No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
NFI-C2 negatively regulates α-sarcoglycan promoter activity in C2C12 myoblasts
Abstract
alpha-Sarcoglycan striated muscle-specific protein is a member of the sarcoglycan-sarcospan complex. Positive and negative transcriptional regulation of sarcoglycan genes are important in sarcoglycan's intracellular localization and sarcolemmal stability. In the present work we assessed the function of NFI transcription factors in the regulation of alpha-sarcoglycan promoter through the C2C12 cell line differentiation. NFI factors act alternatively as activators and negative modulators of alpha-sarcoglycan promoter activity. In myoblasts NFI-A1.1 and NFI-B2 are activators, whereas NFI-C2 and NFI-X2 are negative regulators. In myotubes, all NFI members are activators, being NFI-C2 the less potent. We identified the alpha-sarcoglycan promoter NFI-C2 response element by testing progressive deletion constructs and point mutations in C2C12 cells over-expressing NFI-C2. Gel-shift and chromatin immunoprecipitation experiments demonstrated that NFI factors are indeed interacting in vitro and in vivo with the binding sequence. These results suggest a NFI role in C2C12 cell differentiation.
Supplementary resource (1)
Nucleotide Sequence
June 2004
... Luciferase activity was measured using the Dual Luciferase Reporter Assay System (Promega) according to manufacturer instructions in a TD-20/20 luminometer (Turner Designs). The 2.6-kb α-SG full-length promoter construct (Fig. 2, construct 1) was 6-fold up-regulated in myotubes vs. myoblasts, suggesting that the promoter is activated during C2C12 myogenic differentiation, in agreement with previous reports [16] [17] [18]. Constructs 1–13 presented homogeneous activities in myoblasts (Fig. 2B), suggesting that the α-SG promoter drives basal activity at this differentiation stage. ...
... Construct 6 presented a 2.5-fold activity increase, as compared with construct 5, indicating that the DNA region between nucleotides −1892 and −1710 corresponds to a negative regulatory sequence. This region includes binding sites for NFI, known to negatively and positively regulate the α-SG promoter [17], Sp1, and MyoD family members, which activate muscle-specific gene expression [20]; this fact opens the possibility that E-boxes contained in this region could contribute to its negative activity. Deletion of the promoter region from −1710 to −1522 in construct 7 led to a 3-fold decrease in luciferase activity as compared with construct 6, indicative of a positive regulatory region presence (Fig. 2B, construct 7). ...
... To this end, we performed chromatin immunoprecipitation assays as previously described [17], in C2C12 myoblasts and myotubes using anti-MyoD (M-318), -TFIID (Sl-1) and -TFIIB (Sl-1) antibodies . We utilized an anti-c-myc (9E10) antibody as control. ...
The mouse alpha-sarcoglycan gene is expressed in muscle cells during differentiation, but its transcriptional regulation is not understood. We have characterized the promoter region of the mouse alpha-sarcoglycan gene. This region is composed of positive and negative regulatory elements that respond to the myogenic differentiation environment. Accordingly, MyoD transactivates the alpha-sarcoglycan full-length and the proximal promoter. Chromatin immunoprecipitation assays revealed that MyoD, TFIID, and TFIIB interact with the distal promoter in C2C12 myoblasts, a stage at which the alpha-SG promoter appears to drive basal activity. In myotubes, such factors are located concomitantly at the distal promoter and at a DNA region around the proximal promoter. In agreement with these results, TFIID and TFIIB co-immunoprecipitate with MyoD. We conclude that the alpha-SG promoter is activated by MyoD, which interacts with TFIID and TFIIB in a protein complex differentially located at the distal promoter and around the proximal promoter during myogenic cell differentiation.
... F5 and F6 constructs include nucleotides spanning from +4 to −1892 and from +4 to −1710, respectively [20]. Cell maintenance, differentiation, transfection , as well as luciferase activity assays in myoblasts and myotubes were performed as previously described [19]. Integrity of the constructs employed in this work was confirmed by sequencing with a dye terminator cycle reaction kit (ABI PRISM 310, Applied Biosystems, Foster City, CA, USA). ...
... Previous chromatin immunoprecipitation experiments revealed that MyoD and basal transcription factors interact with a DNA region potentially including E1 and E2 in the C2C12 cell's chromatin context [20]; however, if MyoD directly interacts with such regulatory elements remained uncertain. To address this issue we employed annealed [γ- 32 P] ATP labeled oligonucleotides including E1 and E2 in electrophoretic mobility shift assays (EMSA), in presence of C2C12 myoblast and myotube nuclear extracts (Active Motif) (Fig. 2C), as previously described [19]. The sequence of the oligonucleotides employed as a probe, as well as their position relative to the α-SG transcription start site, is shown in Fig. 2A. ...
... In this regard, it is possible that in the α-SG promoter chromatin context, the MyoD-containing complex associated with E1 and E2 incorporates negative activities from nearby interacting transcriptional repressors. For example, there is evidence suggesting that nuclear factor I-C (NFI-C), which negatively regulates the α-SG promoter, binds to a sequence located downstream from E1 and E2 [19]. In addition , such NFI binding site is adjacent to a consensus sequence for recognition by Sp1, which also plays a negative role in α-SG promoter activity (unpublished results). ...
The alpha-SG promoter is composed of a plethora of cis-regulatory elements, whose individual contribution to alpha-SG gene expression modulation remains unknown. We have identified a negative regulatory element in the alpha-SG distal promoter including two conserved E-boxes (E1 and E2), which interact with MyoD. We found that E1 and E2 negatively modulate the transactivation potential of MyoD on the alpha-SG core promoter. Moreover, such negative effect is mainly mediated by E2, which is surrounded by conserved nucleotides conferring MyoD binding capacity. Our results suggest that modulation of MyoD activity by E1, and particularly E2, contributes to the negative regulation of alpha-SG gene expression during myogenic differentiation.
Background:
Type 2D limb-girdle muscular dystrophy (LGM2D) is a progressive disorder caused by mutations in the alpha sarcoglycan (α-SG) gene. In mice, the α-SG gene contains two promoters that regulate the expression of two different mRNAs (A and B). However, their gene expression pattern during embryonic development has not been explored and their regulation by myogenic and cardiogenic transcription factors has been only partially studied.
Results:
During embryonic development, mRNA A and B of α-SG gene were initially detected in hypaxial muscles, heart, stomach, tongue, and mesenchymal cells, which surround the dorsal region of the somites. Moreover, mRNA B was exclusively expressed in the floor plate and notochord and in the interdigits of limbs. In vitro, MyoD and myogenin positively regulated the transcription of mRNA B during skeletal myogenesis, whereas mRNA A was activated only for MyoD in differentiated skeletal muscle. In addition, Gata-4 together with Mef2c may regulate the expression of mRNA B in heart development, whereas Nkx2.5 and myocardin may activate expression of mRNA A in the differentiated cardiomyocyte.
Conclusions:
The differential expression of α-SG mRNAs during mouse embryonic development may be a consequence of the differential regulation of both promoters by myogenic and cardiogenic factors.
CTCF nuclear factor regulates many aspects of gene expression, largely as a transcriptional repressor or via insulator function.
Its roles in cellular differentiation are not clear. Here we show an unexpected role for CTCF in myogenesis. Ctcf is expressed in myogenic structures during mouse and zebrafish development. Gain- and loss-of-function approaches in C2C12
cells revealed CTCF as a modulator of myogenesis by regulating muscle-specific gene expression. We addressed the functional
connection between CTCF and myogenic regulatory factors (MRFs). CTCF enhances the myogenic potential of MyoD and myogenin
and establishes direct interactions with MyoD, indicating that CTCF regulates MRF-mediated muscle differentiation. Indeed,
CTCF modulates functional interactions between MyoD and myogenin in co-activation of muscle-specific gene expression and facilitates
MyoD recruitment to a muscle-specific promoter. Finally, ctcf loss-of-function experiments in zebrafish embryos revealed a
critical role of CTCF in myogenic development and linked CTCF to broader aspects of development via regulation of Wnt signaling.
