The DUX4 homeodomains mediate inhibition of myogenesis and are functionally exchangeable with the Pax7 homeodomain.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by inappropriate expression of the double homeodomain protein DUX4. DUX4 has bimodal effects, inhibiting myogenic differentiation and blocking MyoD at low levels of expression, and killing myoblasts at high levels. Pax3 and Pax7, which contain related homeodomains, antagonize the cell death phenotype of DUX4 in C2C12 cells, suggesting some type of competitive interaction. Here, we show that the effects of DUX4 on differentiation and MyoD expression require the homeodomains but do not require the C-terminal activation domain of DUX4. We tested the set of equally related homeodomain proteins (Pax6, Pitx2c, OTX1, Rax, Hesx1, MIXL1 and Tbx1) and found that only Pax3 and Pax7 display phenotypic competition. Domain analysis on Pax3 revealed that the Pax3 homeodomain is necessary for phenotypic competition, but is not sufficient, as competition also requires the paired and transcriptional activation domains of Pax3. Remarkably, substitution mutants in which DUX4 homeodomains are replaced by Pax7 homeodomains retain the ability to inhibit differentiation and to induce cytotoxicity.

Development of a test that measures real-time HER2 signaling function in live breast cancer cell lines and primary cells.

BACKGROUND: Approximately 18-20% of all human breast cancers have overexpressed human epidermal growth factor receptor 2 (HER2). Standard clinical practice is to treat only overexpressed HER2 (HER2+) cancers with targeted anti-HER2 therapies. However, recent analyses of clinical trial data have found evidence that HER2-targeted therapies may benefit a sub-group of breast cancer patients with non-overexpressed HER2. This suggests that measurement of other biological factors associated with HER2 cancer, such as HER2 signaling pathway activity, should be considered as an alternative means of identifying patients eligible for HER2 therapies. METHODS: A new biosensor-based test (CELxTM HSF) that measures HER2 signaling activity in live cells is demonstrated using a set of 19 human HER2+ and HER2- breast cancer reference cell lines and primary cell samples derived from two fresh patient tumor specimens. Pathway signaling is elucidated by use of highly specific agonists and antagonists. The test method relies upon well-established phenotypic, adhesion-related, impedance changes detected by the biosensor. RESULTS: The analytical sensitivity and analyte specificity of this method was demonstrated using ligands with high affinity and specificity for HER1 and HER3. The HER2-driven signaling quantified ranged 50-fold between the lowest and highest cell lines. The HER2+ cell lines were almost equally divided into high and low signaling test result groups, suggesting that little correlation exists between HER2 protein expression and HER2 signaling level. Unexpectedly, the highest HER2-driven signaling level recorded was with a HER2- cell line. CONCLUSIONS: Measurement of HER2 signaling activity in the tumor cells of breast cancer patients is a feasible approach to explore as a biomarker to identify HER2-driven cancers not currently diagnosable with genomic techniques. The wide range of HER2-driven signaling levels measured suggests it may be possible to make a distinction between normal and abnormal levels of activity. Analytical validation studies and clinical trials treating HER2- patients with abnormal HER2-driven signaling would be required to evaluate the analytical and clinical validity of using this functional biomarker as a diagnostic test to select patients for treatment with HER2 targeted therapy. In clinical practice, this method would require patient specimens be delivered to and tested in a central lab.

Prospective isolation of human fibroadipogenic progenitors with CD73.

Skeletal muscle relies on coordination between myogenic and non-myogenic interstitial cells for homeostasis and for regeneration and response to injury. Fibroadipogenic progenitors (FAPs) have recently been recognized as key modulators of signaling to promote myogenesis following injury. FAPs are also responsible for the fibrosis and fatty replacement of muscle tissue seen in many diseased states. While extensive use of surface markers to purify FAPs has been undertaken in the mouse system, in particular PDGFRA, markers for human FAPs are less well understood. Here, we show that CD73 can be used as a single positive marker to purify FAPs from the lineage-negative (CD45-neg, CD31-neg) fraction of skeletal muscle mononuclear cells. Although CD73 was previously found to be expressed in cultured myogenic cells, we find that this marker is only acquired upon culture and that the CD73+ fraction of human skeletal muscle has no myogenic activity. We show that Lin-neg CD73+ cells from human muscle undergo fat differentiation as well as fibrogenesis when exposed to appropriate activating signals in vitro. This simple single positive marker approach effectively enables isolation of human FAPs from fresh human skeletal muscle biopsies.

