Molecular Dissection of DNA Sequences and Factors Involved in Slow Muscle-Specific Transcription

ABSTRACT Transcription is a major regulatory mechanism for the generation of slow- and fast-twitch myofibers. We previously identified an upstream region of the slow TnI gene (slow upstream regulatory element [SURE]) and an intronic region of the fast TnI gene (fast intronic regulatory element [FIRE]) that are sufficient to direct fiber type-specific transcription in transgenic mice. Here we demonstrate that the downstream half of TnI SURE, containing E box, NFAT, MEF-2, and CACC motifs, is sufficient to confer pan-skeletal muscle-specific expression in transgenic mice. However, upstream regions of SURE and FIRE are required for slow and fast fiber type specificity, respectively. By adding back upstream SURE sequences to the pan-muscle-specific enhancer, we delineated a 15-bp region necessary for slow muscle specificity. Using this sequence in a yeast one-hybrid screen, we isolated cDNAs for general transcription factor 3 (GTF3)/muscle TFII-I repeat domain-containing protein 1 (MusTRD1). GTF3 is a multidomain nuclear protein related to initiator element-binding transcription factor TF II-I; the genes for both proteins are deleted in persons with Williams-Beuren syndrome, who often manifest muscle weakness. Gel retardation assays revealed that full-length GTF3, as well as its carboxy-terminal half, specifically bind the bicoid-like motif of SURE (GTTAATCCG). GTF3 expression is neither muscle nor fiber type specific. Its levels are highest during a period of fetal development that coincides with the emergence of specific fiber types and transiently increases in regenerating muscles damaged by bupivacaine. We further show that transcription from TnI SURE is repressed by GTF3 when overexpressed in electroporated adult soleus muscles. These results suggest a role for GTF3 as a regulator of slow TnI expression during early stages of muscle development and suggest how it could contribute to Williams-Beuren syndrome.

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Common core sequences are found in skeletal muscle slow- and fast-fiber-type-specific regulatory elements.

Molecular and Cellular Biology ◽

10.1128/mcb.16.5.2408 ◽

1996 ◽

Vol 16 (5) ◽

pp. 2408-2417 ◽

Cited By ~ 59

Author(s):

M Nakayama ◽

J Stauffer ◽

J Cheng ◽

S Banerjee-Basu ◽

E Wawrousek ◽

...

Keyword(s):

Transgenic Mice ◽

Spatial Organization ◽

Molecular Mechanisms ◽

Fiber Type ◽

Regulatory Element ◽

Regulatory Elements ◽

Upstream Region ◽

Regulatory Sequences ◽

Fast Fiber ◽

Core Elements

The molecular mechanisms generating muscle diversity during development are unknown. The phenotypic properties of slow- and fast-twitch myofibers are determined by the selective transcription of genes coding for contractile proteins and metabolic enzymes in these muscles, properties that fail to develop in cultured muscle. Using transgenic mice, we have identified regulatory elements in the evolutionarily related troponin slow (TnIs) and fast (TnIf) genes that confer specific transcription in either slow or fast muscles. Analysis of serial deletions of the rat TnIs upstream region revealed that sequences between kb -0.95 and -0.5 are necessary to confer slow-fiber-specific transcription; the -0.5-kb fragment containing the basal promoter was inactive in five transgenic mouse lines tested. We identified a 128-bp regulatory element residing at kb -0.8 that, when linked to the -0.5-kb TnIs promoter, specifically confers transcription to slow-twitch muscles. To identify sequences directing fast-fiber-specific transcription, we generated transgenic mice harboring a construct containing the TnIs kb -0.5 promoter fused to a 144-bp enhancer derived from the quail TnIf gene. Mice harboring the TnIf/TnIs chimera construct expressed the transgene in fast but not in slow muscles, indicating that these regulatory elements are sufficient to confer fiber-type-specific transcription. Alignment of rat TnIs and quail TnIf regulatory sequences indicates that there is a conserved spatial organization of core elements, namely, an E box, a CCAC box, a MEF-2-like sequence, and a previously uncharacterized motif. The core elements were shown to bind their cognate factors by electrophoretic mobility shift assays, and their mutation demonstrated that the TnIs CCAC and E boxes are necessary for transgene expression. Our results suggest that the interaction of closely related transcriptional protein-DNA complexes is utilized to specify fiber type diversity.