We conclude that CTCF modulates MRF functional interactions in the orchestration of myogenesis.
Nuclear factor I (NFI) is a transcription factor playing wide role in signal transduction pathways and developmental processes in higher eukaryotes. In order to produce recombinant NFI proteins for functional and structural studies, full length cDNAs of individual isoforms were subcloned into pETM30 vector and expressed in Escherichia coli. Although the fusion proteins containing both glutathione S-transferase (GST) and His6 tags at the N-terminus could be overexpressed in detectable amounts, they were found mainly, if not exclusively, in insoluble form. Purification yield was improved by modification of cell disruption procedure and by the use of detergent Tween 20. The final purification strategy represents a triple-helix affinity chromatography consisting of prepurification of bacterial lysate on Heparin-Sepharose with subsequent immobilized metal affinity and glutathione affinity chromatography. Heparin chromatography was crucial for obtaining active NFI proteins, whereas the other steps significantly improved the purity of isolated proteins. As demonstrated by EMSA and DNase I protection assay, the recombinant proteins were able to recognize their cognate DNA sequences.
Alpha sarcoglycan (alpha-SG) is highly expressed in differentiated striated muscle, and its disruption causes limb-girdle muscular dystrophy. Accordingly, the myogenic master regulator MyoD finely modulates its expression. However, the mechanisms preventing alpha-SG gene expression at early stages of myogenic differentiation remain unknown. In this study, we uncovered Sox9, which was not previously known to directly bind muscle gene promoters, as a negative regulator of alpha-SG gene expression. Reporter gene and chromatin immunoprecipitation assays revealed three functional Sox-binding sites that mediate alpha-SG promoter activity repression during early myogenic differentiation. In addition, we show that Sox9-mediated inhibition of alpha-SG gene expression is independent of MyoD. Moreover, we provide evidence suggesting that Smad3 enhances the repressive activity of Sox9 over alpha-SG gene expression in a transforming growth factor-beta-dependent manner. On the basis of these results, we propose that Sox9 and Smad3 are responsible for preventing precocious activation of alpha-SG gene expression during myogenic differentiation.
The transcription factor family of nuclear factor I (NFI) proteins is encoded by four closely related genes: Nfia, Nfib, Nfic, and Nfix. A potential role for NFI proteins in regulating developmental processes has been implicated by their specific expression
pattern during embryonic development and by analysis of NFI-deficient mice. It was shown that loss of NFIA results in hydrocephalus
and agenesis of the corpus callosum and that NFIB deficiency leads to neurological defects and to severe lung hypoplasia,
whereas Nfic knockout mice exhibit specific tooth defects. Here we report the knockout analysis of the fourth and last member of this
gene family, Nfix. Loss of NFIX is postnatally lethal and leads to hydrocephalus and to a partial agenesis of the corpus callosum. Furthermore,
NFIX-deficient mice develop a deformation of the spine, which is due to a delay in ossification of vertebral bodies and a
progressive degeneration of intervertebral disks. Impaired endochondral ossification and decreased mineralization were also
observed in femoral sections of Nfix−/− mice. Consistent with the defects in bone ossification we could show that the expression level of tetranectin, a plasminogen-binding
protein involved in mineralization, is specifically downregulated in bones of NFIX-deficient mice.
The phylogenetically conserved nuclear factor I (NFI) family of transcription/replication proteins is essential both for adenoviral
DNA replication and for the transcription of many cellular genes. We showed previously that the four murine NFI genes (Nfia, Nfib, Nfic, and Nfix) are expressed in unique but overlapping patterns during mouse development and in adult tissues. Here we show that disruption
of the Nfia gene causes perinatal lethality, with >95% of homozygous Nfia−/− animals dying within 2 weeks after birth. Newborn Nfia−/− animals lack a corpus callosum and show ventricular dilation indicating early hydrocephalus. Rare surviving homozygous Nfia−/− mice lack a corpus callosum, show severe communicating hydrocephalus, a full-axial tremor indicative of neurological defects,
male-sterility, low female fertility, but near normal life spans. These findings indicate that while the Nfia gene appears nonessential for cell viability and DNA replication in embryonic stem cells and fibroblasts, loss of Nfia function causes severe developmental defects. This finding of an NFI gene required for a developmental process suggests that
the four NFI genes may have distinct roles in vertebrate development.
Promoter-specific differences in the function of transcription factors play a central role in the regulation of gene expression. We have measured the maximal transcriptional activation potentials of nuclear factor I (NFI) proteins encoded by each of the four identified NFI genes (NFI-A, -B, -C, and -X) by transient transfection in JEG-3 cells using two model NFI-dependent promoters: 1) a simple chimeric promoter containing a single NFI-binding site upstream of the adenovirus major late promoter (NFI-Ad), and 2) the more complex mouse mammary tumor virus long terminal repeat promoter. The relative activation potentials for the NFI isoforms differed between the two promoters, with NFI-X being the strongest activator of NFI-Ad and NFI-B being the strongest activator of the MMTV promoter. To determine if these promoter-specific differences in activation potential were due to the presence of glucocorticoid response elements (GREs), we added GREs upstream of the NFI-binding site in NFI-Ad. NFI-X remains the strongest activator of the GRE containing simple promoter, indicating that differences in relative activation potential are not due solely to the presence of GREs. Since NFI proteins bind to DNA as dimers, we assessed the activation potentials of NFI heterodimers. Here, we show that NFI heterodimers have intermediate activation potentials compared with homodimers, demonstrating one potential mechanism by which different NFI proteins can regulate gene expression.
Chicken TGGCA proteins belong to the ubiquitous, eukaryotic family of NFI-like nuclear proteins, which share an identical
DNA binding specificity. They are involved in viral and cellular aspects of transcriptlonal regulation and they are capable
of stimulating Adenovirus initiation of replication. Using microsequencing data from peptides of isolated proteins and PCR
supported cloning, we have derived four cDNAs for NFI/TGGCA proteins, which are encoded by three separate chicken genes. Sequence
alignments of NFI proteins from chicken and various mammalian species provide evidence for a common genetic equipment among
higher eukaryotes, in which several related genes, employing each differential RNA splicing generate an unexpectedly large
family of diverse NFI proteins. The extensive similarity of the amino acid sequence throughout the complete coding regions
between products of the same gene type in different species indicates a uniform selection pressure on all protein parts, also
on those outside the DNAbinding domain.
The collagen alpha 1(I) promoter, which is efficiently transcribed in NIH 3T3 fibroblasts, contains four binding sites for
trans-acting factors, as demonstrated by DNase I protection assays (D. A. Brenner, R. A. Rippe, and L. Veloz, Nucleic Acids
Res. 17:6055-6064, 1989). This study characterizes the DNA-binding proteins that interact with the two proximal footprinted
regions, both of which contain a reverse CCAAT box and a G + C-rich 12-bp direct repeat. Analysis by DNase I protection assays,
mobility shift assays, competition with specific oligonucleotides, binding with recombinant proteins, and reactions with specific
antisera showed that the transcriptional factors nuclear factor I (NF-I) and Sp1 bind to these two footprinted regions. Because
of overlapping binding sites, NF-I binding and Sp1 binding appear to be mutually exclusive. Overexpression of NF-I in cotransfection
experiments with the alpha 1(I) promoter in NIH 3T3 fibroblasts increased alpha 1(I) expression, while Sp1 overexpression
reduced this effect, as well as basal promoter activity. The herpes simplex virus thymidine kinase promoter, which contains
independent NF-I- and Sp1-binding sites, was stimulated by both factors. Therefore, expression of the collagen alpha 1(I)
gene may depend on the relative activities of NF-I and Sp1.
