gene in the genome, the DNase I hypersensitive sites of the gene were examined. Three sites (DHS-1,2, and 3) were identified upstream and downstream of the gene and these sites were retained in the transgene as well as in the gene observed endogenously.

Developmental alteration of the chromatin state at promoter/replication origin region of the aldolase B locus precedes transcriptional activation in the liver.

Liver-specific expression of the rat aldolase B (AldB) gene is conferred by proximal promoter region (-200 bp to + 1 bp), which is centered on an origin region of DNA replication. Transcriptional activation of the gene in the liver occurs during the late one-third of fetal stage. To know the mechanism involved in such activation, we studied developmental changes in chromatin structure and in the extent of CpG methylation in the promoter/origin region of the gene. At an early fetal stage, when the AldB gene in the liver is not yet activated, the gene chromatin had two DNase 1-hypersensitive sites in the promoter region. One corresponded to that typical of AldB-expressing cells in the adult. The other, located approximately 200 bp upstream of the above site, disappeared as the activation of transcription started. A CpG dinucleotide in the promoter/origin region was heavily methylated at an early stage of gestation, but progressively demethylated as the liver develops. This CpG site is located at the center of an important binding site for a transcription factor. These changes occurred early in the fetal stage, prior to the gene activation, and were thus thought to be associated with differentiation of the liver cell or with cessation of cell proliferation.

Human aldolase C: characterization of the recombinant enzyme expressed in Escherichia coli.

To study the structure/function relationship and enzymatic properties of human aldolase C, we have constructed an Escherichia coli expression plasmid, pHAC11, for the isozyme. E. coli cells carrying this plasmid produced enzymatically active human aldolase C. The kcat and Km values for fructose-1,6-bisphosphate (Fru-1,6-P2) and fructose-1-phosphate (Fru-1-P) of the recombinant enzyme were found to be similar to those of authentic aldolase C from human brain. The Fru-1,6-P2/Fru-1-P activity ratio of the recombinant enzyme is approximately 13.5, which is comparable to that of the recombinant rat aldolase C, but is slightly higher than those of rat brain and hepatoma aldolases C. The substitution of Ser for the carboxyl-terminal Tyr (Tyr-363) of the recombinant enzyme caused a marked decrease in that of Fru-1,6-P2, with little change in that of Fru-1-P. The activity ratio changed from 13.5 for the normal enzyme to 3.8 for the engineered enzyme. Human aldolase C was found to form tetrameric hybrids with aldolase B in vivo when these enzymes were coexpressed in E. coli cells.

Position‐independent, high‐level, and correct regional expression of the rat aldolase C gene in the central nervous system of transgenic mice

Aldolase C is mainly expressed in the central nervous system (CNS). To clarify the regulatory mechanisms for the CNS-specific expression, transgenic mice were created using two constructs of the rat aldolase C gene. A fusion gene comprising the 5' regulatory region of the aldolase C gene was expressed in a CNS-specific manner. However, the expression levels and the cellular localization of the gene varied among transgenic mice. The other construct, including both 5' and 3' regulatory regions of the gene, showed position-independent and high-level expression as well as the correct regional distribution in the CNS. These results indicate that the 13-kb sequence of the rat aldolase C gene contains sufficient information for faithful expression of the gene.

Analysis of a brain-specific isozyme. Expression and chromatin structure of the rat aldolase C gene and transgenes.

Aldolase C mRNA is detected by Northern blot in all fetal tissues in rat; it is very abundant in the adult brain and undetectable in the other adult tissues. However, reverse transcriptase polymerase chain reaction amplification indicates that this gene is not totally repressed in these tissues. A DNase-I hypersensitivity site located in a 115-base pair proximal promoter fragment is detectable in the brain as well as in other adult tissues. Two MspI/HpaII restriction sites located at -3800 and -450 base pairs are demethylated in the brain and totally or partially methylated in other tissues. In transgenic mice, a 12.5-kilobase genomic fragment is strongly and tissue specifically expressed in different lines, with conservation of a methylation pattern similar to that of the endogenous gene. A chloramphenicol acetyltransferase gene directed by either 800 or 115 base pairs of aldolase C 5'-flanking sequences is tissue specifically expressed in transgenic mice, but the level of expression is very low. This level is greatly increased when the transgene consists of a chloramphenicol acetyltransferase hybrid gene directed by 5.5 kilobases of aldolase C 5'-flanking sequences. We propose therefore that the chromatin structure around the aldolase C promoter is accessible in fetal tissues, then remains open in the adult brain, where the gene is very active, as well as in tissues in which it is practically inactive. The specificity of expression in the brain is conferred by a short 115-base pair proximal promoter fragment that needs more upstream sequences to be fully active.

