Mutations in CSPP1, encoding a core centrosomal protein, cause a range of ciliopathy phenotypes in humans.

Ciliopathies are characterized by a pattern of multisystem involvement that is consistent with the developmental role of the primary cilium. Within this biological module, mutations in genes that encode components of the cilium and its anchoring structure, the basal body, are the major contributors to both disease causality and modification. However, despite rapid advances in this field, the majority of the genes that drive ciliopathies and the mechanisms that govern the pronounced phenotypic variability of this group of disorders remain poorly understood. Here, we show that mutations in CSPP1, which encodes a core centrosomal protein, are disease causing on the basis of the independent identification of two homozygous truncating mutations in three consanguineous families (one Arab and two Hutterite) affected by variable ciliopathy phenotypes ranging from Joubert syndrome to the more severe Meckel-Gruber syndrome with perinatal lethality and occipital encephalocele. Consistent with the recently described role of CSPP1 in ciliogenesis, we show that mutant fibroblasts from one affected individual have severely impaired ciliogenesis with concomitant defects in sonic hedgehog (SHH) signaling. Our results expand the list of centrosomal proteins implicated in human ciliopathies.

Control of histone H3 phosphorylation by CaMKIIδ in response to haemodynamic cardiac stress.

Heart failure is associated with the reactivation of a fetal cardiac gene programme that has become a hallmark of cardiac hypertrophy and maladaptive ventricular remodelling, yet the mechanisms that regulate this transcriptional reprogramming are not fully understood. Using mice with genetic ablation of calcium/calmodulin-dependent protein kinase II δ (CaMKIIδ), which are resistant to pathological cardiac stress, we show that CaMKIIδ regulates the phosphorylation of histone H3 at serine-10 during pressure overload hypertrophy. H3 S10 phosphorylation is strongly increased in the adult mouse heart in the early phase of cardiac hypertrophy and remains detectable during cardiac decompensation. This response correlates with up-regulation of CaMKIIδ and increased expression of transcriptional drivers of pathological cardiac hypertrophy and of fetal cardiac genes. Similar changes are detected in patients with end-stage heart failure, where CaMKIIδ specifically interacts with phospho-H3. Robust H3 phosphorylation is detected in both adult ventricular myocytes and in non-cardiac cells in the stressed myocardium, and these signals are abolished in CaMKIIδ-deficient mice after pressure overload. Mechanistically, fetal cardiac genes are activated by increased recruitment of CaMKIIδ and enhanced H3 phosphorylation at hypertrophic promoter regions, both in mice and in human failing hearts, and this response is blunted in CaMKIIδ-deficient mice under stress. We also document that the chaperone protein 14-3-3 binds phosphorylated H3 in response to stress, allowing proper elongation of fetal cardiac genes by RNA polymerase II (RNAPII), as well as elongation of transcription factors regulating cardiac hypertrophy. These processes are impaired in CaMKIIδ-KO mice after pathological stress. The findings reveal a novel in vivo function of CaMKIIδ in regulating H3 phosphorylation and suggest a novel epigenetic mechanism by which CaMKIIδ controls cardiac hypertrophy.

Mutation in PHC1 implicates chromatin remodeling in primary microcephaly pathogenesis.

Primary microcephaly (PM) is a developmental disorder of early neuroprogenitors that results in reduction of the brain mass, particularly the cortex. To gain fresh insight into the pathogenesis of PM, we describe a consanguineous family with a novel genetic variant responsible for the disease. We performed autozygosity mapping followed by exome sequencing to detect the causal genetic variant. Several functional assays in cells expressing the wild-type or mutant gene were performed to understand the pathogenesis of the identified mutation. We identify a novel mutation in PHC1, a human orthologue of the Drosophila polyhomeotic member of polycomb group (PcG), which significantly decreases PHC1 protein expression, increases Geminin protein level and markedly abolishes the capacity to ubiquitinate histone H2A in patient cells. PHC1 depletion in control cells similarly enhances Geminin expression and decreases histone H2A ubiquitination. The ubiquitination defect and accumulation of Geminin with consequent defect in cell cycle are rescued by over-expression of PHC1 in patient cells. Although patients with the PHC1 mutation exhibit PM with no overt progression of the disease, patient cells also show aberrant DNA damage repair, which is rescued by PHC1 overexpression. These findings reveal several cellular defects in cells carrying the PHC1 mutation and highlight the role of chromatin remodeling in the pathogenesis of PM.

