Molecular mechanisms of McArdle's disease (muscle glycogen phosphorylase deficiency). RNA and DNA analysis.

Lack of muscle glycogen phosphorylase activity leads to McArdle's disease, a rare metabolic myopathy. To investigate its molecular basis at the nucleic acid level, we isolated muscle phosphorylase cDNA clones from a human cDNA library in Escherichia coli plasmid pBR 322. Subcloning of one insertion of M13 bacteriophage permitted its definite identification by sequencing. Northern blot experiments revealed one specific messenger RNA of 3.4 kilobases found uniquely in tissues expressing muscle phosphorylase. We show that McArdle's disease exhibits a molecular heterogeneity at the messenger RNA level. In eight unrelated cases of McArdle's disease in which no inactive proteins had been detected, we assayed muscle biopsies for phosphorylase mRNA by Northern blotting. In five cases, no muscle phosphorylase mRNA could be detected, while in three other cases, normal length mRNA was present in lower amounts. Moreover, Southern blot analysis of DNA isolated from white blood cells in four McArdle patients revealed no major deletion or rearrangements of the phosphorylase gene as compared with controls.

A combination of MEF3 and NFI proteins activates transcription in a subset of fast-twitch muscles.

The human aldolase A pM promoter is active in fast-twitch muscles. To understand the role of the different transcription factors which bind to this promoter and determine which ones are responsible for its restricted pattern of expression, we analyzed several transgenic lines harboring different combinations of pM regulatory elements. We show that muscle-specific expression can be achieved without any binding sites for the myogenic factors MyoD and MEF2 and that a 64-bp fragment comprising a MEF3 motif and an NFI binding site is sufficient to drive reporter gene expression in some but, interestingly, not all fast-twitch muscles. A result related to this pattern of expression is that some isoforms of NFI proteins accumulate differentially in fast- and slow-twitch muscles and in distinct fast-twitch muscles. We propose that these isoforms of NFI proteins might provide a molecular basis for skeletal muscle diversity.

Fast-muscle-specific DNA-protein interactions occurring in vivo at the human aldolase A M promoter are necessary for correct promoter activity in transgenic mice.

The human aldolase A tissue-specific M promoter (pM) has served as a model system for identifying pathways that lead to fast-muscle-specialized expression. The current study has delimited the sequences necessary and sufficient for fast-muscle-specific expression in transgenic mice to a short 209-bp fragment extending from bp -164 to +45 relative to the pM transcription start site. Genomic footprinting methods showed that in this proximal region, the same elements that bind muscle nuclear proteins in vitro are involved in DNA-protein interactions in intact muscle nuclei of transgenic mice. Furthermore, these experiments provided the first evidence that different DNA-binding activities exist between slow and fast muscles in vivo. Fast-muscle-specific interactions occur at an element named M1 and at a muscle-specific DNase I-hypersensitive site that was previously detected by in vitro methods. The formation of the muscle-specific DNase I-hypersensitive site reflects binding of proteins to a close element, named M2, which contains a binding site for nuclear factors of the NF1 family. Mutational analysis performed with transgenic mice confirmed the importance of the M1 element for high-level fast-muscle-specific pM activity and suggested that the M2/NF1 element is differently required for correct pM expression in distinct fast muscles. In addition, two other protein binding sites, the MEF3 motif and the USF site, seem to act as stage-specific activators and/or as participants in the establishment of an active chromatin configuration at pM.

Fast-muscle-specific expression of human aldolase A transgenes.