NF1-like proteins play a role in transcription of liver-specific genes. A DNA-binding protein, recognizing half of the canonical NF1 binding site (TGGCA) present on the human albumin and retinol-binding protein genes, has been purified from rat liver. Several peptides deriving from a tryptic digest of the purified protein were sequenced and the sequence was used to synthesize specific oligonucleotides. Two overlapping cDNA clones were obtained from a rat-liver cDNA library; their sequence reveals an open reading frame coding for 505 amino acids, including all the peptides sequenced from the purified protein. The DNA-binding domain, most likely located within the first 250 amino acids, is highly homologous to the sequence of CTF/NF1 purified from HeLa cells. Northern analysis reveals several mRNA species present in different combinations in various rat tissues.
The 50-kDa dystrophin-associated glycoprotein (50-DAG) is a component of the dystrophin-glycoprotein complex, which links the muscle cytoskeleton to the extracellular matrix. 50-DAG is specifically deficient in skeletal muscle of patients with severe childhood autosomal recessive muscular dystrophy and in skeletal and cardiac muscles of BIO 14.6 cardiomyopathic hamsters. The lack of 50-DAG leads to a disruption and dysfunction of the dystrophin-glycoprotein complex in these diseases. The cDNA encoding 50-DAG has now been cloned from rabbit skeletal muscle. The 50-DAG deduced amino acid sequence predicts a novel protein having 387 amino acids, a 17-amino acid signal sequence, one transmembrane domain, and two potential sites of N-linked glycosylation. Affinity-purified antibodies against rabbit 50-DAG fusion proteins or synthetic peptides specifically recognized a 50-kDa protein in skeletal muscle sarcolemma and the 50-kDa component of the dystrophin-glycoprotein complex. In contrast to dystroglycan, which is expressed in a wide variety of muscle and non-muscle tissues, 50-DAG is expressed only in skeletal and cardiac muscles and in selected smooth muscles. Finally, 50-DAG mRNA is present in mdx and Duchenne muscular dystrophy (DMD) muscle, indicating that the down-regulation of this protein in DMD and the mdx mouse is likely a post-translational event.
The identification of potential regulatory motifs in new sequence data is increasingly important for experimental design. Those motifs are commonly located by matches to IUPAC strings derived from consensus sequences. Although this method is simple and widely used, a major drawback of IUPAC strings is that they necessarily remove much of the information originally present in the set of sequences. Nucleotide distribution matrices retain most of the information and are thus better suited to evaluate new potential sites. However, sufficiently large libraries of pre-compiled matrices are a prerequisite for practical application of any matrix-based approach and are just beginning to emerge. Here we present a set of tools for molecular biologists that allows generation of new matrices and detection of potential sequence matches by automatic searches with a library of pre-compiled matrices. We also supply a large library (> 200) of transcription factor binding site matrices that has been compiled on the basis of published matrices as well as entries from the TRANSFAC database, with emphasis on sequences with experimentally verified binding capacity. Our search method includes position weighting of the matrices based on the information content of individual positions and calculates a relative matrix similarity. We show several examples suggesting that this matrix similarity is useful in estimating the functional potential of matrix matches and thus provides a valuable basis for designing appropriate experiments.
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.
The sarcoglycans are transmembrane components of the dystrophin-glycoprotein complex, which links the cytoskeleton to the extracellular matrix in adult muscle fibers. Mutations in all four known sarcoglycan genes (alpha, beta, gamma, and delta) have been found in humans with limb-girdle muscular dystrophy. We have identified a novel protein, epsilon-sarcoglycan, that shares 44% amino acid identity with alpha-sarcoglycan (adhalin). We show that epsilon-sarcoglycan is a membrane-associated glycoprotein and document its expression by Northern blotting, immunoblotting, and immunofluorescence. In contrast to alpha-delta sarcoglycans, which are expressed predominantly or exclusively in striated muscle, epsilon-sarcoglycan is broadly distributed in muscle and nonmuscle cells of both embryos and adults. These results raise the possibility that sarcoglycan-containing complexes mediate membrane-matrix interactions in many cell types.
gamma-Sarcoglycan is a transmembrane, dystrophin-associated protein expressed in skeletal and cardiac muscle. The murine gamma-sarcoglycan gene was disrupted using homologous recombination. Mice lacking gamma-sarcoglycan showed pronounced dystrophic muscle changes in early life. By 20 wk of age, these mice developed cardiomyopathy and died prematurely. The loss of gamma-sarcoglycan produced secondary reduction of beta- and delta-sarcoglycan with partial retention of alpha- and epsilon-sarcoglycan, suggesting that beta-, gamma-, and delta-sarcoglycan function as a unit. Importantly, mice lacking gamma-sarco- glycan showed normal dystrophin content and local- ization, demonstrating that myofiber degeneration occurred independently of dystrophin alteration. Furthermore, beta-dystroglycan and laminin were left intact, implying that the dystrophin-dystroglycan-laminin mechanical link was unaffected by sarcoglycan deficiency. Apoptotic myonuclei were abundant in skeletal muscle lacking gamma-sarcoglycan, suggesting that programmed cell death contributes to myofiber degeneration. Vital staining with Evans blue dye revealed that muscle lacking gamma-sarcoglycan developed membrane disruptions like those seen in dystrophin-deficient muscle. Our data demonstrate that sarcoglycan loss was sufficient, and that dystrophin loss was not necessary to cause membrane defects and apoptosis. As a common molecular feature in a variety of muscular dystrophies, sarcoglycan loss is a likely mediator of pathology.
The sarcoglycans are a complex of four transmembrane proteins (alpha, beta, gamma, and delta) which are primarily expressed in skeletal muscle and are closely associated with dystrophin and the dystroglycans in the muscle membrane. Mutations in the sarcoglycans are responsible for four autosomal recessive forms of muscular dystrophy. The function and the organization of the sarcoglycan complex are unknown. We have used coimmunoprecipitation and in vivo cross-linking techniques to analyze the sarcoglycan complex in cultured mouse myotubes. We demonstrate that the interaction between beta- and delta-sarcoglycan is resistant to high concentrations of SDS and alpha-sarcoglycan is less tightly associated with other members of the complex. Cross-linking experiments show that beta-, gamma-, and delta-sarcoglycan are in close proximity to one another and that delta-sarcoglycan can be cross-linked to the dystroglycan complex. In addition, three of the sarcoglycans (beta, gamma, and delta) are shown to form intramolecular disulfide bonds. These studies further our knowledge of the structure of the sarcoglycan complex. Our proposed model of their interactions helps to explain some of the emerging data on the consequences of mutations in the individual sarcoglycans, their effect on the complex, and potentially the clinical course of muscular dystrophies.
Nuclear factor I (NFI) binds to a region of the phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene promoter adjacent to the cAMP regulatory element (CRE) and inhibits the induction of transcription from the gene promoter caused by the catalytic subunit of protein kinase A. In vivo footprinting studies demonstrated that both the CRE and the NFI-binding site are occupied by transcription factors, regardless of the presence of factors that stimulate (dibutyryl cAMP or dexamethasone) or inhibit (insulin) transcription from the PEPCK gene promoter. The NFI effects on transcription from the PEPCK gene promoter were observed even in the absence of the NFI binding site, suggesting the possibility of other weaker binding sites on the promoter or an interaction of NFI with a transcriptional co-activator. A mammalian two-hybrid system was used to demonstrate direct interaction between the transactivation domain of NFI-C and the CREB binding domain of the CREB-binding protein (CBP). Overexpression of a gene fragment encoding the CREB binding domain of CBP stimulates transcription from the PEPCK gene promoter. The inhibitory effect of NFI on transcription of the PEPCK gene induced by the catalytic subunit of protein kinase A appears to be the result of an interaction between NFI and the CREB-binding protein in which NFI competes with CREB for binding to the CREB-binding site on CBP. In contrast, glucocorticoids and thyroid hormone use the steroid hormone receptor binding domain of CBP to stimulate transcription from the PEPCK gene promoter. NFI-A combines with dexamethasone or thyroid hormone in an additive manner to stimulate PEPCK gene transcription. We conclude that CBP coordinates the action of the multiple factors known to control transcription of the PEPCK gene.
