Diminished Telomeric 3′ Overhangs Are Associated with Telomere Dysfunction in Hoyeraal-Hreidarsson Syndrome

BACKGROUND:Eukaryotic chromosomes end with telomeres, which in most organisms are composed of tandem DNA repeats associated with telomeric proteins. These DNA repeats are synthesized by the enzyme telomerase, whose activity in most human tissues is tightly regulated, leading to gradual telomere shortening with cell divisions. Shortening beyond a critical length causes telomere uncapping, manifested by the activation of a DNA damage response (DDR) and consequently cell cycle arrest. Thus, telomere length limits the number of cell divisions and provides a tumor-suppressing mechanism. However, not only telomere shortening, but also damaged telomere structure, can cause telomere uncapping. Dyskeratosis Congenita (DC) and its severe form Hoyeraal-Hreidarsson Syndrome (HHS) are genetic disorders mainly characterized by telomerase deficiency, accelerated telomere shortening, impaired cell proliferation, bone marrow failure, and immunodeficiency. METHODOLOGY/PRINCIPAL FINDINGS:We studied the telomere phenotypes in a family affected with HHS, in which the genes implicated in other cases of DC and HHS have been excluded, and telomerase expression and activity appears to be normal. Telomeres in blood leukocytes derived from the patients were severely short, but in primary fibroblasts they were normal in length. Nevertheless, a significant fraction of telomeres in these fibroblasts activated DDR, an indication of their uncapped state. In addition, the telomeric 3' overhangs are diminished in blood cells and fibroblasts derived from the patients, consistent with a defect in telomere structure common to both cell types. CONCLUSIONS/SIGNIFICANCE:Altogether, these results suggest that the primary defect in these patients lies in the telomere structure, rather than length. We postulate that this defect hinders the access of telomerase to telomeres, thus causing accelerated telomere shortening in blood cells that rely on telomerase to replenish their telomeres. In addition, it activates the DDR and impairs cell proliferation, even in cells with normal telomere length such as fibroblasts. This work demonstrates a telomere length-independent pathway that contributes to a telomere dysfunction disease

Amontillado is required for Drosophila Slit processing and for tendon-mediated muscle patterning

Slit cleavage into N-terminal and C-terminal polypeptides is essential for restricting the range of Slit activity. Although the Slit cleavage site has been characterized previously and is evolutionally conserved, the identity of the protease that cleaves Slit remains elusive. Our previous analysis indicated that Slit cleavage is essential to immobilize the active Slit-N at the tendon cell surfaces, mediating the arrest of muscle elongation. In an attempt to identify the protease required for Slit cleavage we performed an RNAi-based assay in the ectoderm and followed the process of elongation of the lateral transverse muscles toward tendon cells. The screen led to the identification of the Drosophila homolog of pheromone convertase 2 (PC2), Amontillado (Amon), as an essential protease for Slit cleavage. Further analysis indicated that Slit mobility on SDS polyacrylamide gel electrophoresis (SDS-PAGE) is slightly up-shifted in amon mutants, and its conventional cleavage into the Slit-N and Slit-C polypeptides is attenuated. Consistent with the requirement for amon to promote Slit cleavage and membrane immobilization of Slit-N, the muscle phenotype of amon mutant embryos was rescued by co-expressing a membrane-bound form of full-length Slit lacking the cleavage site and knocked into the slit locus. The identification of a novel protease component essential for Slit processing may represent an additional regulatory step in the Slit signaling pathway

Multiplexin Promotes Heart but Not Aorta Morphogenesis by Polarized Enhancement of Slit/Robo Activity at the Heart Lumen