The expression of the human aldolase A gene is controlled by three alternative promoters. In transgenic mice, pN and pH are active in all tissues whereas pM is activated specifically in adult muscles composed mainly of fast, glycolytic fibers. To detect potential regulatory regions involved in the fast-muscle-specific activation of pM, we analyzed DNase I hypersensitivity in a 4.3-kbp fragment from the 5' end of the human aldolase A gene. Five hypersensitive sites were located near the transcription initiation site of each promoter in those transgenic-mouse tissues in which the corresponding promoter was active. Only one muscle-specific hypersensitive site was detected, mapping near pM. To functionally delimit the elements required for muscle-specific activity of pM, we performed a deletion analysis of the aldolase A 5' region in transgenic mice. Our results show that a 280-bp fragment containing 235 bp of pM proximal upstream sequences together with the noncoding M exon is sufficient for tissue-specific expression of pM. When a putative MEF-2-binding site residing in this proximal pM region is mutated, pM is still active and no change in its tissue specificity is detected. Furthermore, we observed a modulation of pM activity by elements lying further upstream and downstream from pM. Interestingly, pM was expressed in a tissue-specific way in all transgenic mice in which the 280-bp region was present (32 lines and six founder animals). This observation led us to suggest that the proximal pM region contains elements that are able to override to some extent the effects of the surrounding chromatin.

An opportunistic promoter sharing regulatory sequences with either a muscle-specific or a ubiquitous promoter in the human aldolase A gene.

The human aldolase A gene is transcribed from three different promoters, pN, pM, and pH, all of which are clustered within a small 1.6-kbp DNA domain. pM, which is highly specific to adult skeletal muscle, lies in between pN and pH, which are ubiquitous but particularly active in heart and skeletal muscle. A ubiquitous enhancer, located just upstream of pH start sites, is necessary for the activity of both pH and pN in transient transfection assays. Using transgenic mice, we studied the sequence controlling the muscle-specific promoter pM and the relations between the three promoters and the ubiquitous enhancer. A 4.3-kbp fragment containing the three promoters and the ubiquitous enhancer showed an expression pattern consistent with that known in humans. In addition, while pH was active in both fast and slow skeletal muscles, pM was active only in fast muscle. pM activity was unaltered by the deletion of a 1.8-kbp region containing the ubiquitous enhancer and the pH promoter, whereas pN remained active only in fast skeletal muscle. These findings suggest that in fast skeletal muscle, a tissue-specific enhancer was acting on both pN and pM, whereas in other tissues, the ubiquitous enhancer was necessary for pN activity. Finally, a 2.6-kbp region containing the ubiquitous enhancer and only the pH promoter was sufficient to bring about high-level expression of pH in cardiac and skeletal muscle. Thus, while pH and pM function independently of each other, pN, remarkably, shares regulatory elements with each of them, depending on the tissue. Importantly, expression of the transgenes was independent of the integration site, as originally described for transgenes containing the beta-globin locus control region.

Two different species of messenger RNAs specify synthesis of M1 and M2 pyruvate kinase subunits.

A study was performed to determine whether M1 and M2 pyruvate kinases were synthesized under the direction of one or two messenger RNAs. We compared M1 and M2 pyruvate kinases purified from fresh tissues with those neosynthesized under the direction of messenger RNAs from tissues synthesizing either M1 or M2. RNA was isolated from rat muscle, lung, spleen and kidney by ethanol precipitation in 7 M guanidium chloride, translated in rabbit reticulocyte system and newly-synthesized pyruvate kinase subunits were purified by microimmunoaffinity chromatography. Pyruvate kinase from fresh muscle and spleen was purified in one step by a similar process. Muscle and spleen RNA directed the synthesis of M subunits with molecular weights of approx. 61000 and 62000, respectively, the same as those of the corresponding fresh tissue monomers. In addition, peptide maps obtained by partial digestion of neosynthesized M1 and M2 with V8 protease from Staphylococcus aureus confirmed that these polypeptides were clearly different.

Developmental regulation of the aldolase A muscle-specific promoter during in vivo muscle maturation is controlled by a nuclear receptor binding element.