The Nuclear Factor I (NFI) family of site-specific DNA-binding proteins (also known as CTF or CAAT box transcription factor) functions both in viral DNA replication and in the regulation of gene expression. The classes of genes whose expression is modulated by NFI include those that are ubiquitously expressed, as well as those that are hormonally, nutritionally, and developmentally regulated. The NFI family is composed of four members in vertebrates (NFI-A, NFI-B, NFI-C and NFI-X), and the four NFI genes are expressed in unique, but overlapping, patterns during mouse embryogenesis and in the adult. Transcripts of each NFI gene are differentially spliced, yielding as many as nine distinct proteins from a single gene. Products of the four NFI genes differ in their abilities to either activate or repress transcription, likely through fundamentally different mechanisms. Here, we will review the properties of the NFI genes and proteins and their known functions in gene expression and development.
In humans, a subset of cases of Limb-girdle muscular dystrophy (LGMD) arise from mutations in the genes encoding one of the sarcoglycan (alpha, beta, gamma, or delta) subunits of the dystrophin-glycoprotein complex. While adeno-associated virus (AAV) is a potential gene therapy vector for these dystrophies, it is unclear if AAV can be used if a diseased muscle is undergoing rapid degeneration and necrosis. The skeletal muscles of mice lacking gamma-sarcoglycan (gsg-/- mice) differ from the animal models that have been evaluated to date in that the severity of the skeletal muscle pathology is much greater and more representative of that of humans with muscular dystrophy. Following direct muscle injection of a recombinant AAV [in which human gamma-sarcoglycan expression is driven by a truncated muscle creatine kinase (MCK) promoter/enhancer], we observed significant numbers of muscle fibers expressing gamma-sarcoglycan and an overall improvement of the histologic pattern of dystrophy. However, these results could be achieved only if injections into the muscle were prior to the development of significant fibrosis in the muscle. The results presented in this report show promise for AAV gene therapy for LGMD, but underscore the need for intervention early in the time course of the disease process.
The sarcoglycan complex is found normally at the plasma membrane of muscle. Disruption of the sarcoglycan complex, through primary gene mutations in dystrophin or sarcoglycan subunits, produces membrane instability and muscular dystrophy. Restoration of the sarcoglycan complex at the plasma membrane requires reintroduction of the mutant sarcoglycan subunit in a manner that will permit normal assembly of the entire sarcoglycan complex. To study sarcoglycan gene replacement, we introduced transgenes expressing murine gamma-sarcoglycan into muscle of normal mice. Mice expressing high levels of gamma-sarcoglycan, under the control of the muscle-specific creatine kinase promoter, developed a severe muscular dystrophy with greatly reduced muscle mass and early lethality. Marked gamma-sarcoglycan overexpression produced cytoplasmic aggregates that interfered with normal membrane targeting of gamma-sarcoglycan. Overexpression of gamma-sarcoglycan lead to the up-regulation of alpha- and beta-sarcoglycan. These data suggest that increased gamma-sarcoglycan and/or mislocalization of gamma-sarcoglycan to the cytoplasm is sufficient to induce muscle damage and provides a new model of muscular dystrophy that highlights the importance of this protein in the assembly, function, and downstream signaling of the sarcoglycan complex. Most importantly, gene dosage and promoter strength should be given serious consideration in replacement gene therapy to ensure safety in human clinical trials.
Although the existence of repressor protein(s) involved in the regulation of highly inducible metallothionein-I (MT-I) gene expression has been postulated, none has been identified to date. We considered nuclear factor I (NFL) protein as a potential repressor, as three half-sites for NFI binding are present on MT-I promoter and NFI is known to downregulate several cellular gene promoters. Overexpression of all four isoforms of mouse NFI protein (NFI-A, -B, -C, and -X) suppressed both constitutive and heavy metal-induced activation of the MT-I promoter in HepG2 cells. However, unlike other target genes of NFI, direct interaction of NFI with MT-I promoter is not necessary to mediate its repression. Point mutation of the NFI binding sites within the MT-I promoter that abrogates NFI binding in vitro could not alleviate the repression. Similarly, NFI proteins also repress activity of minimal MT-I promoter deficient in the NFI binding sites. Further, an NFI-C deletion mutant lacking the DNA binding domain continued to repress MT-I promoter. Overexpression of MTF-1, the key trails-acting factor involved in MT-I gene transcription, surmounted NFI-mediated repression of the basal and zinc-induced MT-I promoter activity. These data demonstrate that NFI is a repressor of MT-I expression, where its DNA binding activity is not essential to downregulate the MT-I promoter. Interaction of NFI with another protein(s), probably MTF-I, may be involved in this repression.
Embryological and genetic studies of mouse, bird, zebrafish, and frog embryos are providing new insights into the regulatory functions of the myogenic regulatory factors, MyoD, Myf5, Myogenin, and MRF4, and the transcriptional and signaling mechanisms that control their expression during the specification and differentiation of muscle progenitors. Myf5 and MyoD genes have genetically redundant, but developmentally distinct regulatory functions in the specification and the differentiation of somite and head muscle progenitor lineages. Myogenin and MRF4 have later functions in muscle differentiation, and Pax and Hox genes coordinate the migration and specification of somite progenitors at sites of hypaxial and limb muscle formation in the embryo body. Transcription enhancers that control Myf5 and MyoD activation in muscle progenitors and maintain their expression during muscle differentiation have been identified by transgenic analysis. In epaxial, hypaxial, limb, and head muscle progenitors, Myf5 is controlled by lineage-specific transcription enhancers, providing evidence that multiple mechanisms control progenitor specification at different sites of myogenesis in the embryo. Developmental signaling ligands and their signal transduction effectors function both interactively and independently to control Myf5 and MyoD activation in muscle progenitor lineages, likely through direct regulation of their transcription enhancers. Future investigations of the signaling and transcriptional mechanisms that control Myf5 and MyoD in the muscle progenitor lineages of different vertebrate embryos can be expected to provide a detailed understanding of the developmental and evolutionary mechanisms for anatomical muscles formation in vertebrates. This knowledge will be a foundation for development of stem cell therapies to repair diseased and damaged muscles.
The mammalian SWI/SNF-like chromatin-remodeling BAF complex plays several important roles in controlling cell proliferation
and differentiation. Interferons (IFNs) are key mediators of cellular antiviral and antiproliferative activities. In this
report, we demonstrate that the BAF complex is required for the maximal induction of a subset of IFN target genes by alpha
IFN (IFN-α). The BAF complex is constitutively associated with the IFITM3 promoter in vivo and facilitates the chromatin remodeling
of the promoter upon IFN-α induction. Furthermore, we show that the ubiquitous transcription activator Sp1 interacts with
the BAF complex in vivo and augments the BAF-mediated activation of the IFITM3 promoter. Sp1 binds constitutively to the IFITM3
promoter in the absence of the BAF complex, suggesting that it may recruit and/or stabilize the BAF complex binding to the
IFITM3 promoter. Our results bring new mechanistic insights into the antiproliferative effects of the chromatin-remodeling
BAF complex.
