Positive and Negative Intronic Regulatory Elements Control Muscle-Specific Alternative Exon Splicing of Drosophila Myosin Heavy Chain Transcripts

Genetics ◽  
2001 ◽  
Vol 157 (1) ◽  
pp. 259-271 ◽  
Author(s):  
David M Standiford ◽  
Wei Tao Sun ◽  
Mary Beth Davis ◽  
Charles P Emerson
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Alternative Exon ◽  
Regulatory Sequences ◽  
Splice Donor ◽  
Transgenic Expression ◽  
Specific Alternative ◽  
Exon 11

Abstract Alternative splicing of Drosophila muscle myosin heavy chain (MHC) transcripts is precisely regulated to ensure the expression of specific MHC isoforms required for the distinctive contractile activities of physiologically specialized muscles. We have used transgenic expression analysis in combination with mutagenesis to identify cis-regulatory sequences that are required for muscle-specific splicing of exon 11, which is encoded by five alternative exons that produce alternative “converter” domains in the MHC head. Here, we report the identification of three conserved intronic elements (CIE1, -2, and -3) that control splicing of exon 11e in the indirect flight muscle (IFM). Each of these CIE elements has a distinct function: CIE1 acts as a splice repressor, while CIE2 and CIE3 behave as splice enhancers. These CIE elements function in combination with a nonconsensus splice donor to direct IFM-specific splicing of exon 11e. An additional cis-regulatory element that is essential in coordinating the muscle-specific splicing of other alternative exon 11s is identified. Therefore, multiple interacting intronic and splice donor elements establish the muscle-specific splicing of alternative exon 11s.

scholarly journals Splice-Junction Elements and Intronic Sequences Regulate Alternative Splicing of the Drosophila Myosin Heavy Chain Gene Transcript

Genetics ◽  
1997 ◽  
Vol 147 (2) ◽  
pp. 725-741 ◽  
Author(s):  
David M Standiford ◽  
Mary Beth Davis ◽  
Weitao Sun ◽  
Charles P Emerson
Keyword(s):  
Alternative Splicing ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Mutational Analysis ◽  
Regulatory Elements ◽  
Alternative Exon ◽  
Chain Gene ◽  
Gene Transcript ◽  
Specific Alternative ◽  
Exon 11

The Drosophila muscle myosin heavy chain (Mhc) gene primary transcript contains five alternatively spliced exon groups (exons 3, 7, 9, 11 and 15), each of which contains two to five mutually exclusive members. Individual muscles typically select a specific alternative exon from each group for incorporation into the processed message. We report here on the cis-regulatory mechanisms that direct the processing of alternative exons in Mhc exon 11 in individual muscles using transgenic reporter constructs, RT-PCR and directed mutagenesis. The 6.0-kilobase exon 11 domain is sufficient to direct the correct processing of exon 11 alternatives, demonstrating that the alternative splicing cis-regulatory elements are local to Mhc exon 11. Mutational analysis of Mhc exon 11 reveals that the alternative exon nonconsensus 5′-splice donors are essential for alternative splicing regulation in general, but do not specify alternative exons for inclusion in individual muscles. Rather, we show, through exon substitutions and deletion analyses, that a 360-nucleotide intronic domain precisely directs the normal processing of one exon, Mhc exon 11e, in the indirect flight muscle. These and other data indicate that alternative exons are regulated in appropriate muscles through interactions between intronic alternative splice-specificity elements, nonconsensus exon 11 splice donors and, likely, novel exon-specific alternative splicing factors.

Muscle-specific and inducible expression of 293-base pair beta-myosin heavy chain promoter in transgenic mice

1996 ◽  
Vol 271 (3) ◽  
pp. R688-R695 ◽  
Author(s):  
J. L. Wiedenman ◽  
G. L. Tsika ◽  
L. Gao ◽  
J. J. McCarthy ◽  
I. D. Rivera-Rivera ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Specific Activity ◽  
Fiber Type ◽  
Regulatory Element ◽  
Histochemical Staining ◽  
Regulatory Sequences ◽  
Type I ◽  
Adult Mice ◽  
Mechanical Overload

The DNA regulatory element(s) involved in beta-myosin heavy chain (beta-MHC) induction by the physiological stimulus of mechanical overload have not been identified as yet. To delineate regulatory sequences that are required for mechanical overload induction of the beta-MHC gene, transgenic mouse lines were generated that harbor transgenes containing serial deletions of the human beta-MHC promoter to nucleotides -293 (beta 293), -201 (beta 201), and -141 (beta 141) from the transcription start site (+1). Mechanically overloaded adult plantaris and soleus muscles contained 11- and 1.9-fold increases, respectively, in endogenous beta-MHC-specific mRNA transcripts (Northern blot) compared with sham-operated controls. Expression assays (chloramphenicol acetyltransferase specific activity) revealed that only transgene beta 293 expression was muscle specific in both fetal and adult mice and was induced in the plantaris (10- to 27-fold) and soleus (2- to 2.5-fold) muscles by mechanical overload. Histochemical staining for myosin adenosinetriphosphatase activity revealed a fiber-type transition of type II to type I in the overloaded plantaris and soleus muscles. These transgenic data suggest that sequences located between nucleotides -293 and +120 may be sufficient to regulate the endogenous beta-MHC gene in response to developmental signals and to the physiological signals generated by mechanical overload in fast- and slow-twitch muscles.