Deletion of LOX-1 reduces atherogenesis in LDLR knockout mice fed high cholesterol diet.

Atherosclerosis is associated with oxidative stress and inflammation, and upregulation of LOX-1, an endothelial receptor for oxidized LDL (oxLDL). Here, we describe generation of LOX-1 knockout (KO) mice in which binding of oxLDL to aortic endothelium was reduced and endothelium-dependent vasorelaxation preserved after treatment with oxLDL (P<0.01 versus wild-type mice). To address whether endothelial functional preservation might lead to reduction in atherogenesis, we crossed LOX-1 KO mice with LDLR KO mice and fed these mice 4% cholesterol/10% cocoa butter diet for 18 weeks. Atherosclerosis was found to cover 61+/-2% of aorta in the LDLR KO mice, but only 36+/-3% of aorta in the double KO mice. Luminal obstruction and intima thickness were significantly reduced in the double KO mice (versus LDLR KO mice). Expression of redox-sensitive NF-kappaB and the inflammatory marker CD68 in LDLR KO mice was increased (P<0.01 versus wild-type mice), but not in the double KO mice. On the other hand, antiinflammatory cytokine IL-10 expression and superoxide dismutase activity were low in the LDLR KO mice (P<0.01 versus wild-type mice), but not in the double KO mice. Endothelial nitric oxide synthase expression was also preserved in the double KO mice. The proinflammatory signal MAPK P38 was activated in the LDLR KO mice, and LOX-1 deletion reduced this signal. In conclusion, LOX-1 deletion sustains endothelial function leading to a reduction in atherogenesis in association with reduction in proinflammatory and prooxidant signals.

LOX-1 deletion decreases collagen accumulation in atherosclerotic plaque in low-density lipoprotein receptor knockout mice fed a high-cholesterol diet.

AIMS: Collagen, as a component of the extracellular matrix, has been linked to atherosclerotic plaque formation and stability. Activation of LOX-1, a lectin-like oxidized low-density lipoprotein (LDL) receptor-1, exerts a significant role in collagen formation. We examine the hypothesis that LOX-1 deletion may inhibit collagen accumulation in atherosclerotic arteries in LDL receptor (LDLR) knockout (KO) mice. METHODS AND RESULTS: We generated LOX-1 KO and LOX-1/LDLR double KO mice on a C57BL/6 (wild-type mice) background and fed a 4% cholesterol/10% cocoa butter diet for 18 weeks. Vessel wall collagen accumulation was increased in association with atherogenesis in the LDLR KO mice (P < 0.01 vs. wild-type mice), but much less so in the double KO mice (P < 0.01 vs. LDLR KO mice). Collagen accumulation data were corroborated with pro-collagen I measurements. Expression/activity of osteopontin, fibronectin, and matrix metalloproteinases (MMP-2 and MMP-9) was also increased in the LDLR KO mice (P < 0.01 vs. wild-type mice), but not in the mice with LOX-1 deletion (P < 0.01 vs. LDLR KO mice). The expression of NADPH oxidase (p47(phox), p22(phox), gp91(phox), and Nox-4 subunits) and nitrotyrosine was increased in the LDLR KO mice (P < 0.01 vs. wild-type mice) and not in mice with LOX-1 deletion (P < 0.01 vs. LDLR KO mice). Phosphorylation of Akt-1 and endothelial nitric oxide synthase and expression of haem-oxygenase-1 were found to be reduced in the LDLR KO mice (P < 0.01 vs. wild-type mice), but not in the mice with LOX-1 deletion (P < 0.01 vs. LDLR KO mice). CONCLUSION: LOX-1 deletion reduces enhanced collagen deposition and MMP expression in atherosclerotic regions via inhibition of pro-oxidant signals.

LOX-1 abrogation reduces myocardial ischemia-reperfusion injury in mice.

