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Research Notes: Nuclear Respiratory Factor-1 (NRF-1)Nucleic Acids Res. 2005 Aug 22. The imprinted SNRPN locus is a complex transcriptional unit that encodes the SNURF and SmN polypeptides as well as multiple non-coding RNAs. SNRPN is located within the Prader-Willi and Angelman syndrome (PWS/AS) region that contains multiple imprinted genes, which are coordinately regulated by a bipartite imprinting center (IC). The SNRPN 5' region co-localizes with the PWS-IC and contains two DNase I hypersensitive sites, DHS1 at the SNRPN promoter, and DHS2 within intron 1, exclusively on the paternally inherited chromosome. We have examined DHS1 and DHS2 to identify cis- and trans-acting regulatory elements within the endogenous SNRPN 5' region. Analysis of DHS1 by in vivo footprinting and chromatin immunoprecipitation identified allele-specific interaction with multiple regulatory proteins, including NRF-1, which regulates genes involved in mitochondrial and metabolic functions. DHS2 acted as an enhancer of the SNRPN promoter and contained a highly conserved region that showed allele-specific interaction with unphosphorylated RNA polymerase II, YY1, Sp1 and NRF-1, further suggesting a key role for NRF-1 in regulation of the SNRPN locus. We propose that one or more of the regulatory elements identified in this study may also contribute to PWS-IC function. Excerpt from the full text article: In addition, we have identified by sequence analysis a conserved potential NRF-1 binding site in the NDN promoter region, which coincides with a sequence that is in vivo footprinted on the paternal NDN allele only. The fact that NRF-1 may be regulating at least some of the genes in the PWS/AS region is interesting because of the involvement of NRF-1 in the regulation of genes related to mitochondrial biogenesis and function, metabolism (including growth factor receptors and factors involved in glucose homeostasis), DNA replication and transcriptional regulation. This suggests that genes in the AS/PWS region and genes that function in metabolism and in cellular energetics may be co-regulated through the common transcriptional regulator NRF-1. This would further suggest a potential link between energy metabolism and aspects of the PWS phenotype (e.g. obesity and growth factor deficiency). However, the resting metabolic rate of PWS patients does not seem to differ from that of normal obese individuals. FASEB J. 2003 Sep. Nuclear respiratory factor 1 (NRF-1) is a transcriptional activator of nuclear genes that encode a range of mitochondrial proteins including cytochrome c, various other respiratory chain subunits, and delta-aminolevulinate synthase. Activation of NRF-1 in fibroblasts has been shown to induce increases in cytochrome c expression and mitochondrial respiratory capacity. To further evaluate the role of NRF-1 in the regulation of mitochondrial biogenesis and respiratory capacity, we generated transgenic mice overexpressing NRF-1 in skeletal muscle. Cytochrome c expression was increased approximately twofold and delta-aminolevulinate synthase was increased approximately 50% in NRF-1 transgenic muscle. The levels of some mitochondrial proteins were increased 50-60%, while others were unchanged. Muscle respiratory capacity was not increased in the NRF-1 transgenic mice. A finding that provides new insight regarding the role of NRF-1 was that expression of MEF2A and GLUT4 was increased in NRF-1 transgenic muscle. The increase in GLUT4 was associated with a proportional increase in insulin-stimulated glucose transport. These results show that an isolated increase in NRF-1 is not sufficient to bring about a coordinated increase in expression of all of the proteins necessary for assembly of functional mitochondria. They also provide the new information that NRF-1 overexpression results in increased expression of GLUT4. Excerpts from the full text article: Mitochondria contain their own genome, which encodes 13 of the 100 proteins that constitute the enzyme complexes of the respiratory chain. Nuclear genes encode the other respiratory chain proteins as well as the factors that regulate mitochondrial gene expression, enzymes of the citrate cycle, and the enzymes of the fatty acid and ketone oxidation pathways. Research by a number of groups has provided new insights regarding how the expression of individual mitochondrial proteins and the biogenesis of mitochondria are regulated in mammalian tissues. Scarpulla and co-workers discovered nuclear respiratory factor 1 (NRF-1) and nuclear respiratory factor 2 (NRF-2), which are key transcriptional activators of nuclear genes encoding a number of mitochondrial constituents. These include various respiratory chain subunits, delta-aminolevulinate synthase (ALA synthase), mitochondrial transcription factor A (mtTFA), and a range of other mitochondrial and nonmitochondrial proteins. A major breakthrough occurred with the identification and characterization