We have identified a rat cDNA encoding MRF4, a new member of the muscle regulatory factor gene family that includes MyoD1, myogenin, and Myf-5. MRF4 encodes a predicted 27-kD protein that contains a conserved helix-loop-helix motif, which is a common feature of this gene family. Northern analyses indicate that MRF4 is expressed solely in skeletal muscle tissue but is not detected in most embryonic muscle cell lines. Transfection of MRF4 into C3H10T1/2 fibroblasts produces stable myogenic lineages at frequencies that are equal to or greater than those obtained when MyoD1 or myogenin are introduced into these cells. Expression of the MRF4 cDNA leads to expression of the endogenous MyoD1 and myogenin genes, although C3H10T1/2 cells expressing MyoD1 or myogenin cDNAs do not express MRF4. Interestingly, the endogenous MyoD1 and myogenin genes are negatively regulated by serum and by purified growth factors since MRF4-transfected C3H10T1/2 cells activate MyoD1 and myogenin expression only in mitogen-depleted, differentiation-induced muscle cultures. The myofiber-specific expression pattern of MyoD1 and myogenin in these cells suggests that the primary role for this muscle regulatory factor gene family may be in regulating specific terminal differentiation events that are crucial for normal skeletal muscle development.[Key Words: MRF4; muscle regulatory factor; MyoD 1; myogenin; Myf-5: C3H 10T 1/2 cells]

A pituitary LIM homeodomain factor, PLim, is expressed as Rathke's pouch forms and as specific pituitary cell phenotypes are established, suggesting functional roles throughout pituitary development. While selectively expressed in Pituitary organ commitment appears to occur shortly after a region of the somatic ectoderm makes direct contact with neuroectodermal cells in an area of mesenchymal incompetence (1, 2). Subsequently, five distinct cell types, each characterized by the expression of a unique hormone, appear in a spatially and temporally specific fashion (reviewed in refs. 3 and 4). The pituitary-specific POU-domain transcription factor Pit-1 (3, 4) is selectively activated in the caudomedial part of the nascent gland at embryonic day 15.5 (e15.5) in the mouse and is required for activation of the prolactin (Prl), growth hormone (GH), and thyroid-stimulating hormone }3-subunit (TSHI3) genes in this region, as well as for somatotrope, lactotrope, and thyrotrope cell proliferation (5-7). Additional activating factors work with Pit-1 to achieve cellspecific gene activation (8)(9)(10)(11)(12)(13)(14)(15).A second family of homeodomain transcription factors, initially defined by RNA. These RT-PCR-generated DNA fragments were used to screen a mouse pituitary cDNA library and a mouse genomic library. RNase protection assays were performed with an intron 3-containing cDNA clone (no. 11) as template for T7RNA polymerase-directed synthesis of a radiolabeled antisense probe. In situ hybridization of 20-,um sagittal sections of mouse embryos and organs (e9, e9.5, elO.5, and e15.5) fixed in buffered 10% formalin was performed as described (1) by using T7 RNA polymerase to generate two separate 35S-labeled cRNA probes (486 and 550 nt) corresponding to fragments of N-terminal or C-terminal coding sequences of P-Lim.In Vitro Protein-Protein Interaction, DNA Binding, and Transfection Assays. Restriction fragments of P-Lim cDNA were ligated in frame into pGEX-KG (39) to yield glutathione S-transferase (GST) fusion proteins, and PCR was used to generate deletions of each LIM region separately (ALIM-1, A1-87; ALIM-2, A1-29 and A86-154) and of the entire LIM region (P-ALim = A1-151).[35S]Methionine-labeled in vitro translated protein was incubated for 20 min at 37°C with 2-3 jig of GST fusion protein bound to 25 p,l of glutathioneagarose beads in a total volume of 100 ,ul of 20 mM Hepes, pH 7.9/100 mM NaCl/1 mM EDTA/4 mM MgCl2/1 mM dithiothreitol, 0.02% (vol/vol) Nonidet P-40/10% (vol/vol) glycerol/0.5% (wt/vol) nonfat dry milk, with ethidium bromide at 50 ,ug/ml to eliminate potential protein-DNA interactions (32).Protein-mediated gel shift assays, in vitro culture, and transient transfection of cells were performed as described (9,14). For the mouse a-glycoprotein subunit (aGSU) reporter gene, a BamHI-Pst I fragment containing the promoter region of the aGSU starting from bp -440 was ligated to the luciferase gene in the pGL2 basic vector (Promega). Mouse TSH,B (from kb -1.2), rat Prl promoter/enhancer, and mous...