<div><p>The <i>Drosophila</i> heart tube represents a structure that similarly to vertebrates' primary heart tube exhibits a large lumen; the mechanisms promoting heart tube morphology in both <i>Drosophila</i> and vertebrates are poorly understood. We identified Multiplexin (Mp), the <i>Drosophila</i> orthologue of mammalian Collagen-XV/XVIII, and the only structural heart-specific protein described so far in <i>Drosophila</i>, as necessary and sufficient for shaping the heart tube lumen, but not that of the aorta. Mp is expressed specifically at the stage of heart tube closure, in a polarized fashion, uniquely along the cardioblasts luminal membrane, and its absence results in an extremely small heart tube lumen. Importantly, Mp forms a protein complex with Slit, and interacts genetically with both <i>slit</i> and <i>robo</i> in the formation of the heart tube. Overexpression of Mp in cardioblasts promotes a large heart lumen in a Slit-dependent manner. Moreover, Mp alters Slit distribution, and promotes the formation of multiple Slit endocytic vesicles, similarly to the effect of overexpression of Robo in these cells. Our data are consistent with Mp-dependent enhancement of Slit/Robo activity and signaling, presumably by affecting Slit protein stabilization, specifically at the lumen side of the heart tube. This activity results with a Slit-dependent, local reduction of F-actin levels at the heart luminal membrane, necessary for forming the large heart tube lumen. Consequently, lack of Mp results in decreased diastolic capacity, leading to reduced heart contractility, as measured in live fly hearts. In summary, these findings show that the polarized localization of Mp controls the direction, timing, and presumably the extent of Slit/Robo activity and signaling at the luminal membrane of the heart cardioblasts. This regulation is essential for the morphogenetic changes that sculpt the heart tube in <i>Drosophila</i>, and possibly in forming the vertebrates primary heart tube.</p></div

Genetic and physical association between Mp, Slit and Robo.

<p>Cardiac cross sections of wild type (A), <i>slit/+;mp/+</i> (B), <i>robo/+;mp/+</i> (C) double heterozygous, and <i>mp/+</i> (D), <i>slit/+</i> (E), <i>robo/+</i> (F) single heterozygous labeled with anti HOW (green), and anti Dg (red). The cardiac lumens (marked by arrowheads) of the double-heterozygous mutant are smaller relative to the control. G-I: cardiac cross sections of wild type embryos labeled with anti Slit (G,I red) and anti Mp (H,I green), indicating their co-localization along the lumen. J- immunoprecipitation with anti Slit antibodies (or with a control normal mouse serum) of an extract of S2 cells co-transfected with Slit, Robo, and Mp. The same blot was then reacted individually with anti- Slit, Mp, and Robo corresponding to the three upper lanes). The anti Mp antibody reacted with a single band of ∼120 kDa, corresponding to mp cDNA 3hnc1. This immunoprecipitation (IP) is representative of three independent IP experiments. The crude extracts contained comparable amounts of transfected proteins as indicated by the antibody reactivity with each of the transfected cDNA constructs presented in the right panel. K-Immunoprecipitation with anti Slit antibodies of comparable protein extracts taken from stage 16 control (yw), <i>mp^−/−</i>, or embryos overexpressing Mp in heart and muscles (using <i>mef2-GAL4</i> driver). Western blot with anti Slit of the IP material shows elevated levels of Slit in the Mp-overexpressing embryos and reduced Slit levels in <i>mp</i> mutants. Reaction of the same blot with anti Mp antibodies (lower panel) revealed a specific band of ∼39 kDa, corresponding to the Endostatin fragment. Western blot with anti Tropomyosin of the embryo protein extracts before taken to the IP with Slit, is shown in the lower panel, indicating comparable protein levels in each of the samples.</p

Mp activity in cardioblasts depends on Slit.

<p>Cardiac cross sections of wild type (A, A′, A″), <i>slit</i> mutants (B, B′, B″), embryos overexpressing Mp in cardioblasts (C, C′,C″), embryos overexpressing Robo in cardioblasts (D, D′,D″), and embryos overexpressing Mp in <i>slit</i> mutant background (E, E′, E″), all labeled with anti Dg (red) and with anti Armadillo (green). Arrowheads in A–A″ mark the dorsal and ventral junctions and in B–E″ the dorsal junction. Note that overexpression of Mp is not capable of promoting large and curved lumen in <i>slit</i> mutant hearts, and the lack of Armadillo vesicles in these cells. C′ and D′- White arrows mark Armadillo vesicles. C-cardioblast cells. The bar in A is 5 µm and represents the magnification in all panels.</p

Mp enhances Slit/Robo activity in the heart lumen and modulates Slit distribution in the central nervous system.