by Spiegelman's group of peroxisome proliferator-activated receptor (PPAR) coactivator 1 (PGC-1). Overexpression and/or activation of PGC-1 was shown to stimulate mitochondrial biogenesis in C2C12 myocytes, cardiac myocytes, and 3T3 adipocytes and to increase GLUT4 expression in myocytes. The increase in mitochondrial biogenesis induced by PGC-1 appears to be mediated at least in part by the coactivation of NRF-1 by PGC-1. Most of the energy required for muscle contraction during prolonged exercise is provided by generation of ATP via mitochondrial respiration. Endurance exercise induces an increase in skeletal muscle mitochondria, resulting in an enhanced capacity to generate ATP via oxidative phosphorylation. We have found that NRF-1 expression is increased in skeletal muscle 18 h after a bout of exercise, and Murakami et al. have reported that NRF-1 mRNA is increased 6 h after exercise. Although the exercise-induced increase in NRF-1 is relatively small, 50%, its effect on transcription is probably greatly potentiated by the large induction of PGC-1 expression that also occurs in response to a bout of exercise. Herzig et al. found that exposure of BALB 3T3 fibroblasts to serum induces an increase in the expression of cytochrome c and that this effect was mediated by phosphorylation and activation of NRF-1. Despite no increases in other mitochondrial enzymes that were measured, including citrate synthase and cytochrome oxidase, the isolated increase in cytochrome c resulted in a large increase in mitochondrial respiratory capacity. This finding by Herzig et al., which was interpreted as indicating that cytochrome c is rate-limiting for mitochondrial respiration, raised the possibility that in addition to increases in the size and number of mitochondria , exercise might enhance muscle respiratory capacity via a NRF-1-mediated induction of cytochrome c. The purpose of this study was to examine the effect of overexpression of NRF-1 in skeletal muscle. We found that cytochrome c expression was increased twofold in skeletal muscles of NRF-1 transgenic mice. The levels of some other mitochondrial proteins were increased 50–60%, while still others were unchanged. Muscle respiratory capacity was unchanged in the NRF-1 transgenic muscles. An unexpected finding that provides new information regarding the role of NRF-1 is that overexpression of NRF-1 resulted in increased expression of myocyte enhancer factor (MEF) 2A and the GLUT4 isoform of the glucose transporter in muscle. [...] Recent studies have provided evidence that PGC-1 provides the link between adaptive stimuli and increased mitochondrial biogenesis. It has been hypothesized that PGC-1 regulates mitochondrial biogenesis primarily through coactivation of NRF-1. In support of the concept that an increase in NRF-1 activity can mediate an increase in the capacity to generate ATP via mitochondrial respiration, Herzig et al. found that activation of NRF-1 by exposure of fibroblasts to serum induced an increase in mitochondrial respiratory capacity. However, this enhancement of oxidative capacity appeared to be due to an isolated induction of cytochrome c rather than to an increase in mitochondrial biogenesis. Skeletal muscle adapts to the energy demands of endurance exercise with increases in the size and number of mitochondria. Murakami et al. have reported that NRF-1 is increased 6 h after exercise. In contrast, Pilegaard et al. did not observe an increase in NRF-1 mRNA after a bout of exercise that induced a sevenfold increase in PGC-1 mRNA. We have found that a prolonged bout of exercise results in a large induction of PGC-1 expression and modest increases in NRF-1 and NRF-2 that precede the increments in muscle respiratory capacity and mitochondrial enzymes induced by endurance exercise. The purpose of the present study was to further evaluate the role of NRF-1 in the regulation of muscle respiratory capacity using transgenic mice that overexpress NRF-1 in their skeletal muscles. There was a twofold increase in cytochrome c protein in skeletal muscle in the NRF-1 transgenic mice. NRF-1 was originally identified as an activator of cytochrome c expression, and it is now well established that NRF-1 plays a major role in the adaptive increases in cytochrome c induced by stimuli involved in the regulation of cytochrome c expression, such as exposing fibroblasts to serum or electrical stimulation of cardiac myocytes. In addition to the NRF-1 binding site, the cytochrome c promoter contains recognition sites for the cAMP response element binding protein/activating transcription factor (CREB/ATF) family of transcription factors and for SP1; these factors have also been implicated in the regulation of cytochrome c expression. To our knowledge, the present results provide the first evidence that an isolated increase in NRF-1, i.e., in the absence of stimuli that result in increases in CREB/ATF and/or SP1, can bring about increased cytochrome c expression. This finding