Evolution of microRNAs in Amoebozoa and implications for the origin of multicellularity.

MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression in both plants and animals. They are thought to have evolved convergently in these lineages and hypothesized to have played a role in the evolution of multicellularity. In line with this hypothesis, miRNAs have so far only been described in few unicellular eukaryotes. Here, we investigate the presence and evolution of miRNAs in Amoebozoa, focusing on species belonging to Acanthamoeba, Physarum and dictyostelid taxonomic groups, representing a range of unicellular and multicellular lifestyles. miRNAs that adhere to both the stringent plant and animal miRNA criteria were identified in all examined amoebae, expanding the total number of protists harbouring miRNAs from 7 to 15. We found conserved miRNAs between closely related species, but the majority of species feature only unique miRNAs. This shows rapid gain and/or loss of miRNAs in Amoebozoa, further illustrated by a detailed comparison between two evolutionary closely related dictyostelids. Additionally, loss of miRNAs in the Dictyostelium discoideum drnB mutant did not seem to affect multicellular development and, hence, demonstrates that the presence of miRNAs does not appear to be a strict requirement for the transition from uni- to multicellular life.

Monoallelic and biallelic variants in LEF1 are associated with a new syndrome combining ectodermal dysplasia and limb malformations caused by altered WNT signaling.

PURPOSE: LEF1 encodes a transcription factor acting downstream of the WNT-ß-catenin signaling pathway. It was recently suspected as a candidate for ectodermal dysplasia in 2 individuals carrying 4q35 microdeletions. We report on 12 individuals harboring LEF1 variants. METHODS: High-throughput sequencing was employed to delineate the genetic underpinnings of the disease. Cellular consequences were characterized by immunofluorescence, immunoblotting, pulldown assays, and/or RNA sequencing. RESULTS: Monoallelic variants in LEF1 were detected in 11 affected individuals from 4 unrelated families, and a biallelic variant was detected in an affected individual from a consanguineous family. The phenotypic spectrum includes various limb malformations, such as radial ray defects, polydactyly or split hand/foot, and ectodermal dysplasia. Depending on the type and location of LEF1 variants, the inheritance of this novel Mendelian condition can be either autosomal dominant or recessive. Our functional data indicate that 2 molecular mechanisms are at play: haploinsufficiency or loss of DNA binding are responsible for a mild to moderate phenotype, whereas loss of ß-catenin binding caused by biallelic variants is associated with a severe phenotype. Transcriptomic studies reveal an alteration of WNT signaling. CONCLUSION: Our findings establish mono- and biallelic variants in LEF1 as a cause for a novel syndrome comprising limb malformations and ectodermal dysplasia.

Biallelic variants in PCDHGC4 cause a novel neurodevelopmental syndrome with progressive microcephaly, seizures, and joint anomalies.

PURPOSE: We aimed to define a novel autosomal recessive neurodevelopmental disorder, characterize its clinical features, and identify the underlying genetic cause for this condition. METHODS: We performed a detailed clinical characterization of 19 individuals from nine unrelated, consanguineous families with a neurodevelopmental disorder. We used genome/exome sequencing approaches, linkage and cosegregation analyses to identify disease-causing variants, and we performed three-dimensional molecular in silico analysis to predict causality of variants where applicable. RESULTS: In all affected individuals who presented with a neurodevelopmental syndrome with progressive microcephaly, seizures, and intellectual disability we identified biallelic disease-causing variants in Protocadherin-gamma-C4 (PCDHGC4). Five variants were predicted to induce premature protein truncation leading to a loss of PCDHGC4 function. The three detected missense variants were located in extracellular cadherin (EC) domains EC5 and EC6 of PCDHGC4, and in silico analysis of the affected residues showed that two of these substitutions were predicted to influence the Ca2+-binding affinity, which is essential for multimerization of the protein, whereas the third missense variant directly influenced the cis-dimerization interface of PCDHGC4. CONCLUSION: We show that biallelic variants in PCDHGC4 are causing a novel autosomal recessive neurodevelopmental disorder and link PCDHGC4 as a member of the clustered PCDH family to a Mendelian disorder in humans.

A 24-generation-old founder mutation impairs splicing of RBBP8 in Pakistani families affected with Jawad syndrome.

Jawad syndrome is a multiple congenital anomaly and intellectual disability syndrome with mutation in RBBP8 reported only in two families. Here, we report on two new families from Pakistan and identified a previously reported variant in RBBP8, NM_002894.3:c.1808-1809delTA. We could show that this mutation impairs splicing resulting in two different abnormal transcripts. Finally, we could verify a shared haplotype among all four families and estimate the founder event to have occurred some 24 generations ago.