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Fast skeletal muscle-specific expression of a quail troponin I gene in transgenic mice.

Molecular and Cellular Biology ◽

10.1128/mcb.8.12.5072 ◽

1988 ◽

Vol 8 (12) ◽

pp. 5072-5079 ◽

Cited By ~ 7

Author(s):

P L Hallauer ◽

K E Hastings ◽

A C Peterson

Keyword(s):

Skeletal Muscle ◽

Transgenic Mice ◽

Troponin I ◽

Fiber Type ◽

Regulatory Elements ◽

Evolutionary Divergence ◽

Mrna Levels ◽

Fast Skeletal Muscle ◽

Specific Expression ◽

Exon 2

We have produced seven lines of transgenic mice carrying the quail gene encoding the fast skeletal muscle-specific isoform of troponin I (TnIf). The quail DNA included the entire TnIf gene, 530 base pairs of 5'-flanking DNA, and 1.5 kilobase pairs of 3'-flanking DNA. In all seven transgenic lines, normally initiated and processed quail TnIf mRNA was expressed in skeletal muscle, where it accumulated to levels comparable to that in quail muscle. Moreover, in the three lines tested, quail TnIf mRNA levels were manyfold higher in a fast skeletal muscle (gastrocnemius) than in a slow skeletal muscle (soleus). We conclude that the cellular mechanisms directing muscle fiber type-specific TnIf gene expression are mediated by cis-regulatory elements present on the introduced quail DNA fragment and that they control TnIf expression by affecting the accumulation of TnIf mRNA. These elements have been functionally conserved since the evolutionary divergence of birds and mammals, despite the major physiological and morphological differences existing between avian (tonic) and mammalian (twitch) slow muscles. In lines of transgenic mice carrying multiple tandemly repeated copies of the transgene, an aberrant quail TnIf transcript (differing from normal TnIf mRNA upstream of exon 2) also accumulated in certain tissues, particularly lung, brain, spleen, and heart tissues. However, this aberrant transcript was not detected in a transgenic line which carries only a single copy of the quail gene.

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Tissue-specific regulation of mouse hepatocyte nuclear factor 4 expression

Molecular and Cellular Biology ◽

10.1128/mcb.14.11.7276-7284.1994 ◽

1994 ◽

Vol 14 (11) ◽

pp. 7276-7284

Author(s):

W Zhong ◽

J Mirkovitch ◽

J E Darnell

Keyword(s):

Transcription Factor ◽

Transgenic Mice ◽

Transient Transfection ◽

Nuclear Factor ◽

Hepatocyte Nuclear Factor ◽

Distal Enhancer ◽

Specific Expression ◽

Tissue Specific ◽

Hypersensitive Sites ◽

Hepatocyte Nuclear Factor 4

Hepatocyte nuclear factor 4 (HNF-4) is a liver-enriched transcription factor and a member of the steroid hormone receptor superfamily. HNF-4 is required for the hepatoma-specific expression of HNF-1 alpha, another liver-enriched transcription factor, suggesting the early participation of HNF-4 in development. To prepare for further study of HNF-4 in development, the tissue-specific expression of the mouse HNF-4 gene was studied by analyzing the promoter region for required DNA elements. DNase-hypersensitive sites in the gene in liver and kidney tissues were found in regions both distal and proximal to the RNA start that were absent in tissues in which HNF-4 expression did not occur. By use of reporter constructs in transient-transfection assays and with transgenic mice, a region sufficient to drive liver-specific expression of HNF-4 was identified. While an HNF-1 binding site between bp -98 and -68 played an important role in the hepatoma-specific promoter activity of HNF-4 in transient-transfection assays, it was not sufficient for the liver-specific expression of a reporter gene in transgenic mice. Distal enhancer elements indicated by the presence of DNase I-hypersensitive sites at kb -5.5 and -6.5, while not functional in transient-transfection assays, were required for the correct expression of the mouse HNF-4 gene in animals.

