Eva Bober

A novel human muscle factor related to but distinct from MyoD1 induces myogenic conversion in 10T1/2 fibroblasts.

We have isolated the cDNA encoding a novel human myogenic factor, Myf‐5, by weak cross‐hydridization to the mouse MyoD1 probe. Nucleotide sequence analysis and the identification of the corresponding gene indicate that human Myf‐5 is a member of a small gene family which also contains the human homologue to MyoD1. Although structurally related to the mouse factor, the human Myf‐5 constitutes a different protein which nevertheless is capable of inducing the myogenic phenotype in embryonic C3H mouse 10T1/2 ‘fibroblasts’. The existence of more than one MyoD1‐like protein in human skeletal muscle is further suggested by the detection of several similar but distinct cDNA clones. The phenotypic conversion of 10T1/2 cells by the human factor is recognized by the capacity of the cells to form multinucleated syncytia and synthesize sarcomeric myosin heavy chains. Moreover, transient expression of Myf‐5 in 10T1/2 cells leads to the activation of a co‐transfected muscle‐specific CAT reporter gene which by itself is transcriptionally silent in the non‐muscle cell background. The deduced amino acid sequence of clone Myf‐5 reveals a region which is highly similar to myc proteins and the developmental factors from Drosophila encoded by the achaete scute locus and the twist gene. The myc homology region and a preceding cluster of basic amino acids are located in a larger sequence domain with strong similarity to the mouse myogenic factor MyoD1. Two additional short segments with high serine and threonine content are conserved between the two proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Sirt7 Increases Stress Resistance of Cardiomyocytes and Prevents Apoptosis and Inflammatory Cardiomyopathy in Mice

Sirt7 is a member of the mammalian sirtuin family consisting of 7 genes, Sirt1 to Sirt7, which all share a homology to the founding family member, the yeast Sir2 gene. Most sirtuins are supposed to act as histone/protein deacetylases, which use oxidized NAD in a sirtuin-specific, 2-step deacetylation reaction. To begin to decipher the biological role of Sirt7, we inactivated the Sirt7 gene in mice. Sirt7-deficient animals undergo a reduction in mean and maximum lifespans and develop heart hypertrophy and inflammatory cardiomyopathy. Sirt7 mutant hearts are also characterized by an extensive fibrosis, which leads to a 3-fold increase in collagen III accumulation. We found that Sirt7 interacts with p53 and efficiently deacetylates p53 in vitro, which corresponds to hyperacetylation of p53 in vivo and an increased rate of apoptosis in the myocardium of mutant mice. Sirt7-deficient primary cardiomyocytes show a ≈200% increase in basal apoptosis and a significantly diminished resistance to oxidative and genotoxic stress suggesting a critical role of Sirt7 in the regulation of stress responses and cell death in the heart. We propose that enhanced activation of p53 by lack of Sirt7-mediated deacetylation contributes to the heart phenotype of Sirt7 mutant mice.

Myf-6, a new member of the human gene family of myogenic determination factors: evidence for a gene cluster on chromosome 12.

The Myf‐6 gene, a novel member of the human gene family of muscle determination factors has been detected by its highly conserved sequence coding for a putative helix‐loop‐helix domain. This sequence motif is a common feature of all Myf factors and other regulatory proteins. The new Myf gene is located on human chromosome 12, approximately 6.5 Kb upstream of the Myf‐5 locus in a closely linked cluster of myogenic determination genes. Myf‐6 cDNAs were isolated from human and mouse skeletal muscle, the only tissue in which expression of the corresponding mRNA was observed. In contrast to human primary muscle cell cultures which express moderate levels of Myf‐6 mRNA, most established rodent muscle cell lines completely lack this mRNA. Myogenic 10T1/2 cells, however, induced by the expression of either pEMSV‐Myf‐4 or pEMSV‐Myf‐5 activate their endogenous mouse Myf‐6 gene. Constitutive expression of Myf‐6 cDNA in C3H 10T1/2 fibroblasts establishes the muscle phenotype at a similar frequency to the previously characterized myogenic factors. Moreover, muscle‐specific CAT reporter constructs containing either the human myosin light chain (MLC) enhancer or the promoter of the embryonic myosin light chain gene are activated in NIH 3T3 fibroblasts or in CV1 kidney cells by cotransfection of Myf‐6 expression vehicles. This transcriptional activation occurs in the absence of any apparent conversion of the cellular phenotype of the recipient cells. Glutathione‐S‐transferase fusion proteins with Myf‐3, Myf‐4 or Myf‐5 specifically bind to a MEF‐like consensus sequence present in the human MLC enhancer and the MLC1 emb promoter. In contrast, the Myf‐6 hybrid protein interacts weakly with the same sequences showing lower affinity and reduced specificity. Since co‐expressed pEMSV‐Myf‐6, nevertheless, is able to activate transcription of the MLC‐CAT reporter constructs in non‐muscle tissue culture cells, the different DNA binding properties in vitro might suggest that transactivation of gene expression by Myf‐6 involves distinct binding sites and/or additional protein factors.