Functional analyses of Pericentrin and Syne-2 interaction in ciliogenesis.

Pericentrin (Pcnt) is a multifunctional scaffold protein and mutations in the human PCNT gene are associated with several diseases, including ciliopathies. Pcnt plays a crucial role in ciliary development in olfactory receptor neurons, but its function in the photoreceptor-connecting cilium is unknown. We downregulated Pcnt in the retina ex vivo and in vivo via a virus-based RNA interference approach to study Pcnt function in photoreceptors. ShRNA-mediated knockdown of Pcnt impaired the development of the connecting cilium and the outer segment of photoreceptors, and caused a nuclear migration defect. In protein interaction screens, we found that the outer nuclear membrane protein Syne-2 (also known as Nesprin-2) is an interaction partner of Pcnt in photoreceptors. Syne-2 is important for positioning murine photoreceptor cell nuclei and for centrosomal migration during early ciliogenesis. CRISPR/Cas9-mediated knockout of Syne-2 in cell culture led to an overexpression and mislocalization of Pcnt and to ciliogenesis defects. Our findings suggest that the Pcnt-Syne-2 complex is important for ciliogenesis and outer segment formation during retinal development and plays a role in nuclear migration.

Nesprin-1 impact on tumorigenic cell phenotypes.

The largest protein of the nuclear envelope (NE) is Nesprin-1 which forms a network along the NE interacting with actin, Emerin, Lamin, and SUN proteins. Mutations in the SYNE1 gene and reduction in Nesprin-1 protein levels have been reported to correlate with several age related diseases and cancer. In the present study, we tested whether Nesprin-1 overexpression can reverse the malignant phenotype of Huh7 cells, a human liver cancer cell line, which carries a mutation in the SYNE1 gene resulting in reduced Nesprin-1 protein levels, has altered nuclear shape, altered amounts and localization of NE components, centrosome localization and genome stability. Ectopic expression of a mini-Nesprin-1 led to an improvement of the nuclear shape, corrected the mislocalization of NE proteins, the centrosome positioning, and the alterations in the DNA damage response network. Additionally, Nesprin-1 had a profound effect on cellular senescence. These findings suggest that Nesprin-1 may be effective in tumorigenic cell phenotype correction of human liver cancer.

The regulatory subunit phr2AB of Dictyostelium discoideum phosphatase PP2A interacts with the centrosomal protein CEP161, a CDK5RAP2 ortholog.

phr2AB is the regulatory subunit of the Dictyostelium discoideum phosphatase PP2A and is the ortholog of the human B55 regulatory subunit of PP2A. phr2AB was isolated as a binding partner of the centrosomal protein CEP161, an ortholog of mammalian CDK5RAP2. CEP161 is presumably a phosphoprotein and a component of the Hippo pathway. The interaction site was located in the N-terminal half of CEP161 which encompasses the γTURC binding domain in CEP161. This binding domain is responsible for binding of the γ-tubulin ring complex which allows microtubule nucleation at the centrosome. GFP-tagged phr2AB is diffusely distributed throughout the cell and enriched at the centrosome. Ectopic expression of phr2AB as GFP fusion protein led to multinucleation, aberrant nucleus centrosome ratios and an altered sensitivity to okadaic acid. Some of these features were also affected in cells over-expressing domains of CEP161 and in cells from patients suffering from primary microcephaly, which carried a mutated CDK5RAP2 gene.

CDK5RAP2 interaction with components of the Hippo signaling pathway may play a role in primary microcephaly.

Autosomal recessive primary microcephaly (MCPH) is characterized by a substantial reduction in brain size but with normal architecture. It is often linked to mutations in genes coding for centrosomal proteins; however, their role in brain size regulation is not completely understood. By combining homozygosity mapping and whole-exome sequencing in an MCPH family from Pakistan, we identified a novel mutation (XM_011518861.1; c.4114C > T) in CDK5RAP2, the gene associated with primary microcephaly-3 (MCPH3), leading to a premature stop codon (p.Arg1372*). CDK5RAP2 is a component of the pericentriolar material important for the microtubule-organizing function of the centrosome. Patient-derived primary fibroblasts had strongly decreased CDK5RAP2 amounts, showed centrosomal and nuclear abnormalities and exhibited changes in cell size and migration. We further identified an interaction of CDK5RAP2 with the Hippo pathway components MST1 kinase and the transcriptional regulator TAZ. This finding potentially provides a mechanism through which the Hippo pathway with its roles in the regulation of centrosome number is linked to the centrosome. In the patient fibroblasts, we observed higher levels of TAZ and YAP. However, common target genes of the Hippo pathway were downregulated as compared to the control with the exception of BIRC5 (Survivin), which was significantly upregulated. We propose that the centrosomal deficiencies and the altered cellular properties in the patient fibroblasts can also result from the observed changes in the Hippo pathway components which could thus be relevant for MCPH and play a role in brain size regulation and development.

