Vijak Mahdavi

Interaction of myogenic factors and the retinoblastoma protein mediates muscle cell commitment and differentiation

The experiments reported here document that the tumor suppressor retinoblastoma protein (pRB) plays an important role in the production and maintenance of the terminally differentiated phenotype of muscle cells. We show that pRB inactivation, through either phosphorylation, binding to T antigen, or genetic alteration, inhibits myogenesis. Moreover, inactivation of pRB in terminally differentiated cells allows them to reenter the cell cycle. In addition to its involvement in the myogenic activities of MyoD, pRB is also required for the cell growth-inhibitory activity of this myogenic factor. We also show that pRB and MyoD directly bind to each other, both in vivo and in vitro, through a region that involves the pocket and the basic-helix-loop-helix domains, respectively. All the results obtained are consistent with the proposal that the effects of MyoD on the cell cycle and of pRB on the myogenic pathway result from the direct binding of the two molecules.

Thyroid hormone receptor α isoforms generated by alternative splicing differentially activate myosin HC gene transcription

Thyroid hormones are thought to modulate gene expression positively or negatively through interactions with chromatin-associated receptors1. Recently, the c-erbA proto–oncogene products have been shown to be nuclear thyroid hormone (T3) receptors (TR)2–5 by sequence similarity with other steroid receptors and by their ability to bind thyroid hormone. But it has not been shown that these receptors directly activate transcription of the responsive genes in vivo. In addition, the rat TRα gene encodes several messenger RNA (mRNA) species, generated by differential processing of its transcripts (ref. 22). For these reasons we investigated the ability of two major isoforms of the rat TRa gene products to activate transcription of a sarcomeric myosin heavy chain (mHC) gene, because expression of all members of this gene family is responsive to T63. We show here that the rTRα1 receptor is a thyroid hormone-dependent transcriptional factor, which upon binding the T3 responsive element of the α-mHC gene, activates expression of this gene in vivo. The rTRα2 isoform, which is identical to rTRα1 except for its carboxyl terminal portion, is generated by alternative splicing of the rTRα gene transcript. This peptide, when produced in vitro and in vivo failed to bind T3 or other hormones or to trans-activate α-mHC gene expression. Thus, alternative splicing can produce marked differences in the functional properties of a transcriptional factor.

Developmental and hormonal regulation of sarcomeric myosin heavy chain gene family.

Sarcomeric myosin heavy chain (MHC), the main component of the sarcomere, contains the ATPase activity that generates the contractile force of cardiac and skeletal muscles. The different MHC isoforms are encoded by a closely related multigene family. Most members (seven) of this gene family have been isolated and characterized in the rat, including the alpha- and beta-cardiac, skeletal embryonic, neonatal, fast IIA, fast IIB, and extraocular specific MHC. The slow type I skeletal MHC is encoded by the same gene that codes for the cardiac beta-MHC. Each MHC gene studied displays a pattern of expression that is tissue and developmental stage specific, both in cardiac and skeletal muscles. Furthermore, more than one MHC gene is expressed in each muscle while each gene is expressed in more than one tissue. The expression of each MHC gene in cardiac and skeletal muscles is modulated by thyroid hormone. Surprisingly, however, the same MHC gene can be regulated by the hormone in a significantly different manner, even in opposite directions, depending on the muscle in which it is expressed. Moreover, the skeletal embryonic and neonatal MHC genes, so far considered specific to these 2 developmental stages, are normally expressed in certain adult muscles and can be reinduced by hypothyroidism in specific muscles. This complex pattern of expression and regulation of the MHC gene family in cardiac and skeletal muscle sheds new light on the mechanisms involved in determining the biochemical basis of the contractile state. It also indicates that the cardiac contractile system needs to be examined in a broader context, including skeletal muscles, in order to understand fully its developmental and physiologic regulation.