implies that the constitutively expressed levels of these other transcription factors in skeletal muscle are sufficient to permit induction of cytochrome c by an increase in NRF-1 expression. There are two NRF-1 binding sites in the promoter of the ALA synthase gene, and it has been shown that these NRF-1 binding sites are necessary for promoter activity. In fact, the role of NRF-1 in controlling ALA synthase expression is so well established that we used the NRF-1 recognition sequence from the ALAS synthase promoter to quantify NRF-1 DNA binding activity in the EMSA. In the present study, a 10-fold increase in NRF-1 in the muscle of the NRF-1 transgenic mice was associated with only a 50% increase in ALA synthase expression. In contrast, a 50% increase in NRF-1, induced by a bout of exercise or by raising cytosolic Ca2+ by exposing L6 myocytes to caffeine, was associated with a greater than twofold increment in ALA synthase protein. Similar differences in response were seen in a study on HeLa cells in which overexpression of UCP-1 resulted in a modest induction of NRF-1 and a large increase in ALA synthase expression, whereas a high level of NRF-1 overexpression resulted in a smaller increase in ALA synthase. Thus, it seems probable that the relatively small induction of ALA synthase by the massive increase in NRF-1 in the transgenic muscles is due to the requirement for concomitant increases of other transcription factors, NRF-1 phosphorylation, and/or coactivation by PGC-1 for optimal stimulation of ALA synthase expression by NRF-1. The absence of increases in other transcriptional activators or of the coactivating activity of PGC-1 likely also accounts for the relatively weak induction of core protein 1. The finding that mtTFA was not increased in the NRF-1 transgenic muscles, which helps explain why overexpression of NRF-1 did not result in an increase in functional mitochondrial, is likely due to a requirement for both NRF-1 and NRF-2 for stimulation mtTFA expression. The respiratory capacity of the muscles of the NRF-1 transgenic mice was not increased despite a large increase in cytochrome c. Thus, in contrast to the finding that an increase in cytochrome c in fibroblasts in culture increases their respiratory capacity, our results show that cytochrome c concentration is not rate-limiting for mitochondrial respiration in adult skeletal muscle. PGC-1 is a powerful coactivator of NRF-1, and it has been postulated that increased transactivation of NRF-1 regulated genes could be the major mechanism by which PGC-1 induces an increase in mitochondrial biogenesis. However, it is clear from the present results that an isolated increase in NRF-1 is not sufficient to bring about a coordinated increase in the expression of all of the proteins necessary for the assembly of functional mitochondria. In addition to coactivation of NRF-1, PGC-1 coactivates PPAR and induces increased expression of NRF-1 and NRF-2. It seems reasonable that increases in the concentrations and/or transcriptional activity of all three of these factors, and probably additional transcription factors, account for the stimulation of mitochondrial biogenesis by PGC-1. The adaptive stimuli that have been shown to induce increases in skeletal muscle mitochondria, including exercise, hyperthyroidism, lowering of high energy phosphates resulting in activation of AMP kinase, and raising cytosolic Ca2+, also bring about increased expression of the GLUT4 isoform of the glucose transporter in muscle. We therefore routinely also measure muscle GLUT4 concentration in studies of mitochondrial biogenesis and, as a consequence, detected a functionally significant increase in GLUT4 protein concentration in the muscles of the NRF-1 transgenic mice. Pessin's group has shown that the promoter of the GLUT4 gene contains a MEF2 binding site that is essential for activation of transcription and that MEF2A binds to this element as a MEF2A-MEF2D heterodimer. They also found that insulin deficiency, which results in a marked decrease of GLUT4 in insulin-sensitive tissues, causes a selective down-regulation of MEF2A. The addition of MEF2A to nuclear extracts from insulin-deficient, diabetic rat muscle reversed a reduction in binding to the MEF2 element, leading Thai et al. to conclude that MEF2A is necessary for expression of GLUT4 in striated muscle and is responsible for hormonal/metabolic regulation of the GLUT4 gene. Studies of L6 myotubes and rat epitrochlearis muscles showing that activation of AMP kinase or raising cytosolic Ca2+ induces increases in MEF2A, MEF2D, and GLUT4, and the present finding that an isolated increase in NRF-1 results in increased expression of both MEF2A and GLUT4, support this conclusion. On the other hand, Michael et al. found that transfection of myotubes with PGC-1 resulted in an increase in GLUT4 expression mediated by coactivation by PGC-1 of MEF2C rather than MEF2A. In the present study, MEF2C was not increased in muscle in the NRF-1 