Inner nuclear envelope protein SUN1 plays a prominent role in mammalian mRNA export.

Nuclear export of messenger ribonucleoproteins (mRNPs) through the nuclear pore complex (NPC) can be roughly classified into two forms: bulk and specific export, involving an nuclear RNA export factor 1 (NXF1)-dependent pathway and chromosome region maintenance 1 (CRM1)-dependent pathway, respectively. SUN proteins constitute the inner nuclear envelope component of the l I: nker of N: ucleoskeleton and C: ytoskeleton (LINC) complex. Here, we show that mammalian cells require SUN1 for efficient nuclear mRNP export. The results indicate that both SUN1 and SUN2 interact with heterogeneous nuclear ribonucleoprotein (hnRNP) F/H and hnRNP K/J. SUN1 depletion inhibits the mRNP export, with accumulations of both hnRNPs and poly(A)+RNA in the nucleus. Leptomycin B treatment indicates that SUN1 functions in mammalian mRNA export involving the NXF1-dependent pathway. SUN1 mediates mRNA export through its association with mRNP complexes via a direct interaction with NXF1. Additionally, SUN1 associates with the NPC through a direct interaction with Nup153, a nuclear pore component involved in mRNA export. Taken together, our results reveal that the inner nuclear envelope protein SUN1 has additional functions aside from being a central component of the LINC complex and that it is an integral component of the mammalian mRNA export pathway suggesting a model whereby SUN1 recruits NXF1-containing mRNP onto the nuclear envelope and hands it over to Nup153.

A Cdc42- and Rac-interactive binding (CRIB) domain mediates functions of coronin.

The Cdc42- and Rac-interactive binding motif (CRIB) of coronin binds to Rho GTPases with a preference for GDP-loaded Rac. Mutation of the Cdc42- and Rac-interactive binding motif abrogates Rac binding. This results in increased 1evels of activated Rac in coronin-deficient Dictyostelium cells (corA(-)), which impacts myosin II assembly. corA(-) cells show increased accumulation of myosin II in the cortex of growth-phase cells. Myosin II assembly is regulated by myosin heavy chain kinase-mediated phosphorylation of its tail. Kinase activity depends on the activation state of the p21-activated kinase a. The myosin II defect of corA(-) mutant is alleviated by dominant-negative p21-activated kinase a. It is rescued by wild-type coronin, whereas coronin carrying a mutated Cdc42- and Rac-interactive binding motif failed to rescue the myosin defect in corA(-) mutant cells. Ectopically expressed myosin heavy chain kinases affinity purified from corA(-) cells show reduced kinase activity. We propose that coronin through its affinity for GDP-Rac regulates the availability of GTP-Rac for activation of downstream effectors.

A set of genes conserved in sequence and expression traces back the establishment of multicellularity in social amoebae.

BACKGROUND: The developmental cycle of Dictyostelid amoebae represents an early form of multicellularity with cell type differentiation. Mutant studies in the model Dictyostelium discoideum revealed that its developmental program integrates the actions of genes involved in signal transduction, adhesion, motility, autophagy and cell wall and matrix biosynthesis. However, due to functional redundancy and fail safe options not required in the laboratory, this single organism approach cannot capture all essential genes. To understand how multicellular organisms evolved, it is essential to recognize both the conserved core features of their developmental programs and the gene modifications that instigated phenotypic innovation. For complex organisms, such as animals, this is not within easy reach, but it is feasible for less complex forms, such as the Dictyostelid social amoebas. RESULTS: We compared global profiles of gene expression during the development of four social amoebae species that represent 600 mya of Dictyostelia evolution, and identified orthologous conserved genes with similar developmental up-regulation of expression using three different methods. For validation, we disrupted five genes of this core set and examined the phenotypic consequences. CONCLUSION: At least 71 of the developmentally regulated genes that were identified with all methods were likely to be already present in the last ancestor of all Dictyostelia. The lack of phenotypic changes in null mutants indicates that even highly conserved genes either participate in functionally redundant pathways or are necessary for developmental progression under adverse, non-standard laboratory conditions. Both mechanisms provide robustness to the developmental program, but impose a limit on the information that can be obtained from deleting single genes.