No abstract

A small genetic region near the telomere of ovine chromosome 18 was previously shown to carry the mutation causing the callipyge muscle hypertrophy phenotype in sheep. Expression of this phenotype is the only known case in mammals of paternal polar overdominance gene action. A region surrounding two positional candidate genes was sequenced in animals of known genotype. Mutation detection focused on an inbred ram of callipyge phenotype postulated to have inherited chromosome segments identical-by-descent with exception of the mutated position. In support of this hypothesis, this inbred ram was homozygous over 210 Kb of sequence, except for a single heterozygous base position. This single polymorphism was genotyped in multiple families segregating the callipyge locus (CLPG), providing 100% concordance with animals of known CLPG genotype, and was unique to descendants of the founder animal. The mutation lies in a region of high homology among mouse, sheep, cattle, and humans, but not in any previously identified expressed transcript. A substantial open reading frame exists in the sheep sequence surrounding the mutation, although this frame is not conserved among species. Initial functional analysis indicates sequence encompassing the mutation is part of a novel transcript expressed in sheep fetal muscle we have named CLPG1.

In this report, we demonstrate that myogenic cultures inhibited from differentiating by treatment with fibroblast growth factor or transforming growth factor 11 show reduced levels of MyoDl mRNA. Although this repression may contribute to the inhibition of myogenesis by growth factors, additional regulatory pathways must be affected, since inhibition still occurs in cultures engineered to constitutively express MyoDI mRNA.Skeletal muscle differentiation involves the transcriptional activation of the contractile protein gene set and the formation of multinucleated muscle fibers. The contractile protein genes are controlled by cis and tran.s regulatory systems which appear to respond to exogenous growth factors, since addition of fibroblast growth factor (FGF) or transforming growth factor 3 (TGF-3) to differentiating muscle cells in culture inhibits both fusion and the accumulation of these muscle-specific proteins (7,9,13,14,16,21). It has been hypothesized that FGF and TGF-, may influence one or perhaps several master regulatory pathways. Although the regulatory genes that control the induction of skeletal myogenesis have not been defined completely, at least three genes, MyoDl (8,20), inyd (18), and myogenin (22). play an important role in establishing the myogenic lineage and inducing the expression of muscle-specific genes.To determine whether these regulatory genes are modulated by exogenous growth factors, we investigated the transcriptional activity of the MyoDl gene in cells exposed to FGF or TGF-3. 23A2 myoblasts (12) were induced to differentiate in medium containing insulin, transferrin, and selenium (ITS) (23) to establish the normal expression pattern of MyoDl and the contractile protein gene, troponin I (TnI), during skeletal myogenesis. RNA was isolated from proliferating and differentiation-induced 23A2 cultures, electrophoresed through formaldehyde-agarose gels, and hybridized with nick-translated TnI (cM113) (10) and MyoD1 (pEMC11s) (8) probes as described previously (11). TnI mRNA was detected within 24 h after ITS induction and reached maximal levels by 48 h (Fig. 1) (Fig. 2) showed no TnI mRNA (Fig. 2), no myosin heavy-chain protein, and no fusion (data not shown). Interestingly, MyoD1 mRNA levels also were reduced in growth-factortreated cultures so that by 48 h, MyoD1 mRNA was no longer detected. This growth-factor-induced inhibition of differentiation is reversible, however, since removal of FGF from inhibited cultures restored MyoD1 expression, increased TnI mRNA levels (Fig. 2), and led to a resumption of myoblast fusion. Similar results were obtained when TGF-, was removed, although the recovery time was delayed (data not shown). The growth-factor-induced repression of MyoDi mRNA appears to be specific, since the control pAL15 gene (2, 3) was expressed equally in all experimental groups. In addition. the effects of FGF and TGF-3 on myogenesis and MyoD1 expression are not unique to the 23A2 cell line. C2C12 myoblasts (4) behaved in an identical fashion when treated with these growth fac...