During the post-natal period, skeletal muscles undergo important modifications leading to the appearance of different types of myofibers which exhibit distinct contractile and metabolic properties. This maturation process results from the activation of the expression of different sets of contractile proteins and metabolic enzymes, which are specific to the different types of myofibers. The muscle-specific promoter of the aldolase A gene (pM) is expressed mainly in fast-twitch glycolytic fibers in adult body muscles. We investigate here how pM is regulated during the post-natal development of different types of skeletal muscles (slow or fast-twitch muscles, head or body muscles). We show that pM is expressed preferentially in prospective fast-twitch muscles soon after birth; pM is up-regulated specifically in body muscles only later in development. This activation pattern is mimicked by a transgene which comprises only the 355 most proximal sequences of pM. Within this region, we identify a DNA element which is required for the up-regulation of the transgene during post-natal development in body muscles. Comparison of nuclear M1-binding proteins from young or adult body muscles show no qualitative differences. Distinct M1-binding proteins are present in both young and adult tongue nuclear extracts, compared to that present in gastrocnemius extracts.

Myotube-specific activity of the human aldolase A M-promoter requires an overlapping binding site for NF1 and MEF2 factors in addition to a binding site (M1) for unknown proteins.

The human aldolase A gene is expressed in several tissues through the use of three alternative promoters. The activity of one of the promoters, pM, is restricted to skeletal muscle. We reported previously that a proximal 280 bp pM fragment confers tissue-specific expression to a CAT reporter gene in transgenic mice. This small regulatory region directs expression to muscle composed mainly of fast-twitch fibers. Here we show that a minimal promoter fragment from base-pairs -164 to +45 is sufficient to highly active pM during myoblast differentiation in cell culture and demonstrate that two DNA elements play a major role in this activation. These elements consist of a binding site (M1) for unknown ubiquitous proteins and an overlapping binding site for MEF2 and NF1 families of transcription factors. The NF1 factor constitute the main binding activity on the MEF2/NF1 site and, interestingly, some of the DNA-protein complexes that form with muscle nuclear extracts on the NF1 element differ from those that form with non-muscular extracts.

Muscle-type phosphorylase activity present in muscle cells cultured from three patients with myophosphorylase deficiency.

Skeletal muscle fibers cultured from three patients whose mature fibers are deficient in glycogen myophosphorylase (EC 2.4.1.1) were shown to become rather mature, to have no excessive glycogen accumulation, and to develop signifcant myophosphorylase activity. That activity was characterized electrophoretically and immunologically and shown to be muscle phosphorylase rather than a genetically different type, thereby demonstrating true "rejuvenation" in culture of an enzyme genetically programmed ultimately to be deficient.

Isoenzyme pattern of phosphorylase in white blood cells and fibroblasts from patients with liver phosphorylase deficiency.

Isoenzyme patterns of phosphorylase in white blood cells and cultured fibroblasts of a patient affected with liver-type phosphorylase deficiency were studied. Three bands were observed with electrofocusing of white blood cells and liver from controls. In the white blood cells of the patient only two bands were observed. Patient and control fibroblasts showed two bands, probably identical to the two bands observed in the patient's white blood cells. These results indicate that the liver-type phosphorylase is not expressed in the cultured fibroblasts.

Independence and interdependence of the three human aldolase A promoters in transgenic mice.

The human aldolase A gene is transcribed from three alternative promoters, clustered in a small 1.6-kb DNA domain. In transgenic mice, the upstream pN and the downstream pH promoters are ubiquitous, whereas the pM promoter, located between pN and pH, is activated specifically in fast skeletal muscles. A strong ubiquitous enhancer, lying upstream of the pH promoter, is necessary for both pN and pH ubiquitous activities, whereas a fast-muscle-specific enhancer, located upstream of the pM promoter, is required for pM-specific activation. In the present study, we use the transgenic mice model to further investigate the contribution of these two regulatory elements to the overall control of these three promoters. We confirm that the pM and pH promoters are activated independently of each other and, in particular, we show that the activation of pM in fast muscle is not responsible for the downregulation of the downstream pH in this tissue. By contrast, the pN promoter needs the presence of both enhancers to reproduce its correct pattern of activity and is unable to function autonomously in vivo.