The Dictyostelium discoideum GPHR ortholog is an endoplasmic reticulum and Golgi protein with roles during development.

Dictyostelium discoideum GPHR (Golgi pH regulator)/Gpr89 is a developmentally regulated transmembrane protein present on the endoplasmic reticulum (ER) and the Golgi apparatus. Transcript levels are low during growth and vary during development, reaching high levels during the aggregation and late developmental stages. The Arabidopsis ortholog was described as a G protein-coupled receptor (GPCR) for abscisic acid present at the plasma membrane, whereas the mammalian ortholog is a Golgi apparatus-associated anion channel functioning as a Golgi apparatus pH regulator. To probe its role in D. discoideum, we generated a strain lacking GPHR. The mutant had different growth characteristics than the AX2 parent strain, exhibited changes during late development, and formed abnormally shaped small slugs and fruiting bodies. An analysis of development-specific markers revealed that their expression was disturbed. The distributions of the endoplasmic reticulum and the Golgi apparatus were unaltered at the immunofluorescence level. Likewise, their functions did not appear to be impaired, since membrane proteins were properly processed and glycosylated. Also, changes in the external pH were sensed by the ER, as indicated by a pH-sensitive ER probe, as in the wild type.

NKAP is a novel RS-related protein that interacts with RNA and RNA binding proteins.

NKAP is a highly conserved protein with roles in transcriptional repression, T-cell development, maturation and acquisition of functional competency and maintenance and survival of adult hematopoietic stem cells. Here we report the novel role of NKAP in splicing. With NKAP-specific antibodies we found that NKAP localizes to nuclear speckles. NKAP has an RS motif at the N-terminus followed by a highly basic domain and a DUF 926 domain at the C-terminal region. Deletion analysis showed that the basic domain is important for speckle localization. In pull-down experiments, we identified RNA-binding proteins, RNA helicases and splicing factors as interaction partners of NKAP, among them FUS/TLS. The FUS/TLS-NKAP interaction takes place through the RS domain of NKAP and the RGG1 and RGG3 domains of FUS/TLS. We analyzed the ability of NKAP to interact with RNA using in vitro splicing assays and found that NKAP bound both spliced messenger RNA (mRNA) and unspliced pre-mRNA. Genome-wide analysis using crosslinking and immunoprecipitation-seq revealed NKAP association with U1, U4 and U5 small nuclear RNA, and we also demonstrated that knockdown of NKAP led to an increase in pre-mRNA percentage. Our results reveal NKAP as nuclear speckle protein with roles in RNA splicing and processing.

Influence of annexin A7 on insulin sensitivity of cellular glucose uptake.

Insulin sensitivity is decreased by prostaglandin E2 (PGE2), a major product of cyclooxygenase (COX). As shown in erythrocytes, PGE2 formation is inhibited by annexin A7. The present study defined the role of annexin A7 in glucose metabolism. Gene-targeted mice lacking annexin A7 (annexin7 (-/-)) were compared to wild-type mice (annexin7 (+/+)). The serum 6-Keto-prostaglandin-F1α (6-Keto-PGF1α) concentration was measured by ELISA and hepatic COX activity determined by an enzyme assay. Expression of COX-1, COX-2, prostaglandin E synthase, GLUT-4, and insulin receptor was determined by Western blotting. Glucose and insulin serum concentrations were analyzed following an intraperitoneal glucose load and glucose serum levels after intraperitoneal injection of insulin. Experiments were done without and with pretreatment of the mice with COX-inhibitor aspirin. The serum 6-Keto-PGF1α level and hepatic COX activity were significantly higher in annexin7 (-/-) than in annexin7 (+/+) mice. Hepatic COX-1 expression was higher in annexin7 (-/-) mice. Glucose tolerance was decreased in annexin7 (-/-) mice. Intraperitoneal insulin injection decreased the serum glucose level in both genotypes, an effect significantly less pronounced in annexin7 (-/-) mice. Glucose-induced insulin secretion was higher in annexin7 (-/-) mice. GLUT-4 expression in skeletal muscle from annexin7 (-/-) mice was reduced. Aspirin pretreatment lowered the increase in insulin concentration following glucose injection in both genotypes and virtually abrogated the differences in serum insulin between the genotypes. Aspirin pretreatment improved glucose tolerance in annexin7 (-/-) mice. In conclusion, annexin A7 influences insulin sensitivity of cellular glucose uptake and thus glucose tolerance. These effects depend on COX activity.