Disruption of male fertility-critical Dcaf17 dysregulates mouse testis transcriptome.

During mammalian spermatogenesis, the ubiquitin proteasome system maintains protein homoeostasis (proteastasis) and spermatogenic cellular functions. DCAF17 is a substrate receptor in the ubiquitin CRL4 E3 Ligase complex, absence of which causes oligoasthenoteratozoospermia in mice resulting in male infertility. To determine the molecular phenomenon underlying the infertility phenotype caused by disrupting Dcaf17, we performed RNA-sequencing-based gene expression profiling of 3-weeks and 8-weeks old Dcaf17 wild type and Dcaf17 disrupted mutant mice testes. At three weeks, 44% and 56% differentially expressed genes (DEGs) were up- and down-regulated, respectively, with 32% and 68% DEGs were up- and down-regulated, respectively at 8 weeks. DEGs include protein coding genes and lncRNAs distributed across all autosomes and the X chromosome. Gene ontology analysis revealed major biological processes including proteolysis, regulation of transcription and chromatin remodelling are affected due to Dcaf17 disruption. We found that Dcaf17 disruption up-regulated several somatic genes, while germline-associated genes were down-regulated. Up to 10% of upregulated, and 12% of downregulated, genes were implicated in male reproductive phenotypes. Moreover, a large proportion of the up-regulated genes were highly expressed in spermatogonia and spermatocytes, while the majority of downregulated genes were predominantly expressed in round spermatids. Collectively, these data show that the Dcaf17 disruption affects directly or indirectly testicular proteastasis and transcriptional signature in mouse.

Dynamic cytosolic foci of DPPA4 in human pluripotent stem cells.

DPPA4 is essential for the pluripotent stem cell state, yet its function is poorly understood. DPPA4 is localized in the nucleus, where it is associated with active chromatin. We now report that it is also present in the cytosol, where it appears as diffused clouds, distinct foci and sometimes as spaghetti-like structures. This cytosolic localization is dynamic and DPPA4 shuttles between the cytosol and the nucleus. Its presence is almost abolished from the nucleus upon differentiation. Co-immunoprecipitation studies highlighted novel protein interactors, many of which are also found in the cytosol and are implicated in mRNA processing and RNA and protein transport between the cytosol and the nucleus. Finally, the depletion of DPPA4 resulted in cytosolic accumulation of vesicles. The cytosolic presence of DPPA4 highlights unexplored research directions that could significantly advance the understanding of DPPA4 in pluripotent stem cells and in cancer.

Mutation in FBXO32 causes dilated cardiomyopathy through up-regulation of ER-stress mediated apoptosis.

Endoplasmic reticulum (ER) stress induction of cell death is implicated in cardiovascular diseases. Sustained activation of ER-stress induces the unfolded protein response (UPR) pathways, which in turn activate three major effector proteins. We previously reported a missense homozygous mutation in FBXO32 (MAFbx, Atrogin-1) causing advanced heart failure by impairing autophagy. In the present study, we performed transcriptional profiling and biochemical assays, which unexpectedly revealed a reduced activation of UPR effectors in patient mutant hearts, while a strong up-regulation of the CHOP transcription factor and of its target genes are observed. Expression of mutant FBXO32 in cells is sufficient to induce CHOP-associated apoptosis, to increase the ATF2 transcription factor and to impair ATF2 ubiquitination. ATF2 protein interacts with FBXO32 in the human heart and its expression is especially high in FBXO32 mutant hearts. These findings provide a new underlying mechanism for FBXO32-mediated cardiomyopathy, implicating abnormal activation of CHOP. These results suggest alternative non-canonical pathways of CHOP activation that could be considered to develop new therapeutic targets for the treatment of FBXO32-associated DCM.

Advancing male age differentially alters levels and localization patterns of PLCzeta in sperm and testes from different mouse strains.