Determinants of the brain-specific expression of the rat aldolase C gene: ex vivo and in vivo analysis.

A 115-bp promoter fragment of the aldolase C gene is sufficient for conferring neural cell specificity on a reporter gene, in cultured PC12 cells and in transgenic mice. In vitro DNase I protection experiments detected two footprints on the promoter, termed boxes A/A', and B. The 5' A/A' box contains overlapping Sp1 and Krox20/Krox24 binding sites; it binds Sp1 in fibroblasts (box A') and a different complex in brain (box A). Any deletion or mutation of this box that impairs protein recognition also suppresses promoter activity. The replacement of box A/A' by a Sp1 consensus binding site results in the loss of the brain specificity of expression in transgenic mice. Further 3', box B is composed of a 5' direct repeat and a 3' GC box consisting of overlapping Sp1 and Krox20/Krox24 binding sites. Mutation of the direct repeat subregion appears to be more deleterious for the promoter activity than mutation of the G+C-rich subregion.

The MEF-3 motif is required for MEF-2-mediated skeletal muscle-specific induction of the rat aldolase A gene.

The rat aldolase A gene contains two alternative promoters and two alternative first exons. The distal promoter M is expressed at a high level only in skeletal muscle. Previous in vitro transfection studies identified the region from -202 to -85 as an enhancer that is responsible for dramatic activation during the differentiation of chicken primary myoblasts. This enhancer contains an A/T-rich sequence resembling the MEF-2 motif, which is an important element of muscle enhancers and promoters. In this study, we demonstrate that the MEF-2 sequence is essential but not sufficient for the activity of the enhancer. Another region required for the activity was recognized by a nuclear factor, tentatively named MAF1. MAF1 was found in both muscle cells and nonmuscle cells, and MAF1 from both cell types was indistinguishable by gel retardation and DNase I footprint experiments. The sequence required for MAF1 binding is very similar to the MEF-3 motif, which is an element of the skeletal muscle-specific enhancer of the cardiac troponin C gene. Because MAF1 and MEF-3 are closely related in both recognition sequence and distribution, MAF1 and MEF-3 probably represent the same nuclear factor which may play an important role in muscle gene transcription. Thus, the muscle-specific induction of the aldolase A gene is governed by muscle-specific MEF-2 and existing MEF-3 (MAF1).

The MEF-3 motif is required for MEF-2-mediated skeletal muscle-specific induction of the rat aldolase A gene.

The rat aldolase A gene contains two alternative promoters and two alternative first exons. The distal promoter M is expressed at a high level only in skeletal muscle. Previous in vitro transfection studies identified the region from -202 to -85 as an enhancer that is responsible for dramatic activation during the differentiation of chicken primary myoblasts. This enhancer contains an A/T-rich sequence resembling the MEF-2 motif, which is an important element of muscle enhancers and promoters. In this study, we demonstrate that the MEF-2 sequence is essential but not sufficient for the activity of the enhancer. Another region required for the activity was recognized by a nuclear factor, tentatively named MAF1. MAF1 was found in both muscle cells and nonmuscle cells, and MAF1 from both cell types was indistinguishable by gel retardation and DNase I footprint experiments. The sequence required for MAF1 binding is very similar to the MEF-3 motif, which is an element of the skeletal muscle-specific enhancer of the cardiac troponin C gene. Because MAF1 and MEF-3 are closely related in both recognition sequence and distribution, MAF1 and MEF-3 probably represent the same nuclear factor which may play an important role in muscle gene transcription. Thus, the muscle-specific induction of the aldolase A gene is governed by muscle-specific MEF-2 and existing MEF-3 (MAF1).

