Sequence analysis of the confirmed and putative MYND domains.

<p>(A) For the protein sequences indicated on the left, the MYND Zn finger within the interrupted SET domain is compared to the PROSITE signature PS01360 and to the MYND hidden Markov model PFAM signature. For the PROSITE signature, the formula is indicated above with Zn-ligand residues in bold and highlighting the four Zn-ligand pairs. We have highlighted in grey all departures from the signature, including residues that do not match the consensus and stretches of the wrong length. For the PFAM model we indicate whether the sequence is a perfect match (+), a partial match of the right or left portions (+(r) and +(l) respectively), or not a match at all (-, highlighted grey). (B) Scheme of the cross-brace disposition of a MYND-type Zn finger, with the most common Zn-ligand residues depicted in black and Zn ions as empty circles; the numbers indicate the number of aminoacids between Zn-ligand residues according to the PS01360 formula.</p

Species included in the phylogenetic study of Smyd family proteins.

<p>Species included in the phylogenetic study of Smyd family proteins.</p

Proposed evolutionary history of <i>Smyd</i> genes in metazoans.

<p>The schematic tree represents the accepted phylogeny for the phyla/subphyla represented in this work. From an original complement of <i>Smyd</i> genes, comprising Smyd3, Smyd4 and Smyd5, we indicate with white boxes the most likely events of gene gain (+) duplication (Dup.) and expansion (Exp.); and in black boxes the events of gene loss (-).</p

Transcriptional expression profile of <i>Drosophila</i> and mouse <i>Smyd4</i> class genes.

<p>We show a transcriptional study for <i>Drosophila melanogaster</i> genes <i>CG1868</i> (A-A”‘), <i>CG8378</i> (B-B”‘), <i>CG14122</i> (C-C”‘) and <i>CG7759</i> (D-D”‘) and <i>Mus musculus Smyd4</i> (E-G). For the <i>Drosophila</i> genes we show high throughput data from two consortia, the anatomical expression profile of FlyAtlas (A-D) and the temporal expression profile of modENCODE (A’-D’). For these genes we also determined the expression pattern by i<i>n situ</i> hybridization in embryos (anterior to the right, lateral view except where indicated). We show expression in extended germ band (A”-D”) and in stage 16 embryos (A”‘-D”‘). The ventral nerve cord in B”‘ is not visible as the embryo is slightly tilted. We also determined the expression of <i>Smyd4</i> in a 14.5E mouse embryo section. We show sections of the abdomen (E), dorsal trunk (F) and head (G). Abbreviations are used for the tissues where expression is detected as follows: GP, gut primordium; CP, cephalic primordium of the central nervous system; GT, gut; MS, mesoderm; CNS, central nervous system; SP, spinal cord; DRG, dorsal root ganglia; EN, encephalon.</p

Schematic phylogenetic trees of the Smyd proteins.

<p>In all the panels sequences are indicated with abbreviated species name and annotated protein name. Branch lengths and bootstrap values are given as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134106#pone.0134106.g001" target="_blank">Fig 1</a>. (A, B) Phylogenetic trees obtained with the alignment of the metazoan Smyd proteins by the maximum likelihood (A) and neighbor joining (B) methods; the branches corresponding to the four main classes are compressed and the two sequences not included in one of these classes are indicated in grey. (C) Maximum likelihood and neighbor joining (D) phylogenetic trees obtained with the alignment of the extended dataset, which includes the sequences from the metazoans plus unicellular metazoan-related species <i>Capsaspora owczarzaki</i> and <i>Monosiga brevicollis</i>, the plant <i>Arabidopsis thaliana</i> and the yeast <i>Saccharomyces cerevisiae</i>. The non-metazoan species are indicated in black type, and those that are grouped within one of the classes are indicated in the compressed branch with the name preceded by +.</p

ANKK1 is found in myogenic precursors and muscle fibers subtypes with glycolytic metabolism

<div><p>Ankyrin repeat and kinase domain containing 1 (<i>ANKK1</i>) gene has been widely related to neuropsychiatry disorders. The localization of ANKK1 in neural progenitors and its correlation with the cell cycle has suggested its participation in development. However, ANKK1 functions still need to be identified. Here, we have further characterized the ANKK1 localization <i>in vivo</i> and <i>in vitro</i>, by using immunolabeling, quantitative real-time PCR and Western blot in the myogenic lineage. Histologic investigations in mice and humans revealed that ANKK1 is expressed in precursors of embryonic and adult muscles. In mice embryos, ANKK1 was found in migrating myotubes where it shows a polarized cytoplasmic distribution, while proliferative myoblasts and satellite cells show different isoforms in their nuclei and cytoplasm. <i>In vitro</i> studies of ANKK1 protein isoforms along the myogenic progression showed the decline of nuclear ANKK1-kinase until its total exclusion in myotubes. In adult mice, ANKK1 was expressed exclusively in the Fast-Twitch muscles fibers subtype. The induction of glycolytic metabolism in C2C12 cells with high glucose concentration or treatment with berberine caused a significant increase in the <i>ANKK1</i> mRNA. Similarly, C2C12 cells under hypoxic conditions caused the increase of nuclear ANKK1. These results altogether show a relationship between <i>ANKK1</i> gene regulation and the metabolism of muscles during development and in adulthood. Finally, we found ANKK1 expression in regenerative fibers of muscles from dystrophic patients. Future studies in ANKK1 biology and the pathological response of muscles will reveal whether this protein is a novel muscle disease biomarker.</p></div

ANKK1 participates in myogenic differentiation.