LOX-1 is a newly described lectin-like receptor for oxidized-LDL (ox-LDL), which is over-expressed in the ischemic myocardium. To examine the pathogenic role of LOX-1 in the determination of ischemia-reperfusion (I-R) injury to the heart, we developed LOX-1 knockout (KO) mice, and subjected these mice to 60 min of left coronary artery occlusion followed by 60 min of reperfusion. I-R in the LOX-1 KO mice resulted in a significant reduction in myocardial injury as well as in accumulation of inflammatory cells in the I-R myocardium and lipid peroxidation (P<0.01 vs. wild-type mice). Concomitantly, there was significant preservation of cardiac function in the LOX-1 KO mice despite I-R (P<0.01 vs. the wild-type mice). The phosphorylation of oxidative stress-sensitive mitogen-activated protein kinase (p38MAPK) and protein kinase B/Akt-1, expression of nitrotyrosine and inducible nitric oxide synthase (iNOS), and superoxide dismutase activity were enhanced during I-R in the wild-type mice. These alterations in p38MAPK, Akt-1 and iNOS were much less pronounced in the LOX-1 KO mice. The superoxide dismutase activity increased further in the LOX-1 KO mice. These observations provide compelling evidence that LOX-1 may be a key modulator of myocardial I-R injury, and its effect is mediated by pro-oxidant signals. LOX-1 may be a potential target for therapy of myocardial ischemic injury.

Over-expression of angiotensin II type 2 receptor (agtr2) decreases collagen accumulation in atherosclerotic plaque.

Angiotensin (Ang) II, via type 1 receptor activation, exerts a significant role in atherogenesis and collagen synthesis. To test the hypothesis that Ang II type 2 receptor (AT2R) upregulation delivered with adeno-associated virus type 2 (AAV/AT2R) would inhibit collagen synthesis in atherosclerotic arteries, LDLR knockout mice were injected with AAV/AT2R and fed 4% cholesterol diet for 18 weeks. LDLR knockout mice treated with saline or AAV/Neo exhibited extensive vessel wall collagen accumulation, which was reduced by about 50% with AT2R over-expression. AT2R upregulation completely blocked the alterations in the expression of procollagen-I, osteopontin, fibronectin, CD68, and matrix metalloproteinases (MMP-2 and MMP-9), as well as phosphorylation of p38 and p44/42 MAPKs. Activity of superoxide dismutase was reduced in the LDLR KO mice and it increased with AT2R upregulation. This study demonstrates that AT2R over-expression reduces enhanced collagen accumulation, MMP expression and activity in atherosclerotic regions via inhibition of pro-oxidant signals.

Small concentrations of oxLDL induce capillary tube formation from endothelial cells via LOX-1-dependent redox-sensitive pathway.

OBJECTIVE: Vascular endothelial growth factor (VEGF), a key angiogenic growth factor, stimulates angiogenesis. Low levels of reactive oxygen species (ROS) function as signaling molecules for angiogenesis. We postulated that low concentrations of oxLDL might induce low levels of ROS and initiate angiogenesis. METHODS AND RESULTS: An in vitro model of tube formation from human coronary artery endothelial cells (HCAECs) was used. oxLDL (0.1, 1, 2, 5 microg/mL) induced VEGF expression and enhanced tube formation. oxLDL-mediated VEGF expression and tube formation were suppressed by a specific blocking anti-LOX-1 antibody. Anti-LOX-1 antibody also reduced oxLDL-induced increase in the expression of NADPH oxidase (gp91(phox) and p47(phox) subunits) and subsequent intracellular ROS generation, phosphorylation of p38 as well as p44/42MAPK, and NF-kappaB p65 expression. gp91(phox) siRNA had a similar effect. The expression of VEGF and NF-kappaB p65 induced by oxLDL was also inhibited by the specific extracellular signal-regulated kinase (ERK) 1/2 inhibitor U0126 and the p38 MAPK inhibitor SB203580. Importantly, the NADPH oxidase inhibitor apocynin, gp91(phox) siRNA, U0126, and SB203580 all reduced tube formation in response to oxLDL. CONCLUSIONS: These findings suggest that small concentrations of oxLDL promote capillary tube formation by inducing the expression of VEGF via LOX-1-mediated activation of NADPH oxidase- MAPKs-NF-kappaB pathway.

LOX-1 deletion alters signals of myocardial remodeling immediately after ischemia-reperfusion.