Ubiquitous factors that interact simultaneously with two distinct cis-elements on the rat aldolase B gene promoter

The proximal promoter region (nucleotide −202 to −1) of the rat aldolase B gene confers liver-specific transcription, and contains three indispensable protein-binding sites (site A, site B and site C). Site A binds HNF-1 and HNF-3, and site B binds NF-Y and a CCAAT-binding factor AlF-B (Tsutsumi et al. (1989) Mol. Cell. Biol. 9, 4923–4931; Raymondjean et al. (1991) Nucleic Acids Res. 19, 6145–6153), trans-acting factors that interact with site C, however, have not been well characterized. In this study, we identified specific factors that bind site C by two-dimensional gel electrophoretic analyses. The factors interacted with two distinct cis-elements; site C and site B. This observation, together with the fact that site C contains a C/EBP binding motif, implied that the site C-binding factors are members of C/EBP family. However, analyses of their binding characteristics, their relative molecular masses, and their distribution in different cell types showed that the site C binding factors are different from known members of C/EBP family.

A1F-B, a novel CCAAT-binding transcription activator that interacts with the aldolase B promoter

We describe here a 70 kDa transcription factor A1F-B, which preferentially binds to an element encompassing a CCAAT motif on the rat aldolase B promoter. Comparison of binding specificities, relative molecular masses, and subunit compositions with those of other known CCAAT-binding factors indicated that A1F-B is a novel member of CCAAT-binding factors.

Analysis of upstream regulatory regions required for the activities of two promoters of the rat aldolase A gene

Rat aldolase A gene has 2 promoters with different tissue specificities (M- and AH promoters). The M promoter is active only in adult skeletal muscle and induced during myogenesis, whereas the AH promoter is active ubiquitously in many tissues, including various cancer cells. Regulatory sequences for these promoters were investigated through assays for transient expression after introduction into myogenic and nonmyogenic cells. When M promoter—CAT fusion genes were transfected into primary cultures of chicken myoblasts, expression of CAT activity was drastically induced during myotube formation. The region comprising 202 to 85 base pairs (bp) upstream from the transcription initiation site was round to be necessary for the induction and an enhancer activity whose region includes the AT-rich recognition sequence MEF-2 binding site. On the other hand, 2 upstream regions were found to be responsible for AH promoter activity expressed in HepG2 cells. The distal region (−280 to –260) of the promoter includes the AP1 binding sequence, whereas the proximal region (−207 to −180) contains a novel inverted repeat consisting of 22 bp but does not contain known promoter and enhancer sequences.

Expression of the rat aldolase B gene: A liver-specific proximal promoter and an intronic activator

The nature and location of the cis-acting DNA sequences regulating expression of the rat aldolase B gene has been investigated. Two liver-specific DNAse I hypersensitive sites were detected, one located just upstream from the cap site, the second in the middle of the first, 4.8-kbp-long, intron. A fragment of 190 bp 5' to the cap site behaved as a tissue-specific but weak core promoter: it directed a detectable reporter gene expression in the Hep G2 cells and hepatocytes, but not in fibroblasts. The tissue-specific expression was stimulated at least 16 fold when constructs contained the entire first intron. The intronic activating sequences could be ascribed to an inner 2 kbp fragment in which the downstream liver-specific DNAse I hypersensitive site was located.

An additional promoter functions in the human aldolase A gene, but not in rat

The aldolase A gene was isolated from a human DNA library, mapped and sequenced. This gene comprises 12 exons and spans 6.5 kb. From the genomic DNA sequence and from the previous sequence analysis of the cDNA, it was revealed that the first exon L1 and the second exon encode the 5' non-coding sequence of mRNA L1, while the third and forth exons (corresponding to exons M and L2) encode different mRNA, mRNA M and L2, respectively; the following eight exons (exons 5-12) are shared commonly by all the mRNA species. These results indicate that the mRNA species are generated from a single aldolase A gene from one of exons L1, M or L2, in addition to exons 5-12, and also that the usage of a leader exon is similar but clearly distinct from that of rat aldolase A gene which we analyzed [Joh, K., Arai, Y., Mukai, T. & Hori, K. (1986) J. Mol. Biol. 190, 401-410]. By comparing the promoter regions in the human and rat aldolase A genes, we found similar sequences in the rat genome corresponding to those of the human L1, M and L2 promoter. We could not, however, detect any transcripts starting from sequences corresponding to the human L1 promoter in the rat genome, although the products corresponding to human M and L2 were detected. Thus, we conclude that the L1 promoter was either acquired by the human genome or deleted from the rat genome after human and rat diverged during evolution.