Determination of the Consensus Binding Site for MEF2 Expressed in Muscle and Brain Reveals Tissue-specific Sequence Constraints

The myocyte-specific enhancer factor-2 (MEF2) proteins are expressed in the three major types of muscle (skeletal, cardiac, and smooth) and function as transcriptional activators of muscle-specific and growth factor-regulated genes through binding to a canonical A/T-rich cis-element. Although MEF2 proteins are also expressed in brain, MEF2-regulated muscle-specific gene products are not detected in this tissue. To gain insight into the regulation of MEF2 function in vivo, we have selected its optimal DNA targets from a library of degenerate oligonucleotides using anti-MEF2A antibodies and cell extracts from skeletal muscle, heart, and brain. The consensus binding site in these three tissues contains an indistinguishable core motif, 5′-CT(A/t)(a/t)AAATAG-3′. However, the optimal target for MEF2 expressed in the brain shows additional sequence constraints (5′-TGTTACT(A/t)(a/t)AAATAGA(A/t)-3′) that are not observed in the sequences selected with skeletal and cardiac muscle extracts. Thus, differences in DNA binding preferences of MEF2 proteins in muscle and brain may contribute to tissue-specific gene expression during myogenesis and neurogenesis.

Sarcomeric myosin heavy chain gene family: Organization and pattern of expression

The sarcomeric myosin heavy chains (MHCs), which exhibit different levels of ATPase activity, are encoded by a closely related multigene family from which seven members have been identified and characterized in the rat. The MHC genes appear to map to a single chromosome, and at least two of them, alpha- and beta-cardiac, are closely linked in the genome. Each of these genes is approximately 25 kilobases long, and their coding sequences are interrupted by 40 introns. Each MHC gene displays a pattern of expression that is tissue-specific and developmentally regulated, with more than one MHC gene expressed in each muscle and developmental stage. Moreover, with the exception of the extra-ocular muscle MHC gene that has a very specific pattern of expression, the other genes are all expressed in more than one tissue. The expression of all MHC genes can be modulated by thyroid hormone. Surprisingly, however, the same myosin heavy chain gene can be regulated by thyroid hormone in highly different modes, even in opposite directions, depending on the tissue in which it is expressed. Furthermore, the skeletal embryonic and neonatal myosin heavy chain genes, so far considered specific to these two developmental stages, can be re-induced by hypothyroidism in specific adult muscles.

Cardiac Myocyte Terminal Differentiation

The exact mechanism of terminal differentiation in cardiac myocytes is currently unknown. Studies in the skeletal muscle system provided a model where muscle lineage termination gene directly interacts with Rb to produce and maintain the terminally differentiated state. This interaction provided the critical components for the lock in cell cycle arrest in skeletal muscle cell. Cardiac muscle appears on the surface very similar to skeletal muscle especially since they share large numbers of structural and contractile proteins. However, it is clear that cardiac muscle cells are distinct biologically at the regulatory level. First and foremost, differentiation and capacity for hyperplasia (mitosis) is not mutually exclusive, in that the heart being the first functional organ embryologically is able to grow via cell division until shortly after birth. Thereafter further growth is provided by hypertrophy. In skeletal muscle, these two processes, differentiation and ability to undergo mitosis, appear to be mutually exclusive. Second, cardiac muscles have not been shown to express any of the skeletal muscle determination basic helix loop helix factors like myoD or any proteins that are functionally similar. Third, heterokaryons of cardiac myocytes and fibroblasts reveal a lack of dominance of the cardiac muscle phenotype. This is distinctly different in skeletal muscle, whose phenotype is dominant which provided a platform to identify the skeletal muscle determination gene, myoD. Although various basic helix loop helix proteins and homeobox genes have been identified in cardiac myocytes, their function remains to be elucidated. At this time no cardiac determination gene has been identified. Despite these differences, we have shown that the biology of pocket proteins Rb and P107 is similar in skeletal and cardiac myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid and reversible changes in myosin heavy chain expression in response to increased neuromuscular activity of rat fast-twitch muscle

Chronic 10 Hz stimulation of rat fast‐twitch muscle induced rapid and reversible changes in the tissue levels offast myosin heavy chain (HC) mRNA isoforms. These changes consisted of a rapid decrease in HCIIb mRNA and a progressive increase in HCIIa mRNA. After 15 days, the HCIIb mRNA normally amounting to approximately 80%, had decreased to less than 5% of the sum of the two HC mRNA isoforms. HCIIb mRNA was again detectable one day after cessation of stimulation and progressively increased at the expense of HCIIa mRNA with ongoing recovery. These results point to a down‐regulation of the HCIIb gene by the applied stimulus pattern which, conversely, enhances the expression of the HCIIa gene.