Differential expression of myogenic determination genes in muscle cells: possible autoactivation by the Myf gene products.

The development of muscle cells involves the action of myogenic determination factors. In this report, we show that human skeletal muscle tissue contains, besides the previously described Myf‐5, two additional factors Myf‐3 and Myf‐4 which represent the human homologues of the rodent proteins MyoD1 and myogenin. The genes encoding Myf‐3, Myf‐4 and Myf‐5 are located on human chromosomes 11, 1, and 12 respectively. Constitutive expression of a single factor is sufficient to convert mouse C3H 10T1/2 fibroblasts to phenotypically normal muscle cells. The myogenic conversion of 10T1/2 fibroblasts results in the activation of the endogenous MyoD1 and Myf‐4 (myogenin) genes. This observation suggests that the expression of Myf proteins leads to positive autoregulation of the members of the Myf gene family. Individual myogenic colonies derived from MCA C115 cells (10T1/2 fibroblast transformed by methylcholanthrene) express various levels of endogenous MyoD1 mRNA ranging from nearly zero to high levels. The Myf‐5 gene was generally not activated in 10T1/2 derived myogenic cell lines but was expressed in some MCA myoblasts. In primary human muscle cells Myf‐3 and Myf‐4 mRNA but very little Myf‐5 mRNA is expressed. In mouse C2 and P2 muscle cell lines MyoD1 is abundantly synthesized together with myogenin. In contrast, the rat muscle lines L8 and L6 and the mouse BC3H1 cells express primarily myogenin and low levels of Myf‐5 but no MyoD1. Myf‐4 (myogenin) mRNA is present in all muscle cell lines at the onset of differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

SIRT7 Controls Hepatic Lipid Metabolism by Regulating the Ubiquitin-Proteasome Pathway

Sirtuins (SIRT1-7) have attracted considerable attention as regulators of metabolism over the past decade. However, the physiological functions and molecular mechanisms of SIRT7 are poorly understood. Here we demonstrate that Sirt7 knockout mice were resistant to high-fat diet-induced fatty liver, obesity, and glucose intolerance, and that hepatic triglyceride accumulation was also attenuated in liver-specific Sirt7 knockout mice. Hepatic SIRT7 positively regulated the protein level of TR4/TAK1, a nuclear receptor involved in lipid metabolism, and as a consequence activated TR4 target genes to increase fatty acid uptake and triglyceride synthesis/storage. Biochemical studies revealed that the DDB1-CUL4-associated factor 1 (DCAF1)/damage-specific DNA binding protein 1 (DDB1)/cullin 4B (CUL4B) E3 ubiquitin ligase complex interacted with TR4, leading to its degradation, while binding of SIRT7 to the DCAF1/DDB1/CUL4B complex inhibited the degradation of TR4. In conclusion, we propose that hepatic SIRT7 controls lipid metabolism in liver by regulating the ubiquitin-proteasome pathway.

Distinct temporal expression of mouse Nkx-5.1 and Nkx-5.2 homeo☐ genes during brain and ear development

The mouse Nkx-5.1 and Nkx-5.2 genes have been identified by sequence homology to Drosophila NK genes within the homeobox domain. Here, we report the isolation of the Nkx-5.2 cDNA and a detailed comparative analysis of the spatio-temporal expression patterns for Nkx-5.1 and Nkx-5.2 genes. Nkx-5.2 transcripts are first detected in E13.5 embryos where they colocalize with Nkx-5.1 mRNA in the developing central nervous system and the inner ear. However, the onset of Nkx-5.1 transcription begins much earlier in 10 somite stage embryos (E8.5) in the otic placode and the branchial region. Nkx-5.1 expression in the ear persists until birth, whereas in branchial arches it is transient between E8.5 to E11.5. Transcript distribution appears regionalized in the otic vesicle concentrating at the anterior and posterior margin and later at the dorsal side of the otocyst. These domains are distinct from regions expressing Pax-2 and sek, two other early markers for otic development. From E11.5 to birth several Nkx-5.1 expression domains appear in the brain between the ventral diencephalon and the myelencephalon. The same expression domains also exist for Nkx-5.2 beginning at E13.5. The regionally restricted expression pattern of both Nkx-5 genes during mouse development suggests their involvement in cell type specification of neuronal cells.