Pit-1 is a tissue-specific POU domain factor obligatory for the appearance of three cell phenotypes in the anterior pituitary gland. Expression of the pit-1 gene requires the actions of a cell-specific 390-bp enhancer, located 10 kb 5' of the pit-1 transcription initiation site, within sequence that proves essential for effective pituitary targeting of transgene expression during murine development. The enhancer requires the concerted actions of a cell-specific c/s-active element, Pit-1 autoregulatory sites, and atypical morphogen response elements. Pituitary ontogeny in the Pit-l-defective Snell dwarf mouse reveals that pit-1 autoregulation is not required for initial activation or continued expression during critical phases of Pit-1 target gene activation but, subsequently, is necessary for maintenance of pit-1 gene expression following birth. A potent 1,25-dihydroxyvitamin D3-responsive enhancer element defines a physiological site in which a single nucleotide alteration in the sequence of core binding motifs modulates the spacing rules for nuclear receptor response elements. Unexpectedly, the major retinoic acid response element is absolutely dependent on Pit-1 for retinoic acid receptor function. On this DNA element, Pit-1 appears to function as a coregulator of the retinoic acid receptor, suggesting an intriguing linkage between a cell-specific transcription factor and the actions of morphogen receptors that is likely to be prototypic of mechanisms by which other cell-specific transcription factors might confer morphogen receptor responsivity during mammalian organogenesis.

Expression of the mammalian muscle regulatory factors MyoDl, myogenin, and MRF4 will convert C3H1OTl/2 fibroblasts to stable muscle cell lineages. Recent Among these are the Drosophila regulatory proteins twist (48), daughterless (14,17), and the achaete-scute complex (1) as well as the immunoglobulin enhancer-binding proteins E12, E47 (37) and ITF-1, and ITF-2 (26).The initial classification of MyoDl, myogenin, Myf-5, and MRF4 as muscle regulatory factors was based on their ability to convert fibroblasts to stable muscle cell lineages. However, MyoDl, myogenin, and Myf-5 also regulate expression of a number of contractile protein genes (10,22,31,37,50

A screen designed to identify proteins that specifically bind to retinoic acid response elements resulted in the identification of a rat cDNA encoding a novel protein containing six Cys-Cys, His-Cys zinc fingers. This gene is expressed in a restricted fashion exhibiting distinct temporal and spatial patterns in the developing nervous system, primarily brain, spinal cord, sensory ganglia, retina, and nasal epithelia, as well as in the pituitary, and is referred to as neural zinc finger factor 1 (NZF-1). NZF-1 binds specifically to a cis-regulatory element of the ␤-retinoic acid receptor (RAR␤) gene, as well as to other related DNA elements, including two in the upstream enhancer region of the mouse Pit-1 gene. In heterologous cells, NZF-1 activates transcription from promoters containing specific binding sequences and can synergize with other factors, such as Pit-1, to regulate gene expression. These results suggest that NZF-1 may exert regulatory roles in the developing and mature nervous system and in the pituitary gland. Identification of a second mouse gene highly homologous to NZF-1, encoded by a distinct genomic locus, reveals a dispersed gene family encoding proteins containing Cys-Cys, His-Cys motifs.Precise temporal and spatial patterns of development are controlled by sequential activation of a hierarchy of regulatory genes, which encode transcription factors containing multiple classes of DNA binding motifs. Zinc coordinated fingers are one of the most common DNA binding motifs among eukaryotic transcription factors and are classified based on amino acid sequence of the zinc fingers. The Cys-Cys, His-His class, which is typified by the Xenopus transcription factor IIIA (1), contains the largest number of members. These proteins contain two or more fingers in a tandem repeat. In contrast, steroid receptors, such as the glucocorticoid receptor, contain only two zinc coordinated structures with four (C 4 ) and five (C 5 ) conserved cysteines. The third class of zinc fingers, which also binds to single-stranded nucleic acids, has a consensus sequence of Cys-X 2 -Cys-X 4 -His-X 4 -Cys. Such factors are found in transposable element copia, plants, and mammalian cells as well as in retroviruses. Other metal-coordinating proteins have different structures such as C 6 in the yeast GAL4 protein and a cysteinerich structure in the E1A oncoprotein (2).In accordance with their structural diversity, zinc finger proteins play a variety of important roles in cell growth, differentiation, and development. Transcription factor IIIA and the ubiquitous transcription factor SP1 are broadly involved in the regulation of transcription, whereas the Drosophila zinc finger proteins Krü ppel and Hunchback are crucial for proper segmentation of the developing embryo (3-5). In humans, mutations in a kidney zinc finger protein (WT1) result in Wilm's tumor (6, 7). Recently, a zinc finger protein (REST) has been shown to repress neuronal gene expression in non-neuronal tissues (8, 9).Because retinoic acid receptor (RAR) 1 binds ineff...