Nuclear factor I (NFI) genes are expressed in multiple organs throughout development (Chaudhry et al., 1997; for review, see Gronostajski, 2000). All four NFI genes are expressed in embryonic mouse brain, with Nfia, Nfib, and Nfix being expressed highly in developing cortex (Chaudhry et al., 1997). Disruption of the Nfia gene causes agenesis of the corpus callosum (ACC), hydrocephalus, and reduced GFAP expression (das Neves et al., 1999). Three midline structures, the glial wedge, glia within the indusium griseum, and the glial sling are involved in development of the corpus callosum (Silver et al., 1982; Silver and Ogawa, 1983; Shu and Richards, 2001). Because Nfia(-)/- mice show glial abnormalities and ACC, we asked whether defects in midline glial structures occur in Nfia(-)/- mice. NFI-A protein is expressed in all three midline populations. In Nfia(-)/-, mice sling cells are generated but migrate abnormally into the septum and do not form a sling. Glia within the indusium griseum and the glial wedge are greatly reduced or absent and consequently Slit2 expression is also reduced. Although callosal axons approach the midline, they fail to cross and extend aberrantly into the septum. The hippocampal commissure is absent or reduced, whereas the ipsilaterally projecting perforating axons (Hankin and Silver, 1988; Shu et al., 2001) appear relatively normal. These results support an essential role for midline glia in callosum development and a role for Nfia in the formation of midline glial structures.
Polycomb group (PcG) proteins function as high molecular weight complexes that maintain transcriptional repression patterns during embryogenesis. The vertebrate DNA binding protein and transcriptional repressor, YY1, shows sequence homology with the Drosophila PcG protein, pleiohomeotic (PHO). YY1 might therefore be a vertebrate PcG protein. We used Drosophila embryo and larval/imaginal disc transcriptional repression systems to determine whether YY1 repressed transcription in a manner consistent with PcG function in vivo. YY1 repressed transcription in Drosophila, and this repression was stable on a PcG-responsive promoter, but not on a PcG-non-responsive promoter. PcG mutants ablated YY1 repression, and YY1 could substitute for PHO in repressing transcription in wing imaginal discs. YY1 functionally compensated for loss of PHO in pho mutant flies and partially corrected mutant phenotypes. Taken together, these results indicate that YY1 functions as a PcG protein. Finally, we found that YY1, as well as Polycomb, required the co-repressor protein CtBP for repression in vivo. These results provide a mechanism for recruitment of vertebrate PcG complexes to DNA and demonstrate new functions for YY1.
Methylation of specific residues within the N-terminal histone tails plays a critical role in regulating eukaryotic gene expression. Although great advances have been made toward identifying histone methyltransferases (HMTs) and elucidating the consequences of histone methylation, little is known about the recruitment of HMTs to regulatory regions of chromatin. Here we report that the sequence-specific DNA-binding transcription factor Yin Yang 1 (YY1) binds to and recruits the histone H4 (Arg 3)-specific methyltransferase, PRMT1, to a YY1-activated promoter. Our data confirm that histone methylation does not occur randomly but rather is a targeted event and provides one mechanism by which HMTs can be recruited to chromatin to activate gene expression.
Dystroglycan (DG) is an adhesion molecule composed of two subunits, alpha and beta, that are produced by the post-translational cleavage of a single precursor molecule. DG is a pivotal component of the dystrophin-glycoprotein complex (DGC), which connects the extracellular matrix to the cytoskeleton in skeletal muscle and many other tissues. Some muscular dystrophies are caused by mutations of DGC components, such as dystrophin, sarcoglycan or laminin-2, or also of DGC-associated molecules, such as caveolin-3. DG-null mice died during early embriogenesis and no neuromuscular diseases directly associated to genetic abnormalities of DG were identified so far. However, DG plays a crucial role for muscle integrity since its targeting at the sarcolemma is often perturbed in DGC-related neuromuscular disorders.
The eukaryotic genome is partitioned into chromatin domains containing coding and intergenic regions. Insulators have been suggested to play a role in establishing and maintaining chromatin domains. Here we describe the identification and characterization of two separable enhancer blocking elements located in the 5' flanking region of the chicken alpha-globin domain, 11-16 kb upstream of the embryonic alpha-type pi gene in a DNA fragment harboring a MAR (matrix attachment region) element and three DNase I hypersensitive sites (HSs). The most upstream enhancer blocking element co-localizes with the MAR element and an erythroid-specific HS. The second enhancer blocking element roughly co-localizes with a constitutive HS. The third erythroid-specific HS present within the DNA fragment studied harbors a silencing, but not an enhancer blocking, activity. The 11 zinc-finger CCCTC-binding factor (CTCF), which plays an essential role in enhancer blocking activity in many previously characterized vertebrate insulators, is found to bind the two alpha-globin enhancer blocking elements. Detailed analysis has demonstrated that mutation of the CTCF binding site within the most upstream enhancer blocking element abolishes the enhancer blocking activity. The results are discussed with respect to special features of the tissue-specific alpha-globin gene domain located in a permanently open chromatin area.
The mammalian tooth forms by a series of reciprocal epithelial-mesenchymal interactions. Although several signaling pathways
and transcription factors have been implicated in regulating molar crown development, relatively little is known about the
regulation of root development. Four genes encoding nuclear factor I (NFI) transcription-replication proteins are present
in the mouse genome: Nfia, Nfib, Nfic, and Nfix. In order to elucidate its physiological role(s), we disrupted the Nfic gene in mice. Heterozygous animals appear normal, whereas Nfic−/− mice have unique tooth pathologies: molars lacking roots, thin and brittle mandibular incisors, and weakened abnormal maxillary
incisors. Feeding in Nfic−/− mice is impaired, resulting in severe runting and premature death of mice reared on standard laboratory chow. However, a
soft-dough diet mitigates the feeding impairment and maintains viability. Although Nfic is expressed in many organ systems, including the developing tooth, the tooth root development defects were the prominent
phenotype. Indeed, molar crown development is normal, and well-nourished Nfic−/− animals are fertile and can live as long as their wild-type littermates. The Nfic mutation is the first mutation described that affects primarily tooth root formation and should greatly aid our understanding
of postnatal tooth development.
The mouse mammary tumor virus (MMTV) promoter has been used as a model to study how the glucocorticoid receptor (GR) remodels chromatin to allow other transcription factors to bind and activate transcription. To dissect the precise role of nuclear factor 1 (NF1) in chromatin remodeling and transcriptional activation, we used linker-scanning mutants of transcription factor binding sites on the MMTV promoter. We compared the NF1 mutant MMTV promoter in the context of transiently transfected templates (transient transfection) and templates organized as chromatin (stable transfection) to understand the effect of chromatin on factor binding and transcription. We show that on a transiently transfected template, mutation in the NF1 binding site reduces both basal and hormone-dependent transcription. This suggests that NF1 is required for transcription in the absence of organized chromatin. We also found that binding of NF1 on a transiently transfected template is independent of mutation in hormone response elements or the octamer transcription factor (OTF) binding site. In contrast, the binding of OTF proteins to a transiently transfected template was found to be dependent on the binding of NF1, which may imply that NF1 has a stabilizing effect on OTF binding. On a chromatin template, mutation in the NF1 binding site does not affect the positioning of nucleosomes on the promoter. We also show that in the absence of NF1 binding, GR-mediated chromatin remodeling of nucleosome B is reduced and hormone-dependent activation of transcription is abolished. Further, we demonstrate that NF1 is required for both the association of BRG1 chromatin remodeling complex and the GR on the promoter in vivo. These results suggest the novel possibility that NF1 may participate in chromatin remodeling activities in addition to directly enhancing transcription and that in the absence of its binding site the GR is unable to effectively bind the promoter and recruit the remodeling complex.