transgenic mice. It seems possible that either MEF2A or MEF2C can mediate increased GLUT4 expression, depending on metabolic state and the inducing stimulus. PGC-1 increased GLUT4 expression by coactivation of MEF2C in the study by Michael et al. In the present study and in a previous study of the effects of raising cytosolic Ca2+ or activating AMPK, increased expression of GLUT4 was associated with increases in MEF2A protein. Thus, it may be that the initial stimulus for increased GLUT4 expression is provided by coactivation of MEF2C by PGC-1 and the increase in GLUT4 is subsequently maintained by increased expression of MEF2A. It is of course also possible that other transcription factors are involved in mediating the increase in GLUT4 expression found in the NRF-1 transgenic muscles. In addition to their roles in regulating expression of various mitochondrial proteins, NRF-1 and NRF-2 are transcriptional regulators of a number of key enzymes in other pathways involved in substrate metabolism. It is interesting that even though it is not a transcriptional activator of the GLUT4 gene, NRF-1 indirectly regulates the expression of GLUT4, which is rate-limiting for glucose transport into muscle and is therefore another key regulator of substrate metabolism. It is currently not known whether NRF-1 is a transcriptional activator of MEF2A. So, whether the increased expression of MEF2A in NRF-1 transgenic muscles is mediated directly by NRF-1 or is a secondary consequence of another action of NRF-1 is an open question. NRF-1 increases rapidly in skeletal muscle after a bout of exercise. PGC-1, a coactivator of NRF-1, also increases rapidly in muscle after exercise. Our finding that overexpression of NRF-1 results in enhanced GLUT4 expression in muscle helps to explain one of the mechanisms responsible for the concomitant increases in the capacities for glucose transport and oxidative generation of ATP in skeletal muscle adapting to endurance exercise. J Med Genet. 2003 Jun. Selections from the full text article: Fragile X syndrome (FXS) is a triplet repeat disorder caused by a large expansion of the CGG repeat in the 5'-untranslated region (UTR) of the fragile X mental retardation (FMR1) gene. Full mutation alleles are almost always associated with extensive hypermethylation of the repeat and of the upstream CpG island, which correlates with gene silencing and absence of the FMR1 protein. Cognitive function ranging from severe mental retardation to learning disabilities are found in affected people of both sexes. Many mildly affected people show "mosaic" methylation at the FMR1 promoter. Unusual alleles carrying a completely or partially unmethylated full mutation have been described. It was shown that in male patients with FXS with unmethylated alleles in the full mutation range, the FMR1 mRNA level is higher than in normal controls. This finding shows that upregulation of the FMR1 gene occurs in cells with unmethylated full mutation alleles and that the CGG triplet expansion does not suppress transcription directly. Thus, abnormal hypermethylation of the FMR1 promoter suppresses gene transcription. This hypothesis is also supported by the ability of 5-azadeoxycytidine (5-azadC) to restore the FMR1 gene expression in lymphoblastoid cell lines from patients with non-mosaic full mutation FXS by inducing DNA demethylation. The silencing of the hypermethylated FMR1 gene is consistent with a model in which methylation is coupled with the histone acetylation state. It has been found that the 5' end of the FMR1 gene of patients with FXS is associated with deacetylated histones H3 and H410 and that the treatment of fragile X cells with 5-azadC results in the reassociation of acetylated histones with the FMR1 promoter and transcriptional reactivation. This finding suggests that both methylation and histone deacetylation are linked to transcriptional inactivity. In fact, it has been shown that fragile X cell lines treated with histone hyperacetylating drugs can markedly potentiate the effect of 5-azadC on FMR1 gene expression. However, when used alone, such drugs induce only a modest reactivation of the FMR1 gene. The same pattern of dominance of DNA methylation over histone acetylation has also been reported for other genes, the promoter of which resides in a CpG island. Key points We report the effect of acetyl-L-carnitine and L-carnitine on the methylation of the FMR1 promoter in long term cultures. The methylation status of the FMR1 promoter region containing 52 CpG sites was analysed in lymphoblastoid cell lines derived from healthy subjects and patients with FXS by a sensitive bisulphite based technique. We also analysed the 23 CpG sites in exon 1 of the SNRPN gene. CpG sites in control cultures from healthy subjects remained unmethylated in all the experimental conditions described. No changes were seen in the SNRPN gene. The promoter region of the untreated fragile X cell lines remained generally hypermethylated although