OBJECTIVE: Chronic ischemia is associated with alterations in genes that result in myocardial remodeling. An important biochemical basis of cardiac remodeling is generation of reactive oxygen species (ROS). A few studies have suggested that acute ischemia triggers signals for remodeling. We examined the hypothesis that targeted deletion of lectin-like oxidized-LDL receptor (LOX-1) may inhibit signals related to cardiac remodeling. METHODS AND RESULTS: We generated LOX-1 knockout (KO) mice on C57BL/6 (wild-type mice) background, and subjected wild-type and KO mice to ischemia-reperfusion (I-R). The wild-type mice developed a marked reduction in left ventricular systolic pressure and +/-dp/dt(max) and an increase in left ventricular end-diastolic pressure following I-R, and this change was much less in the LOX-1 KO mice, indicating preservation of left ventricular function with LOX-1 deletion. There was evidence for marked oxidative stress (NADPH oxidase expression, malondialdehyde and 8-isoprostane) following I-R in the wild-type mice, much less so in the LOX-1 KO mice (P<0.01). In concert, collagen deposition (Masson's trichrome and Picro-sirius red staining) increased dramatically in the wild-type mice, but only half as much in the LOX-1 KO mice (P<0.01). Collagen staining data was corroborated with procollagen-I expression. Further, fibronectin and osteopontin expression increased in the wild-type mice, but to a much smaller extent in the LOX-1 KO mice (P<0.01). CONCLUSIONS: These findings provide compelling evidence that LOX-1 is a key modulator of cardiac remodeling which starts immediately following I-R.

Life span and stress resistance of Caenorhabditis elegans are differentially affected by glutathione transferases metabolizing 4-hydroxynon-2-enal.

The lipid peroxidation product 4-hydroxynon-2-enal (4-HNE) forms as a consequence of oxidative stress, and acts as a signaling molecule or, at superphysiological levels, as a toxicant. The steady-state concentration of the compound reflects the balance between its generation and its metabolism, primarily through glutathione conjugation. Using an RNAi-based screen, we identified in Caenorhabditis elegans five glutathione transferases (GSTs) capable of catalyzing 4-HNE conjugation. RNAi knock-down of these GSTs (products of the gst-5, gst-6, gst-8, gst-10, and gst-24 genes) sensitized the nematode to electrophilic stress elicited by exposure to 4-HNE. However, interference with the expression of only two of these genes (gst-5 and gst-10) significantly shortened the life span of the organism. RNAi knock-down of the other GSTs resulted in at least as much 4-HNE adducts, suggesting tissue specificity of effects on longevity. Our results are consistent with the oxidative stress theory of organismal aging, broadened by considering electrophilic stress as a contributing factor. According to this extended hypothesis, peroxidation of lipids leads to the formation of 4-HNE in a chain reaction which amplifies the original damage. 4-HNE then acts as an "aging effector" via the formation of 4-HNE-protein adducts, and a resulting change in protein function.

Lifespan extension in hypomorphic daf-2 mutants of Caenorhabditis elegans is partially mediated by glutathione transferase CeGSTP2-2.

Electrophilic stress caused by lipid peroxidation products such as 4-hydroxynonenal (4-HNE) and/or related compounds may contribute to aging. The major mode of 4-HNE metabolism involves glutathione conjugation catalyzed by specialized glutathione transferases. We have previously shown that glutathione transferase CeGSTP2-2, the product of the Caenorhabditis elegans gst-10 gene, has the ability to conjugate 4-HNE, and that its overexpression extends lifespan of C. elegans. We now demonstrate that the expression level of CeGSTP2-2 correlates highly with lifespan in a series of hypomorphic daf-2 mutants of C. elegans. The overexpression of CeGSTP2-2 in daf-2 is abrogated in daf-16; daf-2 mutants, indicating that expression of the gst-10 gene is modulated by insulin-like growth factor signaling. To determine whether the relationship between CeGSTP2-2 and lifespan is causal, we used RNAi to knock down CeGSTP2-2. Treatment with gst-10-specific dsRNA decreased CeGSTP2-2 protein in wild-type N2 and in daf-2 strains to an approximately equal level. The ability to conjugate 4-HNE was similarly decreased by RNAi, suggesting that the increment of that activity in daf-2 over N2 is due largely to the overexpression of CeGSTP2-2. RNAi-mediated knock-down of CeGSTP2-2 led to an increased susceptibility to 4-HNE, paraquat, and heat shock, and to a shortening of lifespan by 13% in both N2 and daf-2 strains. These results indicate that CeGSTP2-2 significantly contributes to the maintenance of the soma, and that this function is augmented in daf-2 mutants concordantly with other longevity assurance genes, probably via insulin-like growth factor signaling.