A ubiquitous enhancer shared by two promoters in the human aldolase A gene.

The human aldolase A gene is transcribed from three different promoters, which are all clustered within a 1.6 kbp DNA domain. Two of these, PN and PH, are ubiquitous and seem to be co-regulated in most tissues while the third one, PM, is specific to adult skeletal muscle. We investigated the sequences involved in the ubiquitous activity of the PN and PH promoters of the human aldolase A gene. Deletion analysis, performed by transient expression assays of chloramphenicol acetyltransferase reporter genes in human HepG2 hepatoma cells, indicated that PH activity results from the interaction of an upstream activating region with two distinct core promoters. The upstream activating region was able to stimulate transcription from the HSV tk promoter as efficiently as the SV40 enhancer in all cell types tested. It appears, therefore, to be a strong ubiquitous enhancer. DNAsel footprinting revealed protections covering sequences scattered along the enhancer, including Sp1 and AP1 motifs. Importantly, we found that this enhancer was also necessary to activity of the other ubiquitous promoter of the aldolase A gene, PN. These studies demonstrate that expression of the human aldolase A gene is mediated by a complex interplay of enhancer and promoter elements.

Reversible extinction of insulin gene expression in insulinoma x fibroblast somatic cell hybrids.

To investigate for the presence of regulator(s) repressing the expression of insulin gene in cells other than pancreatic beta cells, rat insulinoma (RIN) cells secreting insulin were hybridized with fibroblasts from various species. In RIN x L mouse fibroblast hybrids, which maintained most of the parental chromosomes, no insulin transcripts were detected. In three RIN x Indian muntjac fibroblast hybrids and one RIN x human fibroblast hybrid which had lost DNA from the fibroblast parent through subculture, expression of the insulin gene was first extinguished and then reexpressed. This suggests that negative regulator(s), present in fibroblasts, can turn off insulin gene expression in RIN x fibroblast hybrids as long as the gene(s) contributed by the fibroblast are maintained.

Cell-free translation of messenger RNAs from adult and fetal human muscle. Characterization of neosynthesized glycogen phosphorylase, phosphofructokinase and glucose phosphate isomerase.

Using a procedure of ethanol precipitation in concentrated guanidine . HCl solutions followed by chloroform/isoamylic alcohol extraction and washing in 3 M sodium acetate, we isolated high-molecular-weight cellular RNA from human fetal and adult skeletal muscle. About 500 micrograms RNA were obtained/g of fetal muscle and 50 micrograms RNA/g of adult muscle. Both RNA preparations were efficiently translated in a cell-free reticulocyte lysate system and directed synthesis of various polypeptides, one of them of Mr 200,000 probably corresponding to myosin heavy chains. Dodecylsulphate/polyacrylamide gel electrophoretic pattern of polypeptides neosynthesized using either fetal or adult RNA exhibited several differences. Three neosynthesized cytosolic muscle enzymes were purified from the translation mixtures using a micro-method of immunoaffinity chromatography; specificity of the neosynthesized polypeptides purified according to this procedure was checked by immunological competition with the corresponding unlabeled pure muscle enzymes. Successful cell-free translation of RNA from adult skeletal muscle and purification of neosynthesized human enzymes are reported for the first time. These methods, indispensable for further studies on human adult muscle gene expression, could also shed light on the mechanism of some inherited molecular diseases and tumoral or dystrophic processes.