Sperm-specific phospholipase C zeta (PLCζ) initiates intracellular calcium (Ca2+) transients which drive a series of concurrent events collectively termed oocyte activation. Numerous investigations have linked abrogation and absence/reduction of PLCζ with forms of male infertility in humans where oocyte activation fails. However, very few studies have examined potential relationships between PLCζ and advancing male age, both of which are increasingly considered to be major effectors of male fertility. Initial efforts in humans may be hindered by inherent PLCζ variability within the human population, alongside a lack of sufficient controllable repeats. Herein, utilizing immunoblotting, immunofluorescence, and quantitative reverse transcription PCR (qRT-PCR) we examined for the first time PLCζ protein levels and localization patterns in sperm, and PLCζ mRNA levels within testes, from mice at 8 weeks, 12 weeks, 24 weeks, and 36 weeks of age, from two separate strains of mice, C57BL/6 (B6; inbred) and CD1 (outbred). Collectively, advancing male age generally diminished levels and variability of PLCζ protein and mRNA in sperm and testes, respectively, when both strains were examined. Furthermore, advancing male age altered the predominant pattern of PLCζ localization in mouse sperm, with younger mice exhibiting predominantly post-acrosomal, and older mice exhibiting both post-acrosomal and acrosomal populations of PLCζ. However, the specific pattern of such decline in levels of protein and mRNA was strain-specific. Collectively, our results demonstrate a negative relationship between advancing male age and PLCζ levels and localization patterns, indicating that aging male mice from different strains may serve as useful models to investigate PLCζ in cases of male infertility and subfertility in humans.

Rosiglitazone promotes cardiac hypertrophy and alters chromatin remodeling in isolated cardiomyocytes.

Rosiglitazone is an anti-diabetic agent that raised a major controversy over its cardiovascular adverse effects. There is in vivo evidence that Rosiglitazone promotes cardiac hypertrophy by PPAR-γ-independent mechanisms. However, whether Rosiglitazone directly alters hypertrophic growth in cardiac cells is unknown. Chromatin remodeling by histone post-translational modifications has emerged as critical for many cardiomyopathies. Based on these observations, this study was initiated to investigate the cardiac hypertrophic effect of Rosiglitazone in a cellular model of primary neonatal rat cardiomyocytes (NRCM). We assessed whether the drug alters cardiac hypertrophy and its relationship with histone H3 phosphorylation. Our study showed that Rosiglitazone is a mild pro-hypertrophic agent. Rosiglitazone caused a significant increase in the release of brain natriuretic peptide (BNP) into the cell media and also increased cardiomyocytes surface area and atrial natriuretic peptide (ANP) protein expression significantly. These changes correlated with increased cardiac phosphorylation of p38 MAPK and enhanced phosphorylation of H3 at serine 10 globally and at one cardiac hypertrophic gene locus. These results demonstrate that Rosiglitazone causes direct cardiac hypertrophy in NRCM and alters H3 phosphorylation status. They suggest a new mechanism of Rosiglitazone cardiotoxicity implicating chromatin remodeling secondary to H3 phosphorylation, which activate the fetal cardiac gene program.

FBXO32, encoding a member of the SCF complex, is mutated in dilated cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) is a common form of cardiomyopathy causing systolic dysfunction and heart failure. Rare variants in more than 30 genes, mostly encoding sarcomeric proteins and proteins of the cytoskeleton, have been implicated in familial DCM to date. Yet, the majority of variants causing DCM remain to be identified. The goal of the study is to identify novel mutations causing familial dilated cardiomyopathy. RESULTS: We identify FBXO32 (ATROGIN 1), a member of the F-Box protein family, as a novel DCM-causing locus. The missense mutation affects a highly conserved amino acid and is predicted to severely impair binding to SCF proteins. This is validated by co-immunoprecipitation experiments from cells expressing the mutant protein and from human heart tissue from two of the affected patients. We also demonstrate that the hearts of the patients with the FBXO32 mutation show accumulation of selected proteins regulating autophagy. CONCLUSION: Our results indicate that abnormal SCF activity with subsequent impairment of the autophagic flux due to a novel FBXO32 mutation is implicated in the pathogenesis of DCM.