The expression of type I collagen is regulated developmentally and tissue specifically. Two sets of binding sites for nuclear factor I (NF-I) and Sp1 transcription factors arrayed as an imperfect tandem repeat are critical for high activity of the murine alpha-1(I) collagen gene in NIH-3T3 fibroblasts and are conserved in evolution. Gel retardation analysis combined with methylation interference studies show that NF-I and Sp1 bind to overlapping sites in a mutually exclusive manner. Cotransfection studies using Drosophila Schneider L2 cells, which lack both transcription factors, demonstrate that each factor alone trans-activates the gene, while cotransfection of both factors results in the inhibition of the strong Sp1 trans-activation. In contrast, the herpes simplex virus thymidine kinase promoter, which contains functionally independent NF-I and Sp1 binding sites, is maximally transactivated by the cotransfection of both factors. Because the two NF-I/Sp1 binding sites overlap, the ratio of the activities of the two factors rather than their absolute concentrations determine alpha-1(I) gene expression, characterizing these promoter sequences as transcription factor switch elements.
Myogenin is one of four muscle-specific basic helix-loop-helix regulatory factors involved in controlling myogenesis. We here describe various protein complexes that increase the affinity of myogenin for DNA. We mixed an oligonucleotide containing a degenerate center large enough to accommodate multiple binding sites with crude myotube nuclear extracts and used cyclic amplification and selection of targets with an antimyogenin monoclonal antibody to isolate protein-DNA complexes. Since each cycle of selection results in the enrichment for the sequences with the highest affinity, we isolated multicomponent sites in which myogenin binding was increased by its interaction with other DNA binding proteins. Myogenin interacts with members of the nuclear factor 1 family, the muscle-specific factor myocyte-specific enhancer-binding factor 2, and another factor, COMP1 (cooperates with myogenic proteins 1), that binds to the sequence TGATTGAC. Myogenin also exhibits cooperative binding with other proteins that recognize CANNTG motifs, and various constraints on spacing and orientation were observed. The application of this approach to other transcription factors should not only help identify the different functions of myogenin versus other members of the muscle basic helix-loop-helix regulatory family but also help define the general combinatorial mechanisms involved in eukaryotic gene regulation.
This paper describes the structure of a 70-kb porcine gene for nuclear factor I, including its promoter region, comprising a total of 11 exons. Different mRNAs that we have isolated as cDNAs from both porcine liver and human HeLa cells presumably are generated from this gene by differential splicing events. One cDNA species from porcine liver that lacks exon 9 carries coding information for a protein of 439 amino acids. The in vitro translated protein displays all the properties of an NFI-like protein with high affinity toward the sequence element TGG(N)6GCCAA, as shown by gel shift analysis, and no or little affinity toward CCAAT box containing sequences. Cotranslation experiments with full-length and truncated variants of the protein demonstrate that it binds as a dimer to its cognate DNA recognition sequence. Its DNA-binding domain which is retained in all cDNA clones was mapped by deletion analysis to the 250 N-terminal amino acids of the protein. No structural homologies are observed between this protein and other known DNA-binding proteins; instead, the protein contains a novel alpha-helical sequence motif consisting of several lysine residues spaced at intervals of seven amino acids which we have termed the "lysine helix". The C-terminal portion of the protein derived from full-length cDNAs encodes a short amino acid sequence which is identical with the heptapeptide repeat CT7 observed in the C-terminal domain of the largest subunits of yeast and mouse RNA polymerase II. This region is removed by differential splicing in some of the NFI/CTF cDNAs and thus may be of functional significance.
MyoD is a skeletal muscle-specific protein that is able to induce myogenesis in a wide variety of cell types. In this report, we show that MyoD is a DNA binding protein capable of specific interaction with two regions of the mouse muscle creatine kinase gene upstream enhancer, both of which are required for full muscle-specific enhancer activity. MyoD shares antigenicity and DNA binding specificity with MEF1, a myocyte-specific DNA binding factor. The contiguous basic and myc homology regions of MyoD that are necessary and sufficient for specific DNA interaction are the same regions of the protein required to convert 10T1/2 fibroblasts into muscle. These findings suggest that the biological activity of MyoD is mediated via its capacity for specific DNA interaction.
The CTF/NF-I group of cellular DNA binding proteins recognizes the sequence GCCAAT and is implicated in eukaryotic transcription as well as DNA replication. Molecular analysis of human CTF/NF-I complementary DNA clones reveals multiple messenger RNA species containing alternative coding regions, apparently as a result of differential splicing. Expression and functional analysis establish that individual gene products can bind to GCCAAT recognition sites and serve both as promoter-selective transcriptional activators and as initiation factors for DNA replication.
A protein factor that participates in the formation of a covalent complex between the 80,000-dalton precursor of the adenovirus (Ad) terminal protein (pTP) and 5'-dCMP has been isolated and characterized. This 47,000-dalton protein, isolated from nuclear extracts of uninfected HeLa cells, has been designated nuclear factor I. It is free of detectable DNA polymerase alpha, beta, and gamma activities. In the presence of Ad DNA-prot, the Ad-protein fraction (containing the pTP and the Ad-associated DNA polymerase), ATP, Mg2+, and dCTP, nuclear factor I stimulates formation of the pTP-dCMP complex. Addition of the Ad DNA binding protein (Ad DBP) renders the formation of the pTP-dCMP complex completely dependent on the addition of nuclear factor I. When Ad DNA-prot is replaced with phi X174 single-stranded circular DNA, pTP-dCMP complex formation requires only the Ad-protein fraction; Ad DBP and ATP are inhibitory and nuclear factor I has no effect on this reaction. This suggests that the initiation reaction observed with Ad DNA-prot in the absence of Ad DBP occurs at single-stranded DNA sites. In the presence of Ad DBP, these sites are blocked thus creating a requirement for nuclear factor I in pTP-dCMP complex formation.
TFIID is a multisubunit protein complex comprised of the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs). The TAFs in TFIID are essential for activator-dependent transcription. The cloning of a complementary DNA encoding a human TFIID TAF, TAFII55, that has no known homolog in Drosophila TFIID is now described. TAFII55 is shown to interact with the largest subunit (TAFII230) of human TFIID through its central region and with multiple activators--including Sp1, YY1, USF, CTF, adenoviral E1A, and human immunodeficiency virus-type 1 Tat proteins--through a distinct amino-terminal domain. The TAFII55-interacting region of Sp1 was localized to its DNA-binding domain, which is distinct from the glutamine-rich activation domains previously shown to interact with Drosophila TAFII110. Thus, this human TFIID TAF may be a co-activator that mediates a response to multiple activators through a distinct mechanism.
The nuclear factor I (NFI) family of site-specific DNA-binding proteins is required for both the cell-type specific transcription of many viral and cellular genes and for the replication of adenovirus DNA. Although binding sites for NFI proteins within the promoters of several tissue-specific genes have been shown to be essential for their expression, it is unclear which NFI gene products function in specific tissues during development. We have isolated cDNAs from all four murine NFI genes (gene designations Nfia, Nfib, Nfic, and Nfix), assessed the embryonic and postnatal expression patterns of the NFI genes, and determined the ability of specific NFI proteins to activate transcription from the NFI-dependent mouse mammary tumor virus (MMTV) promoter. In adult mice, all four NFI genes are most highly expressed in lung, liver, heart, and other tissues but only weakly expressed in spleen and testis. The embryonic expression patterns of the NFI genes is complex, with NFI-A transcripts appearing earliest-within 9 days postcoitum in the heart and developing brain. The four genes exhibit unique but overlapping patterns of expression during embryonic development, with high level expression of NFI-A, NFI-B, and NFI-X transcripts in neocortex and extensive expression of the four genes in muscle, connective tissue, liver, and other organ systems. The four NFI gene products studied differ in their ability to activate expression of the NFI-dependent MMTV promoter, with the NFI-B protein being most active and the NFI-A protein being least active. These data are discussed in the context of the developmental expression patterns of known NFI-responsive genes. The differential activation of an NFI-dependent promoter, together with the expression patterns observed for the four genes, indicate that the NFI proteins may play an important role in regulating tissue-specific gene expression during mammalian embryogenesis.
