Extradenticle and Homothorax Control Adult Muscle Fiber Identity in Drosophila

SummaryHere we identify a key role for the homeodomain proteins Extradenticle (Exd) and Homothorax (Hth) in the specification of muscle fiber fate in Drosophila. exd and hth are expressed in the fibrillar indirect flight muscles but not in tubular jump muscles, and manipulating exd or hth expression converts one muscle type into the other. In the flight muscles, exd and hth are genetically upstream of another muscle identity gene, salm, and are direct transcriptional regulators of the signature flight muscle structural gene, Actin88F. Exd and Hth also impact muscle identity in other somatic muscles of the body by cooperating with Hox factors. Because mammalian orthologs of exd and hth also contribute to muscle gene regulation, our studies suggest that an evolutionarily conserved genetic pathway determines muscle fiber differentiation

Expression of the Troponin C at 41C Gene in Adult Drosophila Tubular Muscles Depends upon Both Positive and Negative Regulatory Inputs.

Most animals express multiple isoforms of structural muscle proteins to produce tissues with different physiological properties. In Drosophila, the adult muscles include tubular-type muscles and the fibrillar indirect flight muscles. Regulatory processes specifying tubular muscle fate remain incompletely understood, therefore we chose to analyze the transcriptional regulation of TpnC41C, a Troponin C gene expressed in the tubular jump muscles, but not in the fibrillar flight muscles. We identified a 300-bp promoter fragment of TpnC41C sufficient for the fiber-specific reporter expression. Through an analysis of this regulatory element, we identified two sites necessary for the activation of the enhancer. Mutations in each of these sites resulted in 70% reduction of enhancer activity. One site was characterized as a binding site for Myocyte Enhancer Factor-2. In addition, we identified a repressive element that prevents activation of the enhancer in other muscle fiber types. Mutation of this site increased jump muscle-specific expression of the reporter, but more importantly reporter expression expanded into the indirect flight muscles. Our findings demonstrate that expression of the TpnC41C gene in jump muscles requires integration of multiple positive and negative transcriptional inputs. Identification of the transcriptional regulators binding the cis-elements that we identified will reveal the regulatory pathways controlling muscle fiber differentiation

Additional positive regulatory elements exist in the <i>TpnC41C</i> promoter.

<p>(A) Diagram of the aligned <i>TpnC41C</i> and <i>Act79B</i> regulatory regions, showing the similar sequences termed SM3 and SM4. (B-D) Top: diagrams of <i>TpnC41C</i> promoter-<i>nlacZ</i> constructs bearing either wild-type (B), mutated SM3 (C) or double mutant SM3 and MEF2 sites (D). Below the schematics are representative images of cryosections of the transgenic flies stained with: Phalloidin (green, left panels), to reveal the thoracic muscles; anti-β-galactosidase (red, center panels), to reveal nuclear β-galactosidase accumulation; and X-Gal (blue, right panels), to reveal nuclear β-galactosidase activity. The TDT is outlined. Scale bar, 100μm. (E) β-galactosidase activity was measured in lysates from dissected TDT muscles expressing either the <i>nlacZ</i> reporter with no enhancer (vector) or <i>nlacZ</i> reporters controlled by wild type (WT) and mutant <i>TpnC41C</i> promoters (SM3 or SM3/MEF2 indicating the sites that were mutated). Asterisks indicate t-test p-values; * p = 2.2268x10^-12, ** p = 4.49158x10^-18, *** p = 5.39191x10^-19.</p

The <i>TpnC41C</i> promoter is conserved in the Sophophora subgenus and specifically drives reporter expression in the TDT.

<p>(A) The high conservation of the upstream region of the <i>TpnC41C</i> gene is revealed by sequence alignment across five Drosophila species: <i>D</i>. <i>melanogaster (D</i>. <i>mel)</i>, <i>D</i>.<i>simulans (D</i>. <i>sim)</i>, <i>D</i>.<i>sechellia (D</i>. <i>sec)</i>, <i>D</i>. <i>yakuba (D</i>. <i>yak)</i> and <i>D</i>.<i>erecta (D</i>. <i>ere)</i>. Fully conserved nucleotides are highlighted in yellow, and strongly conserved nucleotides are highlighted in blue. Arrow shows the location of the transcription start site, and numbers indicate nucleotide positions relative to the transcription start at +1. Brackets show localization of the conserved binding sites which are discussed later in the paper. (B) Schematic of the <i>D</i>. <i>melanogaster TpnC41C</i> gene, showing exon composition. Protein coding regions are shown in orange, untranslated regions shown in grey. Below are diagrams of the promoter regions tested for activity in the TDT using a <i>lacZ</i> reporter. Promoter activity was evaluated by X-Gal staining on cryosections and was classified as “strong” (+), when the blue staining developed in 5 min; or “weak” (+/-) when it developed in more than 1 h. Right panel shows a representative stained section of the Drosophila thorax expressing the <i>nlacZ</i> reporter controlled by a 300-bp promoter region TC41C-E (spanning the region from -251 to +49). TDT muscles are outlined with a dashed line, asterisks indicate indirect flight muscles. Bar, 100μm.</p

Identification of a negative regulatory element within the <i>TpnC41C</i> enhancer.