the methylation level of individual CpGs was variable. Both acetyl-L-carnitine and L-carnitine induced a modest though evident decrease of the FMR1 promoter hypermethylation in two of the three fragile X cell lines. Our data suggest that long term treatment with the two carnitines has a mild but detectable effect against methylation of the FMR1 alleles. Changes of the methylation patterns over a five year period of alleles from five brothers variably affected by FXS indicate that methylation of individual CpG cytosines is strikingly variable in hypermethylated genotypes obtained from an individual patient. A reduced frequency of hypermethylated alleles occurred in the leucocytes of the two mildly affected brothers. These findings suggest that maintenance of cytosine methylation is a dynamic process that favours unmethylated alleles. It is conceivable that some compounds can be identified that may modulate this process and achieve gene reactivation. Carnitine is a well known naturally occurring compound with an essential role in intermediary metabolism, mainly at the mitochondrial level. Acetyl-L-carnitine (-trimethyl-ß-acetyl-butyrrobetaine) is the carnitine ester naturally present in the central nervous system, differently distributed in the various areas. The enzyme carnitine acetyltransferase catalyses both the formation of acetyl-L-carnitine from carnitine and acetyl-coenzyme A (acetyl-CoA) and the reversible reaction. The modulation of the intracellular concentration of free CoA and acetyl-CoA is recognised to be a common mechanism for the various physiological activities of acetyl-L-carnitine, such as the acetylation of H4 histones. The chemical structure of acetyl-L-carnitine is similar to that of the acetylating agent butyrate. It has been shown that acetyl-L-carnitine, as well as butyrate, inhibits cytogenetic expression of the fragile X site in cultured lymphocytes of patients, suggesting that the interaction of these substances with the chromatin structure at the fragile site was present. Carnitine was also shown to suppress position effect variegation in Drosophila, another indication of a direct effect on chromatin. Finally, recent evidence suggests that acetyl-L-carnitine, the physiological form of carnitine, acts as a histone hyperacetylating agent at the FMR1 locus in fragile X cells. Because transcription of the FMR1 gene in fully mutated patients was obtained by treatment with butyrate, we decided to evaluate the effect of acetyl-L-carnitine and L-carnitine in lymphoblastoid cultures from patients with FXS. In the present study we investigated the effect on the CpG island methylation present in the FMR1 promoter after treatment with L-carnitine and acetyl-L-carnitine. We assessed the methylation status at 52 CpG sites of the FMR1 promoter using bisulphite treated, polymerase chain reaction (PCR) amplified genomic DNA obtained from lymphoblastoid cell cultures from healthy subjects and patients with FXS with CGG repeat expansions of different lengths. Furthermore, we controlled the effect of the two compounds on the methylation status of the putative promoter and exon 1 region of the gene called small nuclear ribonucleoprotein polypeptide N (SNRPN). This gene is reported to be involved in Prader-Willi syndrome and Angelman syndrome. It was chosen because in normal subjects only one allele is methylated, so each of the 23 CpGs present in this region will result in 50% methylated. In this way we could evaluate the effects of L-carnitine and acetyl-L-carnitine on another DNA region and our experimental approach at the same time. [...] Results In this study we investigated the effect of long term treatment with L-carnitine and acetyl-L-carnitine on the methylation status of the CpG island in the promoter region of the FMR1 gene by the bisulphite sequencing technique and on the transcription of the fully mutated FMR1 gene. Lymphoblastoid cell lines from patients with FXS and from healthy subjects were analysed during cell culture progression in the absence or presence either of L-carnitine or acetyl-L-carnitine. The fragile X cell cultures analysed (L, M, and F) were characterised by different CGG repeat expansions, which remained unchanged throughout the long term culturing as detected by Southern blot analysis. [...] The methylation status of the 52 tested CpG sites in cell cultures from patients with FXS is depicted in fig 2. The bisulphite sequencing of the FMR1 promoter was performed for each culture at the start of the cell culturing, after long term culturing, and after long term culturing in the presence of L-carnitine or acetyl-L-carnitine. Each column has been calculated as the ratio between the height of the thymine electropherogram peak and the sum of heights of cytosine and thymine peaks at the individual CpG site. Therefore, the black part of each column reflects the percentage of demethylation seen for each CpG site. A few individual CpG dinucleotides were partially unmethylated in all