CDK6 associates with the centrosome during mitosis and is mutated in a large Pakistani family with primary microcephaly.

Autosomal recessive primary microcephaly (MCPH) is characterized by reduced head circumference, reduction in the size of the cerebral cortex with otherwise grossly normal brain structure and variable intellectual disability. MCPH is caused by mutations of 11 different genes which code for proteins implicated in cell division and cell cycle regulation. We studied a consanguineous eight-generation family from Pakistan with ten microcephalic children using homozygosity mapping and found a new MCPH locus at HSA 7q21.11-q21.3. Sanger sequencing of the most relevant candidate genes in this region revealed a homozygous single nucleotide substitution c.589G>A in CDK6, which encodes cyclin-dependent kinase 6. The mutation changes a highly conserved alanine at position 197 into threonine (p.Ala197Thr). Post hoc whole-exome sequencing corroborated this mutation's identification as the causal variant. CDK6 is an important protein for the control of the cell cycle and differentiation of various cell types. We show here for the first time that CDK6 associates with the centrosome during mitosis; however, this was not observed in patient fibroblasts. Moreover, the mutant primary fibroblasts exhibited supernumerary centrosomes, disorganized microtubules and mitotic spindles, an increased centrosome nucleus distance, reduced cell proliferation and impaired cell motility and polarity. Upon ectopic expression of the mutant protein and knockdown of CDK6 through shRNA, we noted similar effects. We propose that the identified CDK6 mutation leads to reduced cell proliferation and impairs the correct functioning of the centrosome in microtubule organization and its positioning near the nucleus which are key determinants during neurogenesis.

A novel human receptor involved in bitter tastant detection identified using Dictyostelium discoideum.

Detection of substances tasting bitter to humans occurs in diverse organisms including the social amoeba Dictyostelium discoideum. To establish a molecular mechanism for bitter tastant detection in Dictyostelium, we screened a mutant library for resistance to a commonly used bitter standard, phenylthiourea. This approach identified a G-protein-coupled receptor mutant, grlJ(-), which showed a significantly increased tolerance to phenylthiourea in growth, survival and movement. This mutant was not resistant to a structurally dissimilar potent bitter tastant, denatonium benzoate, suggesting it is not a target for at least one other bitter tastant. Analysis of the cell-signalling pathway involved in the detection of phenylthiourea showed dependence upon heterotrimeric G protein and phosphatidylinositol 3-kinase activity, suggesting that this signalling pathway is responsible for the cellular effects of phenylthiourea. This is further supported by a phenylthiourea-dependent block in the transient cAMP-induced production of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in wild-type but not grlJ(-) cells. Finally, we have identified an uncharacterized human protein γ-aminobutyric acid (GABA) type B receptor subunit 1 isoform with weak homology to GrlJ that restored grlJ(-) sensitivity to phenylthiourea in cell movement and PIP3 regulation. Our results thus identify a novel pathway for the detection of the standard bitter tastant phenylthiourea in Dictyostelium and implicate a poorly characterized human protein in phenylthiourea-dependent cell responses.

Balanced cortical stiffness is important for efficient migration of Dictyostelium cells in confined environments.

Dictyostelium discoideum cells resemble in many aspects human leukocytes and serve as a model to study actin cytoskeleton dynamics and cell migration of highly motile cells. Dictyostelium cells deficient in the actin-binding protein filamin (ddFLN) showed a surprisingly subtle change in phenotype with no or only minor effects in single cell motility. These findings were in contrast to the strong actin-crosslinking activities measured for filamin in vitro. In the present study, we set out to revisit the role of ddFLN in cell migration. For this purpose, we examined migration of wild-type, ddFLN-null and ddFLN-overexpressing cells under different conditions. In addition to cyclic-AMP chemotaxis assays using micropipettes, we explored cell migration under more confined conditions: an under-agarose 2D assay and a 3D assay employing a collagen matrix that was adapted from assays for leukocytes. Using 3D migration conditions, cells deficient in ddFLN displayed only a minor impairment of motility, similar to the results obtained for migration in 2D. However, cells overexpressing ddFLN showed a remarkable decrease in the speed of migration in particular in 3D environments. We suggest that these results are in line with an increased stiffening of the cortex due to the crosslinking activity of overexpressed ddFLN. Our conclusion is that the absolute level of ddFLN is critical for efficient migration. Furthermore, our results show that under conditions of increased mechanical stress, Dictyostelium cells, like leukocytes, switch to a bleb-based mode of movement.