Nonsteroidal anti-inflammatory drugs induce apoptosis in cutaneous T-cell lymphoma cells and enhance their sensitivity for TNF-related apoptosis-inducing ligand.

Cutaneous T-cell lymphomas (CTCL) form a heterogeneous group of non-Hodgkin's lymphomas of the skin. In previous studies, we had characterized CTCL cells as resistant to the death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which correlated to pronounced expression of the caspase-8/-10 inhibitor c-FLIP. For identification of proapoptotic strategies in CTCL cells and for overcoming their death ligand resistance, we investigated the effects of nonsteroidal anti-inflammatory drugs (NSAIDs) such as acetylsalicylic acid, sodium salicylate, and diclofenac (DF). These drugs strongly enhanced apoptosis, as well as decreased CTCL cell proliferation and vitality, and DF furthermore sensitized for TRAIL-induced apoptosis. Full activation of the caspase cascade (caspase-3, -8, -9) and decreased mitochondrial membrane potential were characteristic for NSAID treatment, whereas cytochrome c release was seen only for DF. Downregulation of Mcl-1 and enhanced surface expression of TRAIL were seen in response to NSAIDs. Most characteristic for apoptosis induction was the downregulation of c-FLIP. In agreement with the critical role of c-FLIP for apoptosis deficiency of CTCL cells, its overexpression decreased NSAID-mediated apoptosis and its downregulation by small hairpin RNA-enhanced apoptosis. The study provides a rationale for the use of NSAIDs as a new therapeutic option for CTCL patients. Supporting this concept, ex vivo lymphoma cells of CTCL patients also revealed significant sensitivity for NSAID treatment.

Apoptosis induction by SAHA in cutaneous T-cell lymphoma cells is related to downregulation of c-FLIP and enhanced TRAIL signaling.

Suberoylanilide hydroxamic acid (SAHA) has been approved for the treatment of cutaneous T-cell lymphoma (CTCL), but its mode of action remained largely elusive. As shown here in four CTCL cell lines, loss of cell viability correlated with significant time- and dose-dependent induction of apoptosis, whereas cytotoxicity was less pronounced. Both extrinsic and intrinsic apoptosis pathways were activated, as seen by processing of initiator caspases 8 and 9, loss of mitochondrial membrane potential, and cytochrome c release. Characteristically, antiapoptotic mediators such as Mcl-1, XIAP, survivin, and c-FLIP were downregulated. Consistent with its critical function, c-FLIP overexpression resulted in a significant decrease of SAHA-mediated apoptosis. Enhanced sensitivity to TRAIL (TNF-related apoptosis-inducing ligand) and enhanced TRAIL signaling was seen in CTCL cell lines with high sensitivity, whereas cell lines with moderate response were characterized by downregulation of TRAIL-R2 and weaker TRAIL expression. Comparable proapoptotic responses to SAHA and to the combination with TRAIL were seen in ex vivo tumor T cells of CTCL patients. Thus, activation of extrinsic apoptosis pathways, related to c-FLIP downregulation and enhanced TRAIL signaling, appeared as characteristic for CTCL cell responsiveness to SAHA. An improved understanding of the pathways may facilitate its targeted use and the selection of suitable combinations.

Resistance of cutaneous anaplastic large-cell lymphoma cells to apoptosis by death ligands is enhanced by CD30-mediated overexpression of c-FLIP.

Death ligands, including TNF-alpha, CD95L/FasL, and TRAIL, mediate safeguard mechanisms against tumor growth and critically contribute to lymphocyte homeostasis. We investigated death receptor-mediated apoptosis and CD30/CD95 crosstalk in four CD30-positive cell lines of cutaneous anaplastic large-cell lymphoma (cALCL). Whereas CD95 stimulation strongly induced apoptosis in cALCL cells, the pro-apoptotic pathways of TNF-alpha and TRAIL were completely blocked at an early step. Expression of TNF receptor 1 was lost in three of four cell lines, providing an explanation for TNF-alpha unresponsiveness. TRAIL resistance may be explained by the consistent overexpression of cellular flice inhibitory protein (c-FLIP) (four of four cell lines) and frequent loss of the pro-apoptotic Bcl-2 protein Bid (three of four cell lines). Changes at the receptor-expression level were largely ruled out. CD30/CD95 crosstalk experiments showed that CD30 ligation leads to NF-kappaB-mediated c-FLIP upregulation in cALCL cells, which in turn conferred enhanced resistance to CD95-mediated apoptosis. Knockdown of c-FLIP by a lentiviral approach enhanced basic apoptosis rates in cALCL cells and diminished the CD30-mediated suppression of apoptosis, thus proving the significance of c-FLIP in this context. These in vitro findings may be indicative of the clinical situation of cALCL. Further clarifying the defects in apoptosis pathways in cutaneous lymphomas may lead to improved therapies for these disorders.