Interplay of an original combination of factors: C/EBP, NFY, HNF3, and HNF1 in the rat aldolase B gene promoter

The rat aldolase B 5' flanking region (nucleotides - 194 to +41) contains sufficient information for liver-specific expression. A detailed investigation of factors binding to the rat aldolase B 5' flanking region has allowed us to identify three distinct factors that filled different sites of this region (A, B, C). The liver-enriched C/EBP or related factors bind to box C, as demonstrated by the specific interaction with bacterially expressed C/EBP protein. Box B bearing the CCAAT sequence binds the ubiquitous factor NFY. Surprisingly, Box A is able to bind two liver enriched factors, namely HNF1 and HNF3. However, in the context of the intact promoter, as shown by footprinting competition experiments, HNF3 binds solely to this sequence. HNF3, but not HNF1 is a transcriptional activator as demonstrated in the in vitro transcription assay.

The structure of the brain-specific rat aldolase C gene and its regional expression

The rat aldolase C gene was isolated from a rat genomic DNA library. This gene comprises 9 exons and spans 3590 base pairs. A single copy of the gene occurs per haploid rat genome. The initiation of transcription occurs at two different sites. The cellular localiztion of aldolase C mRNA was determined in the central nervous system along with aldolase A mRNA by in situ hybridization. The result indicates the predominant expression of this gene in Purkinje cells of the cerebellar cortex, where aldolase A mRNA was rather repressed.

Two different HNF1-like transcription activators in the liver bind to the same region of the rat aldolase B promoter

Coexistence of two different HNF1-like factors (AlF-A1 and AlF-A2) in rat liver is described. The two factors had similar molecular weight and the same sequence-specificity in DNA-binding. One of these factors AlF-A1 was retained on wheat germ agglutinin-sepharose column and eluted from it with N-acetylglucosamine, while AlF-A2 was not, suggesting that AlF-A1 is glycosylated. Using rat brain nuclear extract, both factors stimulated transcription in vitro, but their stimulatory effects differed from each other.

Transcriptional regulation of an aldolase gene in the regenerating rat liver

The relative abundance of rat aldolase A, B, C, alpha-fetoprotein, and albumin mRNAs was determined by Northern hybridization during liver regeneration after partial hepatectomy. Aldolase A mRNA increased more than 10-fold on the 3rd day after partial resection compared with that of normal adult rat liver. S1 analysis revealed that three species of aldolase A mRNAs (mRNA I, II & III) reappeared. However, transcriptional rate of aldolase A mRNA did not change at all during the regeneration. In contrast, aldolase B, aldolase C and albumin mRNAs did not change at all. These findings suggest that the differentiated hepatocyte maintains a differentiated state during the liver regeneration as seen in aldolase B, whereas "oncofetal" isozymes such as aldolase A resurge after partial hepatectomy under the control of post-transcriptional mechanism.

Construction of rat aldolase C expression plasmid and the hybrid formation between rat aldolase C and human aldolase A or B co-expressed in Escherichia coli

Rat aldolase C cDNA was inserted in an Escherichia coli expression vector to construct the rat aldolase C expression plasmid, pRAC42. This plasmid produces active rat aldolase C in the transfected E. coli host cells. The characteristics of the purified enzyme, e.g. mol. wt, electrophoretic mobilities and kinetic parameters, are indistinguishable from those of authentic rat brain aldolase C. Three different tetrameric hybrid forms, C3A, C2A2 and CA3, in addition to C4 and A4, were found to be produced in the host cell when E.coli was co-transfected with expression plasmids for rat aldolase C and for human aldolase A. Similarly, the hybrid forms, C3B, C2B2 and CB3, in addition to C4 and B4, were also produced in the cells when co-transfected with the plasmids for rat aldolase C and for human aldolase B.

Developmental appearance of transcription factors that regulate liver-specific expression of the aldolase B gene.

The region from -202 to -1 of the rat aldolase B (AldB) promoter directs tissue-specific transcription in vitro. Deletion of the half of this promoter distal to its origin reduced its ability for tissue-specific expression. Two protein-binding sites in this distal element have been identified and characterized. One bound a factor named AIF-A, which is probably identical to previously characterized liver-specific factors HNF-1 and APF. The other, containing a CCAAT motif, bound a factor named AIF-B. These two factors were considerably enriched in liver cells compared with other cells not expressing AldB. In the liver, these two factors increased prior to the activation of the AldB gene during development. Liver-specific in vitro transcription assays indicated that the binding of both factors to their target sequences was required for AldB transcription. These results suggest that AIF-A and AIF-B are positively acting transcription factors that regulate tissue-specific and development stage-specific activation of the AldB gene.