Lifespan and stress resistance of Caenorhabditis elegans are increased by expression of glutathione transferases capable of metabolizing the lipid peroxidation product 4-hydroxynonenal.

Caenorhabditis elegans expresses a glutathione transferase (GST) belonging to the Pi class, for which we propose the name CeGSTP2-2. CeGSTP2-2 (the product of the gst-10 gene) has the ability to conjugate the lipid peroxidation product 4-hydroxynonenal (4-HNE). Transgenic C. elegans strains were generated in which the 5'-flanking region and promoter of gst-10 were placed upstream of gst-10 and mGsta4 cDNAs, respectively. mGsta4 encodes the murine mGSTA4-4, an enzyme with particularly high catalytic efficiency for 4-HNE. The localization of both transgenes was similar to that of native CeGSTP2-2. The 4-HNE-conjugating activity in worm lysates increased in the order: control

High Frequency Hearing Loss and Hyperactivity in DUX4 Transgenic Mice.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by mutations leading to ectopic expression of the transcription factor DUX4, and encompasses both muscle-related and non-muscle phenotypes. Mouse models bearing this gene represent valuable tools to investigate which pathologies are due to DUX4 expression, and how DUX4 leads to these pathologies. The iDUX4(2.7) mouse contains an X-linked doxycycline-inducible DUX4 gene that shows low level basal expression in the absence of doxycycline, leading to male lethality, generally in embryo, but always before 8 weeks of age. Here, we describe additional non-muscle phenotypes in this animal model. We find that iDUX4(2.7) female carriers are extremely hyperactive, spending large amounts of time ambulating and much less time resting. Rare 3-week old males, although hypophagic, runted and extremely fragile, are capable of high activity, but show periods of catatonic torpor in which animals appear dead and respiration is virtually absent. We also examine a non-muscle phenotype of interest to FSHD, high frequency hearing loss. We find that young iDUX4(2.7) females are significantly impaired in their ability to hear at frequencies above 8 kHz. These phenotypes make the iDUX4(2.7) mouse an attractive model in which to study non-muscle activities of DUX4.

GSK3ß inhibition activates the CDX/HOX pathway and promotes hemogenic endothelial progenitor differentiation from human pluripotent stem cells.

WNT/ß-CATENIN signaling promotes the hematopoietic/endothelial differentiation of human embryonic stem cells and human induced pluripotent stem cells (hiPSCs). The transient addition of a GSK3ß inhibitor (GSKi) has been found to facilitate in vitro endothelial cell differentiation from hESCs/hiPSCs. Because hematopoietic and endothelial cells are derived from common progenitors (hemogenic endothelial progenitors [HEPs]), we examined the effect of transient GSKi treatment on hematopoietic cell differentiation from hiPSCs. We found that transient GSKi treatment at the start of hiPSC differentiation induction altered the gene expression profile of the cells. Multiple CDX/HOX genes, which are expressed in the posterior mesoderm of developing embryos, were significantly upregulated by GSKi treatment. Further, inclusion of the GSKi in a serum- and stroma-free culture with chemically defined medium efficiently induced HEPs, and the HEPs gave rise to various lineages of hematopoietic and endothelial cells. Therefore, transient WNT/ß-CATENIN signaling triggers activation of the CDX/HOX pathway, which in turn confers hemogenic posterior mesoderm identity to differentiating hiPSCs. These data enhance our understanding of human embryonic hematopoietic/endothelial cell development and provide a novel in vitro system for inducing the differentiation of hematopoietic cells from hiPSCs.

A functional signal profiling test for identifying a subset of HER2-negative breast cancers with abnormally amplified HER2 signaling activity.