PPARdelta is a type 1 IFN target gene and inhibits apoptosis in T cells.

Peroxisome proliferator-activated receptor beta/delta (PPARdelta) is a nuclear hormone receptor regulating diverse biological processes, including beta-oxidation of fatty acid and epithelial cell differentiation. To date, the role of PPARdelta in the immune system has not been thoroughly studied. Here, we show that PPARdelta is expressed in activated human T cells purified from peripheral blood as well as in T cells isolated from affected psoriasis skin lesions. PPARdelta is induced in T cells on stimulation with type 1 IFN. Functionally, PPARdelta enhances proliferation of primary T cells and blocks apoptosis induced by type 1 IFN and by serum deprivation. We show that these cellular functions are mediated by the activation of extracellular signal-regulated kinase1/2 signaling. Our results (1) establish a direct molecular link between type 1 IFN signaling and PPARdelta, (2) define a functional role for PPARdelta in human T cells, and (3) suggest that the induction of PPARdelta by type 1 IFN contributes to the persistence of activated T cells in psoriasis skin lesions.

PPARdelta enhances keratinocyte proliferation in psoriasis and induces heparin-binding EGF-like growth factor.

Psoriasis is a common skin disease involving keratinocyte proliferation and altered differentiation, as well as T-cell activation. Here, we show that altered gene transcription in psoriatic skin lesions is highly reproducible between independent data sets. Analysis of gene expression confirmed dysregulation in all expected functional categories, such as IFN signaling and keratinocyte differentiation, and allowed molecular fingerprinting of a previously characterized dendritic cell subset associated with psoriasis tumor necrosis factor alpha (TNF-alpha)- and inducible nitric oxide synthase (iNOS)-producing CD11b(INT) DC (Tip-DC). Unexpectedly, a large group of dysregulated transcripts was related to fatty acid signaling and adipocyte differentiation, exhibiting a pattern consistent with the activation of peroxisome proliferator-activated receptor delta (PPARdelta). PPARdelta itself was strongly induced in psoriasis in vivo. In primary keratinocytes, PPARdelta was induced by the transcription factor activator protein 1, in particular by junB, but not by canonical WNT signaling, in contrast to its regulation in colon carcinoma cells. Activation of PPARdelta enhanced proliferation of keratinocytes, while this was inhibited by knockdown of PPARdelta. Finally, heparin-binding EGF-like growth factor (HB-EGF), known to induce epidermal hyperplasia and itself overexpressed in psoriasis, was identified as a direct target gene of PPARdelta. The present data suggest that activation of PPARdelta is a major event in psoriasis, contributing to the hyperproliferative phenotype by induction of HB-EGF.

Optimized production and concentration of lentiviral vectors containing large inserts.

Generation of high titer lentiviral stocks and efficient virus concentration are central to maximize the utility of lentiviral technology. Here we evaluate published protocols for lentivirus production on a range of transfer vectors differing in size (7.5-13.2 kb). We present a modified virus production protocol robustly yielding useful titers (up to 10(7)/ml) for a range of different transfer vectors containing packaging inserts up to 7.5 kb. Moreover, we find that virus recovery after concentration by ultracentrifugation depends on the size of the packaged inserts, heavily decreasing for large packaged inserts. We describe a fast (4 h) centrifugation protocol at reduced speed allowing high virus recovery even for large and fragile lentivirus vectors. The protocols outlined in the current report should be useful for many labs interested in producing and concentrating high titer lentiviral stocks.