A variant octamer motif in a Xenopus H2B histone gene promoter is not required for transcription in frog oocytes

1991 ◽  
Vol 11 (2) ◽  
pp. 641-654
Author(s):  
C Hinkley ◽  
M Perry
Keyword(s):  
Cell Cycle ◽  
Transcription Initiation ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Histone Gene ◽  
Site Directed Mutagenesis ◽  
Regulatory Sequences ◽  
Chromosomal Dna ◽  
Transcriptional Regulatory ◽  
Octamer Motif

Xenopus oocytes, arrested in G2 before the first meiotic division, accumulate histone mRNA and protein in the absence of chromosomal DNA replication and therefore represent an attractive biological system in which to examine histone gene expression uncoupled from the cell cycle. Previous studies have shown that sequences necessary for maximal levels of transcription in oocytes are present within 200 bp at the 5' end of the transcription initiation site for genes encoding each of the five major Xenopus histone classes. We have defined by site-directed mutagenesis individual regulatory sequences and characterized DNA-binding proteins required for histone H2B gene transcription in injected oocytes. The Xenopus H2B gene has a relatively simple promoter containing several transcriptional regulatory elements, including TFIID, CBP, and ATF/CREB binding sites, required for maximal transcription. A sequence (CTTTACAT) in the H2B promoter resembling the conserved octamer motif (ATTTGCAT), the target for cell-cycle regulation of a human H2B gene, is not required for transcription in oocytes. Nonetheless, substitution of a consensus octamer motif for the variant octamer element activates H2B transcription. Oocyte factors, presumably including the ubiquitous Oct-1 factor, specifically bind to the consensus octamer motif but not to the variant sequence. Our results demonstrate that a transcriptional regulatory element involved in lymphoid-specific expression of immunoglobulin genes and in S-phase-specific activation of mammalian H2B histone genes can activate transcription in nondividing amphibian oocytes.

scholarly journals Functional domains of the Drosophila melanogaster muscle myosin heavy-chain gene are encoded by alternatively spliced exons.

1989 ◽  
Vol 9 (7) ◽  
pp. 2957-2974 ◽  
Author(s):  
E L George ◽  
M B Ober ◽  
C P Emerson
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Rna Splicing ◽  
Single Copy ◽  
Alternative Exon ◽  
Chain Gene ◽  
Drosophila Genome ◽  
Alternatively Spliced ◽  
Muscle Myosin ◽  
Exon Structure

The single-copy Drosophila muscle myosin heavy-chain (MHC) gene, located at 36B(2L), has a complex exon structure that produces a diversity of larval and adult muscle MHC isoforms through regulated alternative RNA splicing. Genomic and cDNA sequence analyses revealed that this 21-kilobase MHC gene encodes these MHC isoforms in 19 exons. However, five sets of these exons, encoding portions of the S1 head and the hinge domains of the MHC protein, are tandemly repeated as two, three, four, or five divergent copies, which are individually spliced into RNA transcripts. RNA hybridization studies with exon-specific probes showed that at least 10 of the 480 possible MHC isoforms that could arise by alternative RNA splicing of these exons are expressed as MHC transcripts and that the expression of specific members of alternative exon sets is regulated, both in stage and in muscle-type specificity. This regulated expression of specific exons is of particular interest because the alternatively spliced exon sets encode discrete domains of the MHC protein that likely contribute to the specialized contractile activities of different Drosophila muscle types. The alternative exon structure of the Drosophila MHC gene and the single-copy nature of this gene in the Drosophila genome make possible transgenic experiments to test the physiological functions of specific MHC protein domains and genetic and molecular experiments to investigate the mechanisms that regulate alternative exon splicing of MHC and other muscle gene transcripts.

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

1996 ◽  
Vol 16 (5) ◽  
pp. 2408-2417 ◽  
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.

scholarly journals Multiple positive and negative 5' regulatory elements control the cell-type-specific expression of the embryonic skeletal myosin heavy-chain gene.