The results of clinical trials evaluating the efficacy of HER2 inhibitors in patients with breast cancer indicate that the correlation between HER2 receptor levels and patient outcomes is as low as 50%. The relatively weak correlation between HER2 status and response to HER2-targeting drugs suggests that measurement of HER2 signaling activity, rather than absolute HER2 levels, may more accurately diagnose HER2-driven breast cancer. A new diagnostic test, the CELx HER2 Signaling Profile (CELx HSP) test, is demonstrated to measure real-time HER2 signaling function in live primary cells. In the present study, epithelial cells extracted fresh from breast cancer patient tumors classified as HER2 negative (HER2-, n = 34 of which 33 were estrogen receptor positive) and healthy subjects (n = 16) were evaluated along with reference breast cancer cell lines (n = 19). Live cell response to specific HER2 agonists (NRG1b and EGF) and antagonist (pertuzumab) was measured. Of the HER2- breast tumor cell samples tested, 7 of 34 patients (20.5%; 95% CI = 10%-37%) had HER2 signaling activity that was characterized as abnormally high. Amongst the tumor samples there was no correlation between HER2 protein status (by cell cytometry) and HER2 signaling activity (hyperactive or normal) (Regression analysis P = 0.144, R2 = 0.068). One conclusion is that measurement of HER2 signaling activity can identify a subset of breast cancers with normal HER2 receptor levels with abnormally high levels of HER2 signaling. This result constitutes a new subtype of breast cancer that should be considered for treatment with HER2 pathway inhibitors.

Dominant lethal pathologies in male mice engineered to contain an X-linked DUX4 transgene.

Facioscapulohumeral muscular dystrophy (FSHD) is an enigmatic disease associated with epigenetic alterations in the subtelomeric heterochromatin of the D4Z4 macrosatellite repeat. Each repeat unit encodes DUX4, a gene that is normally silent in most tissues. Besides muscular loss, most patients suffer retinal vascular telangiectasias. To generate an animal model, we introduced a doxycycline-inducible transgene encoding DUX4 and 3' genomic DNA into a euchromatic region of the mouse X chromosome. Without induction, DUX4 RNA was expressed at low levels in many tissues and animals displayed a variety of unexpected dominant leaky phenotypes, including male-specific lethality. Remarkably, rare live-born males expressed DUX4 RNA in the retina and presented a retinal vascular telangiectasia. By using doxycycline to induce DUX4 expression in satellite cells, we observed impaired myogenesis in vitro and in vivo. This mouse model, which shows pathologies due to FSHD-related D4Z4 sequences, is likely to be useful for testing anti-DUX4 therapies in FSHD.

Expression of the human FSHD-linked DUX4 gene induces neurogenesis during differentiation of murine embryonic stem cells.

Misexpression of the double homeodomain protein DUX4 in muscle is believed to cause facioscapulohumeral muscular dystrophy (FSHD). Although strategies are being devised to inhibit DUX4 activity in FSHD, there is little known about the normal function of this protein. Expression of DUX4 has been reported in pluripotent cells and testis. To test the idea that DUX4 may be involved in initiating a germ lineage program in pluripotent cells, we interrogated the effect of expressing the human DUX4 gene at different stages during in vitro differentiation of murine embryonic stem (ES) cells. We find that expression of even low levels of DUX4 is incompatible with pluripotency: DUX4-expressing ES cells downregulate pluripotency markers and rapidly differentiate even in the presence of leukemia inhibitory factor (LIF) and bone morphogenetic protein 4 (BMP4). Transcriptional profiling revealed unexpectedly that DUX4 induced a neurectodermal program. Embryoid bodies exposed to a pulse of DUX4 expression displayed severely inhibited mesodermal differentiation, but acquired neurogenic potential. In a serum-containing medium in which neurogenic differentiation is minimal, DUX4 expression served as a neural-inducing factor, enabling the differentiation of Tuj1+ neurites. These data suggest that besides effects in muscle and germ cells, the involvement of DUX4 in neurogenesis should be considered as anti-DUX4 therapies are developed.

Aspirin inhibits oxidant stress, reduces age-associated functional declines, and extends lifespan of Caenorhabditis elegans.