the starting cultures. In the F cell line the number of the sites that showed a partial demethylation increased from two to four in the control culture and to five in cells treated with L-carnitine and acetyl-L-carnitine. In this culture we found a striking spontaneous demethylation of sites 28 and 29. In the M cell line the CpG sites that are partially unmethylated increased from six and eight in the starting and untreated cultures, to 10 and 14 in cells treated with L-carnitine and acetyl-L-carnitine, respectively. In the L cell line the number of unmethylated CpG sites increased from four in the starting culture to six in the untreated culture, and to 13 in cells treated with L-carnitine and to 11 in cells treated with acetyl-L-carnitine. Only sites 28 and 42 were partially unmethylated in all experimental conditions. In particular, position 28 was unmethylated in up to 70% of the cells in the starting culture and in cultures treated with the two compounds. The mean methylation value for each culture, averaged on the 52 CpG sites, is summarised in table 1. The overall hypermethylated status of the L and M cell lines did not change after long term culturing with any added compound, but we found a decrease of mean methylation both with L-carnitine and acetyl-L-carnitine. The methylation degree of the F cell line did not change in long term cultures in the presence of the two compounds, although it showed a tendency to decrease methylation as well. The marked reduction of the mean methylation value in the untreated long term culture is the result of the unexpected high demethylation of only two sites (70% for site 28 and 60% for site 29). None of the 52 cytosines analysed in control cultures from healthy subjects was methylated in all the experimental conditions described confirming that methylation over time of the normal FMR1 gene is a very unlikely event. To control for the effect of our treatments on a non-pathologically methylated sequence, the human SNRPN sequence in the putative promoter, exon 1, and 5' region of intron 1 was also analysed. The methylation pattern of all individual 23 CpG sites in the DNA fragment analysed was evaluated. As expected, a 50% methylation was found in both control and fragile X derived starting cultures. The quite striking similarity between related electropherograms enables us to infer that not even a minimal change occurred after long term culturing either with or without the two carnitines (data not shown). The RT-PCR designed for detecting transcriptional reactivation of the FMR1 gene in the three tested fragile X cell lines was negative before treatment, as expected, but remained negative even after long term culturing with both acetyl-L-carnitine and L-carnitine; meanwhile FMR1 gene expression was detected in all cultures from normal subjects. Discussion The clinical features of fragile X syndrome are the result of the hampered transcriptional activity of the FMR1 promoter secondary to its hypermethylated status. Attempts to reactivate gene expression in cells with a CGG expansion in the full mutation range have been successful. This was obtained by reducing DNA methylation with 5-azadC and, although to a lesser extent, by inducing histone hyperacetylation with drugs such as trichostatin A, 4-phenylbutyrate, and sodium butyrate. A general view recently emerged that histones regulate the access of proteins to the DNA and post-transcriptional histone modifications such as lysine acetylation or methylation mediate the epigenetic effects of DNA methylation patterns. It has become apparent that histone deacetylation follows DNA hypermethylation in a cascade process leading to chromatin inactivation and it was shown by chromatin immunoprecipitation that the inactive fully mutated FMR1 gene is associated with relatively hypoacetylated histones. It has been shown that USF1, USF2, and -Pal/Nrf-1 are the major transcription factors that bind the FMR1 promoter at the E box and -Pal/Nrf-1 DNA binding sites, respectively, as indicated in fig 1. The binding of -Pal/Nrf-1 is abolished by methylation, whereas the binding of USF1 and USF2 is only reduced. Thus, DNA methylation can have a direct, as well as an indirect, effect on the transcription of the FMR1 gene and seems to be dominant over histone acetylation, that is, FMR1 reactivation cannot take place efficiently only with histone hyperacetylating drugs, if DNA is not previously demethylated, as suggested by Chiurazzi et al. However, data obtained from studies on Neurospora crassa suggested that, at least in some instances, histone hyperacetylation may eventually cause DNA demethylation, just as DNA demethylation would induce histone reacetylation (and gene reactivation). We hypothesised that a long term treatment with histone hyperacetylating drugs may therefore be effective in reactivating FMR1 expression in fragile X cell cultures. Unfortunately, butyrate cannot be added for too long in culture because it readily causes cell cycle