In vivo developmental modifications of the expression of genes encoding muscle-specific enzymes in rat.

cDNA clones for rat muscle-type creatine kinase and glycogen phosphorylase and aldolase A were isolated from a rat muscle cDNA library. An additional clone recognizing an unidentified 2.7-kilobase pair mRNA species was also isolated. These cDNA clones were used as probes to investigate the expression of the corresponding mRNAs during muscle development. Two aldolase A mRNA species were detected, one of 1650 bases expressed in non-muscle tissues, fetal muscle, and adult slow-twitch muscle, the other of 1550 bases was highly specific of adult fast-twitch skeletal muscle differentiation. These aldolase A mRNAs were shown by primer extension to differ by their 5' ends. The accumulation of muscle-type phosphorylase and creatine kinase and muscle-specific aldolase A mRNA accumulation during muscle development seems to be a coordinate process occurring progressively from the 17th day of intrauterine life up to the 30th day after birth. In contrast, the 2.7-kilobase pair RNA species is maximally expressed at the 1st week after birth as is the neonatal form of myosin heavy chain mRNA.

Expression of aldolase A messenger RNAs in human adult and foetal tissues and in hepatoma.

3 specific cDNA clones for human aldolase A were isolated from a human muscle library. One of them was subcloned in M 13 phage, then used as a probe to investigate the patterns and the levels of aldolase A mRNA in various human tissues. Two mRNA species differing in length were observed. The lighter one -1550 bases- was found specific to skeletal muscle; its amount increased during muscle development. The heavier aldolase A mRNA -1650 bases- accounted for foetal and ubiquitous presence of aldolase A isozyme. The resurgence of aldolase A in hepatomas occurred through this latter mRNA species.

Extinction of the human insulin gene expression in insulinoma x fibroblast somatic cell hybrids involves cis-acting DNA elements.

Insulin gene expression in rat insulinoma (RIN) cells is extinct in RIN x fibroblast hybrids and can reappear upon loss of DNA contributed by the fibroblast parent. (Besnard et al., Exp. Cell Res. 185:101-108, 1989). In the present study, we looked for the role of 5'-flanking sequences of the human insulin gene in the negative control observed in the hybrids. RIN cells were transformed with composite genes which consisted of the coding sequence of the gpt gene placed under the control of 5'-flanking regions of the human insulin gene (Ins.gpt gene). Upon hybridization of these cells with mouse fibroblasts, the expression of both Ins.gpt and endogenous rat insulin genes were suppressed together. The results obtained indicate that cis-acting DNA elements are involved in the negative control of the gene. These elements are located in a fragment spread from -258 to +241 of the transcription origin of the human insulin gene.

Functional analysis of DNase-I hypersensitive sites at the mouse porphobilinogen deaminase gene locus. Different requirements for position-independent expression from its two promoters.

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

Testis expression of hormone-sensitive lipase is conferred by a specific promoter that contains four regions binding testicular nuclear proteins.

The testicular isoform of hormone-sensitive lipase (HSLtes) is encoded by a testis-specific exon and 9 exons common to the testis and adipocyte isoforms. In mouse, HSLtes mRNA appeared during spermiogenesis in round spermatids. Two constructs containing 1.4 and 0.5 kilobase pairs (kb) of the human HSLtes gene 5'-flanking region cloned upstream of the chloramphenicol acetyltransferase gene were microinjected into mouse oocytes. Analyses of enzyme activity in male and female transgenic mice showed that 0.5 kb of the HSLtes promoter was sufficient to direct expression only in testis. Cell transfection experiments showed that CREMtau, a testis-specific transcriptional activator, does not transactivate the HSLtes promoter. Using gel retardation assays, four testis-specific binding regions (TSBR) were identified using testis and liver nuclear extracts. The testis-specific protein binding on TSBR4 was selectively competed by a probe containing a SRY/Sox protein DNA recognition site. Sox5 and Sox6 which are expressed in post-meiotic germ cells bound TSBR4. Mutation of the AACAAAG motif in TSBR4 abolished the binding. Moreover, binding of the high mobility group domain of Sox5 induced a bend within TSBR4. Together, our results showed that 0.5 kb of the human HSLtes promoter bind Sox proteins and contain cis-acting elements essential for the testis specificity of HSL.