Reduced Mobility of Fibroblast Growth Factor (FGF)-Deficient Myoblasts Might Contribute to Dystrophic Changes in the Musculature of FGF2/FGF6/mdx Triple-Mutant Mice

ABSTRACT Development and regeneration of muscle tissue is a highly organized, multistep process that requires cell proliferation, migration, differentiation, and maturation. Previous data implicate fibroblast growth factors (FGFs) as critical regulators of these processes, although their precise role in vivo is still not clear. We have explored the consequences of the loss of multiple FGFs (FGF2 and FGF6 in particular) for muscle regeneration in mdx mice, which serve as a model for chronic muscle damage. We show that the combined loss of FGF2 and FGF6 leads to severe dystrophic changes in the musculature. We found that FGF6 mutant myoblasts had decreased migration ability in vivo, whereas wild-type myoblasts migrated normally in a FGF6 mutant environment after transplantation of genetically labeled myoblasts from FGF6 mutants in wild-type mice and vice versa. In addition, retrovirus-mediated expression of dominant-negative versions of Ras and Ral led to a reduced migration of transplanted myoblasts in vivo. We propose that FGFs are critical components of the muscle regeneration machinery that enhance skeletal muscle regeneration, probably by stimulation of muscle stem cell migration.

FGFs control the patterning of the inner ear but are not able to induce the full ear program.

FGF2 or FGF8 applied ectopically, close to the developing otic placode enhances transcription of a subset of ear marker genes such as Nkx5-1, SOHo1 and Pax2. Other ear expressed genes (Dlx5 and BMP4) are not up-regulated by FGFs. Ectopic FGFs lead to an increase in size of the vestibulo-cochlear ganglion. This phenotypic change is due to an increased recruitment of epithelial cells to the neuronal fate rather than to an enhanced proliferation. We also observed an induction of additional, vesicle-like structures upon ectopic FGF treatment, but this induction never led to enrolment of a full ear program. We further demonstrate that FGF8 is expressed in two separate, short waves, first at the otic placode stage and later at the vesicle stage. Both activities correspond to critical morphogenetic events in ear development. We propose that FGF8 is an important regulator of otocyst patterning.

Regionalized expression of Nkx5-1, Nkx5-2, Pax2 and sek genes during mouse inner ear development

Nkx5-1 and Nkx5-2 are two highly related homeobox genes which are expressed during mouse development in the inner ear. Here, we present the detailed expression of both genes within the developing ear and a comparison to the expression of other potential control genes in this organ. Both genes are active between E13.5 and birth in non-sensory epithelium of the semicircular canals, utricle and saccule. Nkx5-1 and Nkx5-2 are also expressed in the cochlea, where the expression is restricted to the stria vascularis. The endolymphatic duct is devoid of any Nkx5 transcripts. Pax2 is expressed in epithelial cells of the ventral part of the membranous labyrinth where it overlaps with the Nkx5 expression domain. sek shows a complementary pattern to Nkx5 in the vestibular epithelium. In the cochlea sek is expressed throughout the mesenchyme and epithelium but not in the stria vascularis. In the vestibulum Pax2 and sek is limited to the ventral part whereas Nkx5 genes are active throughout. These data suggest that Nkx5 genes, Pax2 and sek play different roles in the patterning of inner ear structures.

Inner ear and lateral line expression of a zebrafish Nkx5-1 gene and its downregulation in the ears of FGF8 mutant, ace.

An orthologue of the mouse homeobox gene Nkx5-1 was cloned and characterized in the zebrafish. As in the mouse and chick, the zebrafish Nkx5-1 gene is expressed in the ear placode and vesicle and in cells forming the vestibulo-acoustic ganglion. In addition, a novel expression domain, the lateral line, appears in the zebrafish, supporting a common precursor hypothesis for these two organs. In the FGF8 zebrafish mutant ace, expression of Nkx5-1 in the otic structures is diminished. The most significant reduction of zfNkx5-1 expression was observed in cells of the vestibulo-acoustic ganglion.