arrest, and previous experiments with sodium butyrate or 4-phenylbutyrate alone had to be limited to short treatments of 24-48 hours. Previous results from our groups have shown that acetyl-L-carnitine increases levels of H4 acetylation, also in the FMR1 gene itself. Therefore we were interested in exploring the DNA methylation status of the FMR1 promoter after long term treatment with acetyl-L-carnitine and L-carnitine. In the present study, no change in the CGG repeat expansion was detected by Southern blotting and long term treatment with the two carnitines that were well tolerated by cells that possibly seemed to benefit in growth and viability. However, no reactivation of FMR1 gene transcription has been shown in treated fragile X cell lines compared with the untreated ones. Experiments with RT-PCR were negative at the start of treatment in cell lines F, M, and L, but remained negative after the three month treatment with the two compounds. We found that the overall methylation status of the FMR1 promoter in fragile X lymphoblastoid cell lines is quite stable after culturing for about 100 cell duplications without any treatment. However, as previously reported, we found that methylation of individual CpG cytosine is variable in hypermethylated cell lines. On the other hand, we found a modest though measurable reduction of the hypermethylated status of the promoter in two of the three fragile X cell lines (L and M) grown with L-carnitine or acetyl-L-carnitine. It is noteworthy that the two compounds were less efficient in the F cell line, which harbours an expansion of more than 2.5 kb suggesting that acetyl-L-carnitine and L-carnitine may be somewhat effective in reversing the hypermethylation present in full mutations with smaller CGG expansions. The SNRPN gene is monoallelically expressed and has been used as an internal control for testing the potential demethylating effect of carnitines on another locus as well as for checking the efficiency of our experimental approach. The SNRPN gene is expressed from the paternal allele and hypermethylation is present only in the maternal allele. We consistently obtained a 1:1 ratio between the cytosine and thymine peaks at each of the 23 CpG sites investigated, before any treatment, confirming that our bisulphite sequencing approach was working well. Treatment with carnitines did not change the hemimethylated status of the SNRPN promoter region. These data suggest that carnitines do not affect the methylation status of the cell itself but may be effective in the abnormal hypermethylation of the FMR1 gene to move the methylation equilibrium towards the unmethylated status. Overall these data show that methylation of individual CpGs in the FMR1 gene is a dynamic process that seems to favour unmethylated alleles (see sites 28 and 29 of F cell lines). This trend seems to be favoured by the carnitines we used. We suppose that L-carnitine and acetyl-L-carnitine, acting on the histone hyperacetylation process, favour demethylation of the FMR1 gene. It is conceivable to suppose that much longer treatments with acetyl-L-carnitine and L-carnitine might further decrease the methylation status. Any future pharmacological attempt at reactivating the FMR1 gene in vivo should therefore contemplate the use of safe DNA demethylating drugs ideally targeted to the FMR1 promoter region. Biochim Biophys Acta. 1998 Jun 5. Nuclear respiratory factor 1 (NRF-1) is a regulatory factor of nuclear genes for respiratory subunits and for components of the mitochondrial transcription and replication machinery. This study investigated the effects of an acute bout of aerobic exercise on the postexercise expression of mRNA for NRF-1 and RNA moiety of endonuclease for mitochondrial RNA processing (MRP-RNA) in soleus muscle of 5 days-trained and untrained rats. In the trained group, rats were run on a motor-driven treadmill at a speed of 25 m/min for 90 min/day for 5 days. On the final day, rats were run by the same procedures and were sacrificed at various postexercise time points (0.5, 3, 6, and 24 h). The basal level of cytochrome oxidase activity was increased by the training, which was associated with the increase in the expression of mRNAs for subunit VIc and III of the enzyme. The NRF-1 mRNA expression was transiently increased by approximately 35% at the time point of 6 h after exercise, although the basal level of the expression was not altered by training. A similar transient increase (approximately 50%) in NRF-1 expression by the acute bout of exercise was also observed in untrained rats. In contrast to the NRF-1 expression, the basal level of MRP-RNA abundance was not altered by 5 days training and was not affected by the single exercise bout in either 5 days-trained or untrained rats. These results suggest that the postexercise increase in NRF-1 mRNA expression in rat skeletal muscle may be an early response to endurance exercise for an enhancement of the mitochondrial oxidative capacity. |