<p>Cross sections through the heart (A–F′″) or the aorta (G–H′) of wild type embryos (A, A′, G, G′); <i>robo</i> mutant (B, B′), embryos overexpressing Robo in cardioblasts (C, C′); <i>mp</i> mutant (D, D′), or embryos overexpressing Mp in cardioblasts (E, E′, H, H′) labeled with anti Slit (red) and anti Dg (green). F–F′″ show embryos expressing dominant-negative Rab5-YFP in cardioblasts labeled with Slit (F, red), YFP (F′, green), Slit and YFP (F″), or Slit and Dg (white, F′″). Panels A–H′ are single optical confocal sections to enable comparison of the extent of cytoplasmic Slit vesicles in each genotype. Insets are 2.5 folds enlargement of the heart luminal domain. White arrows in each inset, indicate Slit vesicle/s, with the exception of E′″ where the white arrow indicates Slit vesicle position. Note the reduction in the number and size of Slit endocytic vesicles observed in both <i>mp</i> and robo mutants, and their elevation following Mp and Robo overexpression. Represented schemes of Slit vesicles distribution are indicated for each genotype. Note the alteration in Slit distribution following Rab5 dominant negative overexpression (the majority of the protein overlaps Dg at the luminal membrane, and the cytoplasmic Slit is associated with the luminal membrane). I and I′ show wild type (I), or embryo overexpressing Mp in the midline using <i>sim-GAL4</i> driver (I′), both labeled for Fasciclin II (red). Note the shortening of the distance between the longitudinal commissures and the midline (marked by the white arrow), following overexpression of Mp. Cross sections through the ventral nerve cord of wild type (J,J′) or an embryo overexpressing Mp in the midline cells (K,K′) labeled with anti Slit (red) and with anti HOW (which marks the midline glia, green J′,K′). High Slit accumulation in the midline (marked by white arrowheads) is observed following overexpression of Mp. Bar in A is 5 µm and represents the magnification in panels A–H′. Bar in I is 20 µm, and represents magnification in I,I′. Bar in J is 10 µm and represents magnification in J–K′).</p

Mp exhibits heart lumen-specific distribution and is necessary and sufficient for cardiac lumen formation.

<p>Upper panel: a scheme of the three stages of cardiac tube closure: I- cardioblasts approach the dorsal midline. II- formation of the dorsal junction and the inward curvature of the luminal membrane. III- formation of the ventral junction and tube closure. Mp initial expression is observed between stage II to stage III (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003597#pgen.1003597.s001" target="_blank">Figure S1</a>). A whole mount wild type embryo at stage 16 labeled for Mp (A,A′, green), and Mef-2 (A, red). The heart and aorta domains are indicated. The arrowhead in A indicates a pair of ostia cells which do not express Mp. B-I are cross sections of stage 16 embryos. Wild type heart (B,B′) or aorta (C,C′) labeled with Dystroglycan (Dg, red, B,C) and Mp (green B,B′,C,C′) demonstrate the cardiac specific distribution of Mp in the heart lumen. D-I are cross sections of: wild type heart (D), <i>mp</i> mutant heart (E), heart cardioblasts overexpressing Mp (F), wild type aorta (G) <i>mp</i> mutant aorta (H), aorta cardioblasts overexpressing Mp (I), labeled with Dg (red) and with anti HOW, which labels the cardioblast cytoplasm (green). Arrowheads in D-I mark the cardiac lumen. Note the formation of large cardiac lumen (29 µm², F), following Mp overexpression in the heart and the formation of a heart-like lumen in the aorta following Mp overexpression in the aorta. J - quantification of the lumen cross section area, K- quantification of the luminal perimeter, L- quantification of cardioblast cross section area measured from 3–4 cross sections per embryo in multiple number of embryos (n). A statistically significant reduction (indicated by three stars) in both cardiac luminal area (reduction of 55%, p = 8.6E-0.6) and perimeter (reduction of 27%, p = 3.8E-0.50) was observed. A slight reduction in cardioblasts total area was also detected (13%, p = 0.02, one star). C- cardioblast cell. Scale bars represent 20 µm in A,A′, and 5 µm in all cross sections (B,C, D–L).</p