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Rbfox1 is required for myofibril development and maintaining fiber type–specific isoform expression in Drosophila muscles

Life Science Alliance ◽

10.26508/lsa.202101342 ◽

2022 ◽

Vol 5 (4) ◽

pp. e202101342

Author(s):

Elena Nikonova ◽

Amartya Mukherjee ◽

Ketaki Kamble ◽

Christiane Barz ◽

Upendra Nongthomba ◽

...

Keyword(s):

Alternative Splicing ◽

Muscle Development ◽

Rna Binding ◽

Troponin I ◽

Rna Binding Proteins ◽

Fiber Type ◽

Structural Defects ◽

Specific Gene ◽

Fiber Types ◽

Isoform Expression

Protein isoform transitions confer muscle fibers with distinct properties and are regulated by differential transcription and alternative splicing. RNA-binding Fox protein 1 (Rbfox1) can affect both transcript levels and splicing, and is known to contribute to normal muscle development and physiology in vertebrates, although the detailed mechanisms remain obscure. In this study, we report that Rbfox1 contributes to the generation of adult muscle diversity in Drosophila. Rbfox1 is differentially expressed among muscle fiber types, and RNAi knockdown causes a hypercontraction phenotype that leads to behavioral and eclosion defects. Misregulation of fiber type–specific gene and splice isoform expression, notably loss of an indirect flight muscle–specific isoform of Troponin-I that is critical for regulating myosin activity, leads to structural defects. We further show that Rbfox1 directly binds the 3′-UTR of target transcripts, regulates the expression level of myogenic transcription factors myocyte enhancer factor 2 and Salm, and both modulates expression of and genetically interacts with the CELF family RNA-binding protein Bruno1 (Bru1). Rbfox1 and Bru1 co-regulate fiber type–specific alternative splicing of structural genes, indicating that regulatory interactions between FOX and CELF family RNA-binding proteins are conserved in fly muscle. Rbfox1 thus affects muscle development by regulating fiber type–specific splicing and expression dynamics of identity genes and structural proteins.

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Transgenic Mice with Cardiac-Specific Expression of Activating Transcription Factor 3, a Stress-Inducible Gene, Have Conduction Abnormalities and Contractile Dysfunction

American Journal Of Pathology ◽

10.1016/s0002-9440(10)61735-x ◽

2001 ◽

Vol 159 (2) ◽

pp. 639-650 ◽

Cited By ~ 71

Author(s):

Yoshichika Okamoto ◽

Alysia Chaves ◽

Jingchun Chen ◽

Robert Kelley ◽

Keith Jones ◽

...

Keyword(s):

Transcription Factor ◽

Transgenic Mice ◽

Contractile Dysfunction ◽

Activating Transcription Factor 3 ◽

Specific Expression ◽

Conduction Abnormalities ◽

Activating Transcription Factor ◽

Inducible Gene ◽

Transcription Factor 3

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Calcineurin Is Necessary for the Maintenance but Not Embryonic Development of Slow Muscle Fibers

Molecular and Cellular Biology ◽

10.1128/mcb.25.15.6629-6638.2005 ◽

2005 ◽

Vol 25 (15) ◽

pp. 6629-6638 ◽

Cited By ~ 74

Author(s):

Misook Oh ◽

Igor I. Rybkin ◽

Victoria Copeland ◽

Michael P. Czubryt ◽

John M. Shelton ◽

...

Keyword(s):