<p><b>(A)</b> ANKK1 (α-STk2) and ACTININ immunostaining of C2C12 cells during myogenic differentiation. D0: proliferative myoblast; D3: myoblasts in differentiation; D6: myotubes. <b>(B)</b> Quantification of nuclear ANKK1 fluorescence intensity (a.u.) (N = 3) <b>(C)</b> Western blot analysis of C2C12 subcellular fractions. β-ACTIN was used as control and α-LAMIN A/C as nuclear marker (N = 1). <b>(D)</b> Rhabdomyosarcoma (RD) myoblasts differentiation, <b>(E)</b> quantification of nuclear ANKK1 (N = 3) and <b>(F)</b> Western blot of subcellular fractions (N = 1). <b>(G)</b> Immunostaining of ANKK1 with PAX7 and MYOD in C2C12 cells, (left) and quantification of positive nuclei for ANKK1, PAX7 and MYOD along myogenic differentiation (right, N = 3). Images were taken from confocal optical sections that are representative for the group averages. Scale bar: 25 μm <i>p</i> < 0.05: *; <i>p</i> < 0.01: **; <i>p</i> < 0.001: ***. D: Days after induction of differentiation; a.u: arbitrary units; N: Nucleus; C: Cytoplasm.</p

<i>ANKK1</i> is expressed exclusively in Fast-Twitch fibers.

<p><b>(A)</b> Representative images for the group averages of IHC analysis of ANKK1, MyHC1 and MyHC2 in mice adult skeletal gastrocnemius serial sections and <b>(B)</b> colocalization of ANKK1 and MyHC2 in triceps and soleus muscles. The black asterisk indicates the same fiber. Scale bar: 50 μm. <b>(C)</b> Glycolysis induction in C2C12 myoblasts using HG and berberine during 24 h treatments. Oxygen consumption was assessed using a Clark-type oxygen electrode (N = 7) and lactic acid production was measured (N = 3). Relative oxygen and L-lactic acid production values are shown (±SD). <b>(D)</b> <i>ANKK1</i> expression analysis by reverse transcriptase polymerase chain reaction (N = 3). Relative mRNA values (±SD) are shown. <b>(E)</b> Western blot of subcellular C1C12 fractions using α-STk2 (N = 4). β-ACTIN was used as control and α-TUBULIN as cytosolic marker. Western blot bands were quantified using the ImageJ 1.45v software and the relative expression was represented related to β-Actin value. <i>p</i> < 0.05: *; <i>p</i> < 0.01: **; <i>p</i> < 0.001: ***.N: nucleus; C: cytoplasm; RE: Relative Expression; Ber: berberine.</p

ANKK1 is located in SCs and in a fiber subtype in adult muscles.

<p>ANKK1 (detected with α-STk, α-STk2 and α-STk3), PAX7 and MYOD immunostaining of mouse FDB isolated fibers and gastrocnemius sections of adult mice. Nuclei were stained with DAPI in IF and with hematoxylin in IHC. <b>(A)</b> ANKK1+ (α-STk2)/PAX7+ SCs. Boxed area in ANKK1 images are amplified [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197254#pone.0197254.ref025" target="_blank">25</a>]. The green arrows show ANKK1− nuclei. <b>(B)</b> ANKK1+(α-STk2)/MYOD+ SCs showing ANKK1 cytoplasmic expression. <b>(C)</b> ANKK1+(α-STk3)/PAX7+ and <b>(D)</b> ANKK1+(α-STk3)/MYOD+ SCs showing ANKK1 cytoplasmic expression. Scale bar: 25 μm. <b>(E)</b> α-STk, α-STk2 and α-STk3 antibodies recognize the same pattern for ANKK1 in a serial of transversal gastrocnemius sections. <b>(F)</b> ANKK1 is expressed in the cytoplasm of some fibers and in SCs (black arrow). Scale bar: 50 μm. Images are representative for the group averages.</p

ANKK1 is expressed in regenerating fibers in neuromuscular dystrophies.

<p>ANKK1 and a mix of embryonic and neonatal myosin heavy chains (eMyHC/nMyHC) immunostaining of adult skeletal muscle sections. The samples were non-dystrophic (control) and dystrophic patients (DMD, LGDM2A, LGMD2B and FSHD). ANKK1 and eMyHC/nMyHC were studied in serial muscle sections. The black asterisk indicates the same fiber. The black arrows indicate ANKK1+ nuclei. Scale bar: 50 μm. All regenerating fibers from each tissue section were counted and analyzed. Images are representative for the group averages.</p