Mp activity reduces F-actin levels at the cardiac luminal membrane in a <i>slit</i> dependent manner.

<p>Cardiac cross sections of embryos at stage 15 (A, A′) or stage 16 (B–G′) labeled with phalloidin (green) and Dg (red), of the following genotypes: wild type (A, A′, B, B′), <i>slit</i> mutant (C, C′), <i>mp</i> mutant (D, D′), embryos overexpressing Mp (<i>mef2-GAL4>UAS-mp</i>, E, E′), embryos overexpressing Robo (<i>mef-2-GAL4>UAS-robo</i>, F, F′) or <i>slit</i> mutant embryos overexpressing Mp (G, G′). White arrows indicate the luminal membrane, while the luminal membrane of stage 16 wild type and Mp overexpressing embryos displays reduced F-actin levels, the luminal membrane of <i>slit</i> and <i>mp</i> mutant exhibits elevated F-actin levels. Note that overexpression of Mp in <i>slit</i> mutant background did not reduce F-actin levels at the luminal membrane although a small lumen was detected. The scheme in H summarizes the results; in wild type (WT) heart a constitutive activation of Slit/Robo at the luminal membrane, promoted by Mp reduces F-actin levels at the luminal membrane. In <i>mp</i> mutants Slit/Robo signaling is reduced, and consequently F-actin levels are elevated leading to a small lumen. In contrast, overexpression of Mp (Mp OE) leads to elevated Slit/Robo signaling, reducing luminal F-actin levels and enhancing lumen size. Mp overexpression in the absence of Slit exhibited elevated levels of luminal F-actin and a small lumen. Bar in A is 5 µm and represents magnification of all panels.</p

Reduced telomeric overhang signal in the HHS-affected cells.

<p>(A) Genomic DNA samples prepared from S2 and control LCLs (PDL of 44 and 50, respectively; 2 and 4 µg) or fibroblasts (PDL of 14 and 16 for S2 and C, respectively; 2 µg) were digested with MboI and AluI and electrophoresed in a 0.7% agarose gel. The average length of the 3′ overhang was estimated by in-gel hybridization of native DNA to a C-rich telomeric probe (native panels). The DNA was subsequently denatured <i>in situ</i> and re-hybridized to the same probe to measure the total TTAGGG repeat signal (denatured panels). (B) The histograms below the images represent the quantified native (overhang) signals normalized to the denatured (total) signals and presented as percentage of the normalized overhang signals of the controls.</p

TIF formation in HHS-affected fibroblasts with normal telomere length.

<p>(A) Control (C) and HHS-affected (S2) primary fibroblast cultures (established at the ages of 30 and 17 years, and grown to PDL of 14 and 10, respectively) were immunostained for TRF1 (green) and γ-H2AX (red), and with DAPI for the nuclei (blue), as indicated above the images. The bottom panels show enlarged images that include several telomeres. The images of the affected and control cells were obtained and processed in the same way, side by side. (B) The number of TIFs (defined as colocalized TRF1 and γ-H2AX foci) was counted in randomly-chosen 67 affected and 58 control cells. The graph shows the percentage of cells with at least five such foci. (C) Genomic DNA was prepared from these cultures and the average length of telomeres estimated by Southern analysis.</p