Molecular mechanisms of cardiac gene expression

Although the physiological properties of the myocardium and their dynamic character have been the focus of intense research during the past three decades, the biochemical and molecular correlates underlying cardiac development and performance have, until recently, remained poorly understood. The development of modern cellular and molecular biology has provided the necessary tools to undertake the study of the mechanisms involved in cardiac development and to understand the basis for important clinical and experimental problems in cardiovascular physiology. Most of the gene encoding contractile proteins have been cloned and characterized. The availability of molecular probes and the ability to introduce genes into individual cell types and tissues of living animals, are the most important breakthroughs of molecular and cell biology. This permits not only to analyze basic mechanisms of gene expression but has also significant practical applications for gene therapy. It is now possible to analyze the role of different regulatory gene sequences and identify their corresponding trans-active factors. In addition, direct gene injection makes it possible to study gene expression in a natural context, under conditions that are physiologically relevant and controllable.

Molekulare Mechanismen der kardialen Genexpression

Obwohl die physiologischen Eigenschaften des Myokards und ihr dynamischer Charakter in den letzten drei Jahrzehnten im Mittelpunkt intensiver Forschung standen, war uber die biochemischen und molekularen Korrelate, die der kardialen Entwicklung und Leistung zugrunde liegen, bis vor kurzem wenig bekannt. Die Entwicklung der modernen Zell- und Molekularbiologie ermoglichte es, die Mechanismen zu untersuchen, die bei der kardialen Entwicklung eine Rolle spielen, und die Basis fur wichtige klinische und experimentelle Probleme der kardiovaskularen Physiologie zu verstehen. Der Grosteil des Gens, das die kontraktilen Proteine verschlusselt, konnte kloniert und charakterisiert werden. Die Verfugbarkeit von Molekularsonden und die Fahigkeit, Gene in individuelle Zelltypen und in Gewebe lebender Tiere einzubringen, sind die wichtigsten Durchbruche der Molekular- und Zellbiologie. Dies erlaubt es nicht nur, Grundmechanismen der Genexpression zu analysieren, sondern es bietet auch bedeutende praktische Anwendungsmoglichkeiten im Bereich der Gentherapie. Es ist jetzt moglich, die Rolle der verschiedenen regulatorischen Gensequenzen zu analysieren und ihre entsprechenden transaktiven Faktoren zu identifizieren. Zusatzlich ermoglicht es die direkte Geninjektion, die Genexpression in einem naturlichen Kontext zu studieren, unter Bedingungen, die physiologisch relevant und kontrollierbar sind.

PROMOTER SELECTION AND ALTERNATIVE PRE-mRNA SPLICING ARE USED TO GENERATE COMPLEX CONTRACTILE PROTEIN PHENOTYPES

Publisher Summary This chapter describes the use of promoter selection and alternative pre-mRNA splicing to generate complex contractile protein phenotypes. The regulated expression of structurally distinct developmentally regulated and cell type-specific protein isoforms is a fundamental characteristic of eukaryotic cells. The molecular mechanisms responsible for generating this protein diversity might be broadly categorized into two main systems: those that select a particular gene among the members of a multigene family for expression in a particular cell and those that generate several different proteins from a single gene. The latter mechanism includes DNA rearrangement and alternative pre-mRNA splicing, each producing the differential use of intragenic sequences that lead to the production of multiple protein isoforms from a single gene. DNA rearrangement appears to be restricted to a very limited set of genes coding for immunoglobulins and T-cell receptors. In contrast, the increasing numbers of genes in organisms ranging from Drosophila to human, including their RNA and DNA viruses, are known to be alternatively spliced. The restricted combinatorial use of the different members of these multigene families allows for the generation of a moderate number of qualitatively different sarcomere types that, at least in some cases, exhibit significantly different physiological characteristics.