Unusual combinatorial involvement of poly-A/T tracts in organizing genes and chromatin in Dictyostelium.

Dictyostelium discoideum is an amoebozoa that exists in both a free-living unicellular and a multicellular form. It is situated in a deep branch in the evolutionary tree and is particularly noteworthy in having a very A/T-rich genome. Dictyostelium provides an ideal system to examine the extreme to which nucleotide bias may be employed in organizing promoters, genes, and nucleosomes across a genome. We find that Dictyostelium genes are demarcated precisely at their 5' ends by poly-T tracts and precisely at their 3' ends by poly-A tracts. These tracts are also associated with nucleosome-free regions and are embedded with precisely positioned TATA boxes. Homo- and heteropolymeric tracts of A and T demarcate nucleosome border regions. Together, these findings reveal the presence of a variety of functionally distinct polymeric A/T elements. Strikingly, Dictyostelium chromatin may be organized in di-nucleosome units but is otherwise organized as in animals. This includes a +1 nucleosome in a position that predicts the presence of a paused RNA polymerase II. Indeed, we find a strong phylogenetic relationship between the presence of the NELF pausing factor and positioning of the +1 nucleosome. Pausing and +1 nucleosome positioning may have coevolved in animals.

Contribution of SUN1 mutations to the pathomechanism in muscular dystrophies.

Mutations in several genes encoding nuclear envelope (NE) associated proteins cause Emery-Dreifuss muscular dystrophy (EDMD). We analyzed fibroblasts from a patient who had a mutation in the EMD gene (p.L84Pfs*6) leading to loss of Emerin and a heterozygous mutation in SUN1 (p.A203V). The second patient harbored a heterozygous mutation in LAP2alpha (p.P426L) and a further mutation in SUN1 (p.A614V). p.A203V is located in the N-terminal domain of SUN1 facing the nucleoplasm and situated in the vicinity of the Nesprin-2 and Emerin binding site. p.A614V precedes the SUN domain, which interacts with the KASH domain of Nesprins in the periplasmic space and forms the center of the LINC complex. At the cellular level, we observed alterations in the amounts for several components of the NE in patient fibroblasts and further phenotypic characteristics generally attributed to laminopathies such as increased sensitivity to heat stress. The defects were more severe than observed in EDMD cells with mutations in a single gene. In particular, in patient fibroblasts carrying the p.A203V mutation in SUN1, the alterations were aggravated. Moreover, SUN1 of both patient fibroblasts exhibited reduced interaction with Lamin A/C and when expressed ectopically in wild-type fibroblasts, the SUN1 mutant proteins exhibited reduced interactions with Emerin as well.

Annexin A7 deficiency potentiates cardiac NFAT activity promoting hypertrophic signaling.

Annexin A7 (Anxa7) is a cytoskeletal protein interacting with Ca(2+) signaling which in turn is a crucial factor for cardiac remodeling following cardiac injury. The present study explored whether Anxa7 participates in the regulation of cardiac stress signaling. To this end, mice lacking functional Anxa7 (anxa7(-/-)) and wild-type mice (anxa7(+/+)) were investigated following pressure overload by transverse aortic constriction (TAC). In addition, HL-1 cardiomyocytes were silenced with Anxa7 siRNA and treated with isoproterenol. Transcript levels were determined by quantitative RT-PCR, transcriptional activity by luciferase reporter assay and protein abundance by Western blotting and confocal microscopy. As a result, TAC treatment increased the mRNA and protein levels of Anxa7 in wild-type mice. Moreover, TAC increased heart weight to body weight ratio and the cardiac mRNA levels of αSka, Nppb, Col1a1, Col3a1 and Rcan1, effects more pronounced in anxa7(-/-) mice than in anxa7(+/+) mice. Silencing of Anxa7 in HL-1 cardiomyocytes significantly increased nuclear localization of Nfatc1. Furthermore, Anxa7 silencing increased NFAT-dependent transcriptional activity as well as αSka, Nppb, and Rcan1 mRNA levels both, under control conditions and following ß-adrenergic stimulation by isoproterenol. These observations point to an important role of annexin A7 in the regulation of cardiac NFAT activity and hypertrophic response following cardiac stress conditions.