1987 ◽  
Vol 7 (12) ◽  
pp. 4377-4389 ◽  
Author(s):  
P F Bouvagnet ◽  
E E Strehler ◽  
G E White ◽  
M A Strehler-Page ◽  
B Nadal-Ginard ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Simian Virus 40 ◽  
Muscle Cells ◽  
Regulatory Elements ◽  
Simian Virus ◽  
Chain Gene ◽  
Specific Element

To identify the DNA sequences that regulate the expression of the sarcomeric myosin heavy-chain (MHC) genes in muscle cells, a series of deletion constructs of the rat embryonic MHC gene was assayed for transient expression after introduction into myogenic and nonmyogenic cells. The sequences in 1.4 kilobases of 5'-flanking DNA were found to be sufficient to direct expression of the MHC gene constructs in a tissue-specific manner (i.e., in differentiated muscle cells but not in undifferentiated muscle and nonmuscle cells). Three main distinct regulatory domains have been identified: (i) the upstream sequences from positions -1413 to -174, which determine the level of expression of the MHC gene and are constituted of three positive regulatory elements and two negative ones; (ii) a muscle-specific regulatory element from positions -173 to -142, which restricts the expression of the MHC gene to muscle cells; and (iii) the promoter region, downstream from position -102, which directs transcription initiation. Introduction of the simian virus 40 enhancer into constructs where subportions of or all of the upstream sequences are deleted (up to position -173) strongly increases the level of expression of such truncated constructs but without changing their muscle specificity. These upstream sequences, which can be substituted for by the simian virus 40 enhancer, function in an orientation-, position-, and promoter-dependent fashion. The muscle-specific element is also promoter specific but does not support efficient expression of the MHC gene. The MHC promoter in itself is not muscle specific. These results underline the importance of the concerted action of multiple regulatory elements that are likely to represent targets for DNA-binding-regulatory proteins.

scholarly journals An M-CAT binding factor and an RSRF-related A-rich binding factor positively regulate expression of the alpha-cardiac myosin heavy-chain gene in vivo.

1994 ◽  
Vol 14 (8) ◽  
pp. 5056-5065 ◽  
Author(s):  
J D Molkentin ◽  
B E Markham
Keyword(s):  
Dna Binding ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Rat Heart ◽  
Simian Virus 40 ◽  
Regulatory Elements ◽  
Developmental Expression ◽  
Adult Rat ◽  
Binding Factor ◽  
Enhancer Factor

Cardiac muscle-restricted expression of the alpha-myosin heavy-chain (alpha-MHC) gene is regulated by multiple elements in the proximal enhancer/promoter. Within this region, an M-CAT site and an A-rich site were identified as potential regulatory elements. Site-specific mutations in each site, individually, reduced activity from the wild-type promoter by approximately 85% in the adult rat heart, demonstrating that these sites were positive regulatory elements. alpha-MHC, beta-MHC, and chicken cardiac troponin T (cTnT) M-CAT sites interacted with an M-CAT-binding factor (MCBF) from rat heart nuclear extracts that was immunologically related to transcriptional enhancer factor 1, a factor that binds within the simian virus 40 enhancer. The factor that bound the A-rich region (ARF) was antigenically related to the RSRF family of proteins, ARF was distinct from myocyte-specific enhancer factor 2 (MEF-2) on the basis of DNA-binding specificity and developmental expression. Like MEF-2, ARF DNA-binding activity was present in the heart and brain; however, no ARF activity was detected in extracts from skeletal muscle or C2C12 myotubes. MCBF and ARF DNA-binding activities were developmentally regulated with peak levels in the 1- to 2-day neonatal heart. The activity of both factors increased nearly fivefold in adult rat hearts subjected to a pressure overload. By comparison, the levels of alpha-MHC binding factor 2 did not change during hypertrophy. Binding sites for MCBF and ARF are present in several genes that are upregulated during cardiac hypertrophy. Our results suggest that these factors participate in the alterations in gene expression that occur during cardiac development and hypertrophy.

scholarly journals A conserved GATA motif in a tissue-specific DNase I hypersensitive site of the cardiac α-myosin heavy chain gene

1997 ◽  
Vol 325 (1) ◽  
pp. 47-51 ◽  
Author(s):  
Weei-Yuarn HUANG ◽  
Choong-Chin LIEW
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Developmental Stages ◽  
Regulatory Elements ◽  
Dnase I ◽  
Upstream Region ◽  
Chain Gene ◽  
Nuclear Extracts ◽  
Hypersensitive Sites ◽  
Gata Motif

Transgenic analysis has indicated that far upstream regulatory elements of the cardiac α-myosin heavy chain (MyHC) gene are required for appropriate transgene expression [Subramaniam, Gulick, Neumann, Knotts and Robbins (1993) J. Biol. Chem. 268, 4331–4336]. In an attempt to identify these as-yet-undefined regulatory elements, we mapped the DNase I hypersensitive sites (DHSs) in the 4 kb upstream region of the hamster cardiac α-MyHC gene. When using nuclei isolated from late-gestational and adult heart ventricles, a strong DHS was identified in the -1.9 kb region (α-1.9 kb site). It cannot be detected in kidney, liver or cardiofibroblast nuclei. Within this site, we found a conserved GATA-motif that interacts specifically with GATA-binding factors in nuclear extracts of cardiomyocytes at various developmental stages. These data provide further evidence to support the role of GATA factors in the regulation of cardiac α-MyHC gene expression.

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