<p>(A) The upper panel shows an electrophoretic mobility shift assay resulting from combination of pupal nuclear extracts with dsDNA probes. For each probe, lanes contain radioactively-labeled probe alone (lanes 1, 4, 7, 10, 13, 16, 19), or probe plus nuclear extract (lanes 2, 5, 8, 11, 14, 17, 20), or probe plus nuclear extract plus 100-fold excess of unlabeled probe (lanes 3, 6, 9, 12, 15, 18). NS–non-specific band. Probe sequences are shown in the lower panel. Red sequences indicate mutated nucleotides. Brackets indicate boundaries of the R1 and the truncated MEF2 sites.(B) Mutation of R1 boosts promoter activity and compromises specificity of the <i>TpnC41C</i> promoter fragment. Upper diagrams show relative position of the R1 site within the promoter-<i>nlacZ</i>. Below the diagrams are representative images of cryosections of the transgenic flies stained with: Phalloidin (green, left panels), to reveal the thoracic muscles; anti-β-galactosidase (red, center panels), to reveal nuclear β-galactosidase accumulation; and X-Gal (blue, right panels), to reveal nuclear β-galactosidase activity. The TDT is outlined, and asterisks indicate the flight muscles. Scale bar, 100μm.</p

MEF2 binds to and activates the <i>TpnC41C</i> promoter.

<p>(A) MEF2 binding to the <i>TpnC41C</i> promoter was analyzed by electrophoretic mobility shift assay. On the left: the sequence of the 30-bp probe, containing a putative MEF2 site (underlined) and its mutated version (mutated nucleotides shown in red). The double-stranded, radioactively-labeled wild-type probe was incubated with <i>in vitro</i> translated MEF2 protein, resulting in a shifted DNA-MEF2 complex (lane 1). The specificity of binding was tested by adding to the reaction mixture a 100-fold excess of the same unlabeled oligonucleotide (“competitor”, lane 2), or unlabeled oligonucleotide with the nucleotide substitution in the MEF2 site (“mut. competitor”, lane 3). Lysate containing no MEF2 protein only resulted in a non-specific interaction between protein in the lysate and the DNA probe (lane 4, NS). (B) Activation of the <i>TpnC41C</i> promoter in Drosophila S2 cells by MEF2. Cells were transfected with either wild type (dark blue) or mutated (light blue) <i>TpnC41C</i> promoter-<i>nlacZ</i> constructs, alongside either empty expression vector (VECTOR) or expression vector for MEF2 (+MEF2). β-galactosidase activity was analyzed in cell lysates 24 hours post-transfection, and the results are expressed as normalized β-galactosidase activity. Asterisks indicate t-test p-values; * p = 2.18x10^-5, ** p = 0.003283.</p

Genetic Evidence for the Role of the Vacuole in Supplying Secretory Organelles with Ca2+ in Hansenula polymorpha.

Processes taking place in the secretory organelles require Ca2+ and Mn2+, which in yeast are supplied by the Pmr1 ion pump. Here we observed that in the yeast Hansenula polymorpha Ca2+ deficiency in the secretory pathway caused by Pmr1 inactivation is exacerbated by (i) the ret1-27 mutation affecting COPI-mediated vesicular transport, (ii) inactivation of the vacuolar Ca2+ ATPase Pmc1 and (iii) inactivation of Vps35, which is a component of the retromer complex responsible for protein transport between the vacuole and secretory organelles. The ret1-27 mutation also exerted phenotypes indicating alterations in transport between the vacuole and secretory organelles. These data indicate that ret1-27, pmc1 and vps35 affect a previously unknown Pmr1-independent route of the Ca2+ delivery to the secretory pathway. We also observed that the vacuolar protein carboxypeptidase Y receives additional modifications of its glycoside chains if it escapes the Vps10-dependent sorting to the vacuole

Prion properties of the Sup35 protein of yeast Pichia methanolica

The Sup35 protein (Sup35p) of Saccharomyces cerevisiae is a translation termination factor of the eRF3 family. The proteins of this family possess a conservative C–terminal domain responsible for translation termination and N–terminal extensions of different structure. The N–terminal domain of Sup35p defines its ability to undergo a heritable prion-like conformational switch, which is manifested as the cytoplasmically inherited [PSI(+)] determinant. Here, we replaced the N–terminal domain of S.cerevisiae Sup35p with an analogous domain from Pichia methanolica. Overexpression of hybrid Sup35p induced the de novo appearance of cytoplasmically inherited suppressor determinants manifesting key genetic and biochemical traits of [PSI(+)]. In contrast to the conventional [PSI(+)], ‘hybrid’ [PSI(+)] showed lower mitotic stability and preserved their suppressor phenotype upon overexpression of the Hsp104 chaperone protein. The lack of Hsp104 eliminated both types of [PSI(+)]. No transfer of prion state between the two Sup35p variants was observed, which reveals a ‘species barrier’ for the [PSI(+)] prions. The data obtained show that prion properties are conserved within at least a part of this protein family

Functional redundancy and nonredundancy between two Troponin C isoforms in adult muscles

We investigated the functional overlap of two muscle Troponin C (TpnC) genes that are expressed in the adult fruit fly, : is predominantly expressed in the indirect flight muscles (IFMs), whereas is the main isoform in the tergal depressor of the trochanter muscle (TDT; jump muscle). Using CRISPR/Cas9, we created a transgenic line with a homozygous deletion of and compared its phenotype to a line lacking functional We found that the removal of either of these genes leads to expression of the other isoform in both muscle types. The switching between isoforms occurs at the transcriptional level and involves minimal enhancers located upstream of the transcription start points of each gene. Functionally, the two TpnC isoforms were not equal. Although ectopic TpnC4 in TDT muscles was able to maintain jumping ability, TpnC41C in IFMs could not effectively support flying. Simultaneous functional disruption of both TpnC genes resulted in jump-defective and flightless phenotypes of the survivors, as well as abnormal sarcomere organization. These results indicated that TpnC is required for myofibril assembly, and that there is functional specialization among TpnC isoforms in