AIMS: Oxidative stress and inflammation are leading risk factors for age-associated functional declines. We assessed aspirin effects on endogenous oxidative-stress levels, lifespan, and age-related functional declines, in the nematode Caenorhabditis elegans. RESULTS: Both aspirin and its salicylate moiety, at nontoxic concentrations (0.5-1 mM), attenuated endogenous levels of reactive oxygen species (p<0.001), and upregulated antioxidant genes encoding superoxide dismutases (especially sod-3, p<0.001), catalases (especially ctl-2, p<0.0001), and two glutathione-S-transferases (gst-4 and gst-10; each p<0.005). Aspirin, and to a lesser degree salicylate, improved survival of hydrogen peroxide, and in the absence of exogenous stress aspirin extended lifespan by 21%-23% (each p<10(-9)), while salicylate added 14% (p<10(-6)). Aspirin and salicylate delayed age-dependent declines in motility and pharyngeal pumping (each p<0.005), and decreased intracellular protein aggregation (p<0.0001)-all established markers of physiological aging-consistent with slowing of the aging process. Aspirin fails to improve stress resistance or lifespan in nematodes lacking DAF-16, implying that it acts through this FOXO transcription factor. INNOVATION: Studies in mice and humans suggest that aspirin may protect against multiple age-associated diseases by reducing all-cause mortality. We now demonstrate that aspirin markedly slows many measures of aging in the nematode. CONCLUSIONS: Aspirin treatment is associated with diminished endogenous oxidant stress and enhanced resistance to exogenous peroxide, both likely mediated by activation of antioxidant defenses. Our evidence indicates that aspirin attenuates insulin-like signaling, thus protecting against oxidative stress, postponing age-associated functional declines and extending C. elegans lifespan under benign conditions.

Mesp1 patterns mesoderm into cardiac, hematopoietic, or skeletal myogenic progenitors in a context-dependent manner.

Mesp1 is regarded as the master regulator of cardiovascular development, initiating the cardiac transcription factor cascade to direct the generation of cardiac mesoderm. To define the early embryonic cell population that responds to Mesp1, we performed pulse inductions of gene expression over tight temporal windows following embryonic stem cell differentiation. Remarkably, instead of promoting cardiac differentiation in the initial wave of mesoderm, Mesp1 binds to the Tal1 (Scl) +40 kb enhancer and generates Flk-1+ precursors expressing Etv2 (ER71) and Tal1 that undergo hematopoietic differentiation. The second wave of mesoderm responds to Mesp1 by differentiating into PDGFRα+ precursors that undergo cardiac differentiation. Furthermore, in the absence of serum-derived factors, Mesp1 promotes skeletal myogenic differentiation. Lineage tracing revealed that the majority of yolk sac and many adult hematopoietic cells derive from Mesp1+ precursors. Thus, Mesp1 is a context-dependent determination factor, integrating the stage of differentiation and the signaling environment to specify different lineage outcomes.

A focal domain of extreme demethylation within D4Z4 in FSHD2.

OBJECTIVE: Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disease with an unclear genetic mechanism. Most patients have a contraction of the D4Z4 macrosatellite repeat array at 4qter, which is thought to cause partial demethylation (FSHD1) of the contracted allele. Demethylation has been surveyed at 3 restriction enzyme sites in the first repeat and only a single site across the entire array, and current models postulate that a generalized D4Z4 chromatin alteration causes FSHD. The background of normal alleles has confounded the study of epigenetic alterations; however, rare patients (FSHD2) have a form of the disease in which demethylation is global, i.e., on all D4Z4 elements throughout the genome. Our objective was to take advantage of the global nature of FSHD2 to identify where disease-relevant methylation changes occur within D4Z4. METHODS: Using bisulfite sequencing of DNA from blood and myoblast cells, methylation levels at 74 CpG sites across 3 disparate regions within D4Z4 were measured in FSHD2 patients and controls. RESULTS: We found that rates of demethylation caused by FSHD2 are not consistent across D4Z4. We identified a focal region of extreme demethylation within a 5' domain, which we named DR1. Other D4Z4 regions, including the DUX4 ORF, were hypomethylated but to a much lesser extent. CONCLUSIONS: These data challenge the simple view that FSHD is caused by a broad "opening" of D4Z4 and lead us to postulate that the region of focal demethylation is the site of action of the key D4Z4 chromatin regulatory factors that go awry in FSHD.