The sarcoglycan complex consists of four membrane-spanning proteins and was shown to be exclusively distributed in striated muscles. In this study, we analyzed the pattern of expression of the mRNAs and proteins of the sarcoglycan subunits during cell differentiation in a culture of myocytes. All four sarcoglycan mRNAs were detectable in proliferating cells, and expression of the alpha- and gamma-subunits was up-regulated by 20- and 50-fold following muscle cell fusion. However, sarcoglycan proteins were scarcely detectable in proliferating cells and were first detected 2 days after the induction to be differentiated. The accumulation of the sarcoglycan protein subunits was accompanied by cell differentiation. The discrepancy between the expression of the mRNAs and proteins of the sarcoglycan subunits in proliferating cells may be ascribed to rapid degradation of the protein.
The transcription factor YY1 is a complex protein that is involved in repressing and activating a diverse number of promoters. Numerous studies have attempted to understand how this one factor can act both as a repressor and an activator in such a wide set of different contexts. The fact that YY1 interacts with a number of key regulatory proteins (e.g. TBP, TFIIB, TAFII55, Sp1, and E1A) has suggested that these interactions are important for determining which particular function of YY1 is displayed at a specific promoter. Two groups of proteins, previously known to function as corepressors and coactivators, that now seem likely to modulate YY1's functions, are the histone deacetylases (HDAC) and histone acetyltransferases (HAT). These two groups of enzymes modify histones, and this modification is proposed to alter chromatin structure. Acetylated histones are typically localized to active chromatin while deacetylated histones colocalize with transcriptionally inactive chromatin. When these enzymes are directed to a promoter through a DNA binding factor such as YY1, that promoter can be activated or repressed. This review will discuss the recent work dealing with the different proteins that interact with YY1, with particular emphasis on ones that modify chromatin, and how they could be involved in regulating YY1's activities.
The upstream promoter of the rat androgen receptor (AR) gene contains a strong negative regulatory region located at the -388 to -340 nucleotide position. The distal part (-388/-373) of this regulatory region binds NFI, a ubiquitous transcription factor, while the proximal portion (-372/-340) contains an overlapping binding site for two nuclear proteins. This composite regulatory region (-388/-340) was initially defined by deoxyribonuclease I footprinting as the continuous stretch of a nuclease-protected site. NFI specificity of the distal portion (-388/-373) of the footprint was established through cross-competition in electrophoretic mobility shift assay (EMSA) using the well characterized NFI element of the adenovirus major late promoter and by immunoreactivity to the NFI antibody. EMSA with oligonucleotide duplexes corresponding to the proximal domain (-372/-340) indicated multiple retarded bands with at least two major DNA-protein complexes. Further analysis with truncated oligonucleotide duplexes showed that these two major proteins bind to this domain in an overlapping manner. Within this overlapping area, the position spanning -359 to -347 is essential for the formation of either of these two complexes. Substitution of four G with T residues in the overlapping area totally abolished all protein binding at the downstream -372/-340 site. Point mutations that abolish specific binding at either the NFI or immediately downstream multiprotein-binding site caused about a 10-fold increase in AR promoter activity in transfected HepG2 cells. Double mutation involving both the NFI and proximal overlapping protein-binding sites failed to cause any additional increase in promoter function. From these results we conclude that the AR promoter contains a composite negative regulatory region at -388/-340, and the repressor function may involve a coordinate interaction between NFI and at least two other nuclear factors.
Muscular dystrophy is a heterogeneous genetic disease that affects skeletal and cardiac muscle. The genetic defects associated with muscular dystrophy include mutations in dystrophin and its associated glycoproteins, the sarcoglycans. Furthermore, defects in dystrophin have been shown to cause a disruption of the normal expression and localization of the sarcoglycan complex. Thus, abnormalities of sarcoglycan are a common molecular feature in a number of dystrophies. By combining biochemistry, molecular cell biology, and human and mouse genetics, a growing understanding of the sarcoglycan complex is emerging. Sarcoglycan appears to be an important, independent mediator of dystrophic pathology in both skeletal muscle and heart. The absence of sarcoglycan leads to alterations of membrane permeability and apoptosis, two shared features of a number of dystrophies. beta-sarcoglycan and delta-sarcoglycan may form the core of the sarcoglycan subcomplex with alpha- and gamma-sarcoglycan less tightly associated to this core. The relationship of epsilon-sarcoglycan to the dystrophin-glycoprotein complex remains unclear. Animals lacking alpha-, gamma- and delta-sarcoglycan have been described and provide excellent opportunities for further investigation of the function of sarcoglycan. Dystrophin with dystroglycan and laminin may be a mechanical link between the actin cytoskeleton and the extracellular matrix. By positioning itself in close proximity to dystrophin and dystroglycan, sarcoglycan may function to couple mechanical and chemical signals in striated muscle. Sarcoglycan may be an independent signaling or regulatory module whose position in the membrane is determined by dystrophin but whose function is carried out independent of the dystrophin-dystroglycan-laminin axis.
Muscular dystrophies represent a heterogeneous group of disorders, which have been largely classified by clinical phenotype. In the last 10 years, identification of novel skeletal muscle genes including extracellular matrix, sarcolemmal, cytoskeletal, cytosolic, and nuclear membrane proteins has changed the phenotype-based classification and shed new light on the molecular pathogenesis of these disorders. A large number of genes involved in muscular dystrophy encode components of the dystrophin-glycoprotein complex (DGC) which normally links the intracellular cytoskeleton to the extracellular matrix. Mutations in components of this complex are thought to lead to loss of sarcolemmal integrity and render muscle fibers more susceptible to damage. Recent evidence suggests the involvement of vascular smooth muscle DGC in skeletal and cardiac muscle pathology in some forms of sarcoglycan-deficient limb-girdle muscular dystrophy. Intriguingly, two other forms of limb-girdle muscular dystrophy are possibly caused by perturbation of sarcolemma repair mechanisms. The complete clarification of these various pathways will lead to further insights into the pathogenesis of this heterogeneous group of muscle disorders.
Four sarcoglycan subunit proteins, alpha-, beta-, gamma- and delta-sarcoglycans, form a complex on the skeletal muscle cell surface membrane and a gene defect in any one of them causes the loss or marked decrease of whole sarcoglycan complex, resulting in an autosomal recessive muscular dystrophy, sarcoglycanopathy. To characterize the regulation of sarcoglycan transcription during myocyte differentiation, we isolated the promoter regions for all sarcoglycan transcripts and measured the level of transcriptional activity of these promoter regions in the C2C12 skeletal muscle cell line. The promoters of gamma-sarcoglycan and one of two promoters of alpha-sarcoglycan exhibited marked transcriptional activation following differentiation to myotubes. Then, we characterized the 1.5-kb region of the gamma-sarcoglycan promoter by generating reporter-constructs having various deletions and measuring their transcriptional activities. In this promoter, we identified a basal promoter region and two enhancer regions dependent on differentiation. We also showed that A/T-rich and E box elements in the upstream enhancer region are essential for the activation of gamma-sarcoglycan transcription following myotube formation. Furthermore, from the identification of binding proteins to these elements together with the cotransfection experiments with the gamma-sarcoglycan promoter reporter construct and cDNAs encoding these binding factors to 10T1/2 fibroblast cell line, it was suggested that MyoD directs the transcription of gamma-sarcoglycan gene as one of the trans activators.