Skeletal Muscle ◽

Fiber Type ◽

Oxidative Capacity ◽

Muscle Fiber Types ◽

Fiber Types ◽

Specific Expression ◽

Interacting Protein ◽

Type Composition ◽

Slow Fiber ◽

Fast Twitch

ABSTRACT Skeletal muscles are a mosaic of slow and fast twitch myofibers. During embryogenesis, patterns of fiber type composition are initiated that change postnatally to meet physiological demand. To examine the role of the protein phosphatase calcineurin in the initiation and maintenance of muscle fiber types, we used a “Flox-ON” approach to obtain muscle-specific overexpression of the modulatory calcineurin-interacting protein 1 (MCIP1/DSCR1), an inhibitor of calcineurin. Myo-Cre transgenic mice with early skeletal muscle-specific expression of Cre recombinase were used to activate the Flox-MCIP1 transgene. Contractile components unique to type 1 slow fibers were absent from skeletal muscle of adult Myo-Cre/Flox-MCIP1 mice, whereas oxidative capacity, myoglobin content, and mitochondrial abundance were unaltered. The soleus muscles of Myo-Cre/Flox-MCIP1 mice fatigued more rapidly than the wild type as a consequence of the replacement of the slow myosin heavy chain MyHC-1 with a fast isoform, MyHC-2A. MyHC-1 expression in Myo-Cre/Flox-MCIP1 embryos and early neonates was normal. These results demonstrate that developmental patterning of slow fibers is independent of calcineurin, while the maintenance of the slow-fiber phenotype in the adult requires calcineurin activity.

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An upstream region of the rat spermatogenesis-specific heat-shock-like Hst70 gene confers testis-specific expression in transgenic mice

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1993.tb17643.x ◽

1993 ◽

Vol 212 (1) ◽

pp. 137-143 ◽

Cited By ~ 9

Author(s):

Jan WISNIEWSKI ◽

Marek MALEZEWSKI ◽

Zdzislaw KRAWCZYK ◽

Lashitew GEDAMU

Keyword(s):

Heat Shock ◽

Transgenic Mice ◽

Specific Heat ◽

Upstream Region ◽

Specific Expression ◽

Rat Spermatogenesis ◽

Testis Specific Expression

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A 5'-upstream region of a bovine keratin 6 gene confers tissue-specific expression and hyperproliferation-related induction in transgenic mice.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.92.11.4783 ◽

1995 ◽

Vol 92 (11) ◽

pp. 4783-4787 ◽

Cited By ~ 35

Author(s):

A. Ramirez ◽

M. Vidal ◽

A. Bravo ◽

F. Larcher ◽

J. L. Jorcano

Keyword(s):

Transgenic Mice ◽

Upstream Region ◽

Specific Expression ◽

Tissue Specific ◽

Tissue Specific Expression ◽

Keratin 6

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Rbfox1 is required for myofibril development and maintaining fiber-type specific isoform expression in Drosophila muscles

10.1101/2021.05.09.443278 ◽

2021 ◽

Author(s):

Elena Nikonova ◽

Ketaki Kamble ◽

Amartya Mukherjee ◽

Christiane Barz ◽

Upendra Nongthomba ◽

...

Keyword(s):

Muscle Development ◽

Rna Binding ◽

Troponin I ◽

Rna Binding Proteins ◽

Fiber Type ◽

Specific Gene ◽

Fiber Types ◽

Transcript Levels ◽

Splice Isoform ◽

Isoform Expression

Protein isoform transitions confer distinct properties on muscle fibers and are regulated predominantly by differential transcription and alternative splicing. RNA-binding Fox protein 1 (Rbfox1) can affect both transcript levels and splicing, and is known to control skeletal muscle function. However, the detailed mechanisms by which Rbfox1 contributes to normal muscle development and physiology remain obscure. In this study, we report that Rbfox1 contributes to the generation of adult muscle diversity in Drosophila. Rbfox1 is differentially expressed in tubular and fibrillar muscle fiber types. RNAi knockdown of Rbfox1 leads to a loss of flight, climbing and jumping ability, as well as eclosion defects. Myofibers in knockdown muscle are frequently torn, and sarcomeres are hypercontracted. These defects arise from mis-regulation of fiber-type specific gene and splice isoform expression, notably loss of an IFM-specific isoform of Troponin-I that is critical for regulating myosin activity. We find that Rbfox1 influences mRNA transcript levels through 1) direct binding of 3'-UTRs of target transcripts as well as 2) through regulation of myogenic transcription factors, including Mef2, Exd and Salm. Moreover, Rbfox1 modulates splice isoform expression through 1) direct regulation of target splice events in structural genes and 2) regulation of the CELF-family RNA-binding protein Bruno1. Our data indicate that cross-regulatory interactions observed between FOX and CELF family RNA-binding proteins in vertebrates are conserved between their counterparts, Rbfox1 and Bruno1 in flies. Rbfox1 thus affects muscle development by regulation of both fiber-type specific gene and gene isoform expression dynamics of identity genes and structural proteins.

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