The studies cited above provide strong evidence that core promoter diversity is an important contributor to combinatorial regulation. In the future, it will be important to subject this hypothesis to more stringent tests. With respect to the study by Butler and Kadonaga (2001) in this issue, it will be important to identify the enhancers that exhibit core promoter preferences, as well as the promoters that may be relevant targets of those enhancers. Evidence that the endogenous core promoters possess the anticipated structures would provide considerable support for the hypothesis. In addition to confirming the importance of core promoter preferences for combinatorial regulation, it will be important to explore in greater depth the mechanistic basis of these preferences. In some respects, this goal will be difficult to achieve until current controversies regarding the basic mechanisms of transcriptional activation have been resolved. On the other hand, because the core promoter preferences of transcriptional activators lead to a number of testable predictions, further exploration of the mechanisms underlying these preferences may contribute to the resolution of the controversies.
Mutations of different components of the dystrophin-glycoprotein complex (DGC) cause muscular dystrophies that vary in terms of severity, age of onset, and selective involvement of muscle groups. Although the primary pathogenetic processes in the muscular dystrophies have clearly been identified as apoptotic and necrotic muscle cell death, the pathogenetic mechanisms that lead to cell death remain to be determined. Studies of components of the DGC in muscle and in nonmuscle tissues have revealed that the DGC is undoubtedly a multifunctional complex and a highly dynamic structure, in contrast to the unidimensional concept of the DGC as a mechanical component in the cell. Analysis of the DGC reveals compelling analogies to two other membrane-associated protein complexes, namely integrins and caveolins. Each of these complexes mediates signal transduction cascades in the cell, and disruption of each complex causes muscular dystrophies. The signal transduction cascades associated with the DGC, like those associated with integrins and caveolins, play important roles in cell survival signaling, cellular defense mechanisms, and regulation of the balance between cell survival and cell death. This review focuses on the functional components of the DGC, highlighting the evidence of their participation in cellular signaling processes important for cell survival. Elucidating the link between these functional components and the pathogenetic processes leading to cell death is the foremost challenge to understanding the mechanisms of disease expression in the muscular dystrophies due to defects in the DGC.
The dystrophin glycoprotein complex (DGC) is found at the plasma membrane of muscle cells, where it provides a link between the cytoskeleton and the extracellular matrix. A subcomplex within the DGC, the sarcoglycan complex, associates with dystrophin and mediates muscle membrane stability. Mutations in sarcoglycan genes lead to muscular dystrophy and cardiomyopathy in both humans and mice. In invertebrates, there are three sarcoglycan genes, while in mammals there are additional sarcoglycan genes that probably arose from gene duplication events. We identified a novel mammalian sarcoglycan, zeta-sarcoglycan, that is highly related to gamma-sarcoglycan and delta-sarcoglycan. We generated a zeta-sarcoglycan-specific antibody and found that zeta-sarcoglycan associated with other members of the sarcoglycan complex at the plasma membrane. Additionally, zeta-sarcoglycan was reduced at the membrane in muscular dystrophy, consistent with a role in mediating membrane stability. zeta-Sarcoglycan was also found as a component of the vascular smooth muscle sarcoglycan complex. Together, these data demonstrate that zeta-sarcoglycan is an integral component of the sarcoglycan complex and, as such, is important in the pathogenesis of muscular dystrophy.
Most of the genes that are central to the process of skeletal muscle differentiation remain in a transcriptionally silent or "off" state until muscle cells (myoblasts) are induced to differentiate. Although the mechanisms that contribute to this phenomenon are still unclear, it is likely that histone deacetylases (HDACs), which play an important role in the repression of genes, are principally involved. Recent studies indicate that the initiator of the myogenic program, namely MyoD, can associate with the deacetylase HDAC1 in vivo, and because HDACs are usually recruited to promoters by specific proteins, we considered the possibility that these two proteins might be acting together at the promoters of muscle-specific genes to repress their transcription in myoblasts. In this work, we show by chromatin immunoprecipitation (ChIP) assays that MyoD and HDAC1 are both occupying the promoter of myogenin and that this gene is in a region of repressed chromatin, as revealed by enrichment in histone H3 lysine 9 (Lys-9) methylation and the underacetylation of histones. Surprisingly, after the myoblasts are induced to differentiate, the promoter becomes absent of HDAC1, and eventually the acetyltransferase P/CAF takes it place alongside MyoD. In addition, enrichment of histone H3 acetylation (Lys-9/14) and phosphorylation of Ser-10 can now be observed at the myogenin promoter. These data strongly suggest that in addition to its widely accepted role as an activator of differentiation-specific genes, MyoD also can perform as a transcriptional repressor in proliferating myoblasts while in partnership with a HDAC.
The dystrophin-glycoprotein complex (DGC) is a multisubunit complex that connects the cytoskeleton of a muscle fiber to its surrounding extracellular matrix. Mutations in the DGC disrupt the complex and lead to muscular dystrophy. There are a few naturally occurring animal models of DGC-associated muscular dystrophy (e.g. the dystrophin-deficient mdx mouse, dystrophic golden retriever dog, HFMD cat and the delta-sarcoglycan-deficient BIO 14.6 cardiomyopathic hamster) that share common genetic protein abnormalities similar to those of the human disease. However, the naturally occurring animal models only partially resemble human disease. In addition, no naturally occurring mouse models associated with loss of other DGC components are available. This has encouraged the generation of genetically engineered mouse models for DGC-linked muscular dystrophy. Not only have analyses of these mice led to a significant improvement in our understanding of the pathogenetic mechanisms for the development of muscular dystrophy, but they will also be immensely valuable tools for the development of novel therapeutic approaches for these incapacitating diseases.
Transcription of the mouse Ren-1(c) gene in kidney tumor-derived As4.1 cells, which express high levels of renin mRNA, is dependent on a proximal promoter element and a 242-bp enhancer region located 2.6 kb upstream of the transcription start site. We showed previously that the enhancer contains a cAMP responsive element (CRE) and an E-box. Mutation of either element resulted in almost complete loss of the Ren-1(c) expression. In this report we show that there are additional transcription factor-binding sites within the Ren-1(c) enhancer contributing to the enhancer activity. Electrophoretic mobility shift and supershift assays have identified four nuclear factor I (NFI)-binding sites, an Sp1/Sp3 site and an unidentified transcription factor-binding site (Ei) located upstream of the CRE and E-box. Mutation of the Sp1/Sp3 site or Ei reduced Ren-1(c) expression by 40% or 30%, respectively, while mutations of four NFI-binding sites resulted in an 89% decrease in expression. Thus, these protein-DNA interaction sites are essential for transcription of mouse renin genes. There are four homologous NFI genes (NFI-A, -B, -C and -X) in vertebrates and multiple alternatively spliced isoforms from each gene. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assays have demonstrated that NFI-X is the predominant NFI mRNA expressed in As4.1 cells. Direct study of involvement of NFI-X in regulation of renin genes is underway.
A family of human CCAAT-box-binding proteins active in transcription and [31] Developmental expression of sarcoglycan gene products in cultured myocytes
- C Santoro
- N Mermod
- P C Andrews
- R Tjian
- S Noguchi
- E Wakabayashi
- M Imamura
- M Yoshida
- E Ottawa
C. Santoro, N. Mermod, P.C. Andrews, R. Tjian, A family of human CCAAT-box-binding proteins active in transcription and [31] S. Noguchi, E. Wakabayashi, M. Imamura, M. Yoshida, E. Ottawa, Developmental expression of sarcoglycan gene products in cultured myocytes, Biochem. Biophys. Res. Commun. 262 (1999) 88–93.
Negative regulation of the androgen receptor gene promoter by NFI and an adjacently located multiprotein-binding site
- Song