Update TRENDS in Molecular Medicine Vol.12 No.10

Research Focus Cooperation to amplify -dosage-imbalance effects

Susana de la Luna1 and Xavier Estivill2

1 ICREA and Gene Function Group, and Disease Program, Center for Genomic Regulation-CRG, 08003-Barcelona, Spain 2 Genetic Causes of Disease Group, Genes and Disease Program, Center for Genomic Regulation-CRG and Pompeu Fabra University, Barcelona Biomedical Research Park, 08003-Barcelona, Spain

Trisomy 21, also known as (DS), is a From gene-dosage imbalance to pathology complex developmental disorder that affects many ThepresenceofanextracopyofHSA21 genes predicts an organs, including the brain, heart, skeleton and increased expression of 1.5-fold at the RNA level for immune system. A working hypothesis for understand- those genes in trisomy. Experiments in which this effect ing the consequences of trisomy 21 is that the over- has been evaluated indicate that this is indeed the case expression of certain genes on 21, alone for most HSA21 genes in DS samples and for their or in cooperation, is responsible for the clinical features orthologs in mouse trisomic models [3].Inthesimplest of DS. There is now compelling evidence that the scenario, the overexpression of one specific gene would products of two genes on , lead to the disturbance of a biological process and, as a Down syndrome candidate region 1 (DSCR1)and result, a single gene would be responsible for each patho- dual-specificity tyrosine-(Y)-phosphorylation regulated logical feature of DS. However, it is more probable that kinase 1A (DYRK1A), interact functionally, and that the overexpression of several of the 250 HSA21 genes their increased dosage cooperatively leads to dysregu- would contribute to alter a functional pathway in a lation of the signaling pathways that are controlled by specific cell at a specific time. In some cases, functional the nuclear factor of activated T cells (NFAT) family of interactions would result in additive or even synergistic factors, with potential consequences for effects, leading to signal amplification. In others, the several organs and systems that are affected in DS positive effect of one gene might counteract the detri- individuals. mental effects of other dosage-sensitive genes that act on the same pathway and, hence, result in no net change. Whatever the case might be, the final phenotypic out- Trisomy 21 and Down syndrome: genotype and come cannot be foreseen on the basis of analyzing the overexpression of single genes. Humans have two copies of each of the 23 autosomic in all their somatic cells. In some genetic A novel altered pathway in Down syndrome abnormalities that are known as trisomies, an extra A clear example of a cooperative interaction between chromosome is present due to defective chromosome two HSA21 genes on a signaling pathway has recently segregation during germ-cell meiosis. Trisomy of been presented for the nuclear factor of activated human chromosome 21 (HSA21)isthemostcommon T cells (NFAT) pathway (see Glossary), as the result of trisomy at birth, the only one for which individuals the collaborative effort of several groups at Stanford survive beyond childhood and the cause of a develop- University and the Genetic Engineering Unit at Kyoto mental and dysmorphic disorder known as Down syndrome University [4]. (DS). NFAT transcription factors are well-known regulators The clinical presentation of DS is complex because the of development and normal physiology in mammals [5]. normal physiology of many organs and systems is Their activity is controlled by cycles of dephosphorylation affected. Apart from the distinctive facial features, all and phosphorylation that determine the amount of the DS individuals present intellectual disabilities, hypoto- transcription factor in the nucleus and, therefore, the nia and early development of the pathological changes final transcriptional response [6]. The phosphatase that are typical of Alzheimer’s disease (AD). Other clin- is responsible, following increases in intra- ical features not observed in all DS individuals include cellular Ca2+, for the NFAT dephosphorylation step heart defects, abnormalities of the gut and the immune and subsequent nuclear entry. Several kinases, including system, and increased risk of leukaemia, among many glycogen synthase 3 and casein kinase 1, participate in others [1,2]. The severity of each feature, including the phosphorylation step that acts as a switch-off mental retardation, is highly variable among indivi- mechanism [6]. duals, suggesting that there is an important contribution One HSA21 gene, Down syndrome candidate region 1 of other influences such as stochastic, environmental or (DSCR1), acts as a negative regulator of this pathway genetic factors. by virtue of its inhibitory activity on calcineurin [7]. Given the significant connection of calcineurin with

Corresponding author: de la Luna, S. ([email protected]) disease, the DSCR1 gene has received a lot of attention, Available online 17 August 2006. and many names have been given to its protein www.sciencedirect.com 452 Update TRENDS in Molecular Medicine Vol.12 No.10

that were engineered not to express one or several NFAT Glossary family members or to lack calcineurin activity. The pre- Calcineurin (also known as protein phosphatase 2B): it is an enzyme that sence of DS-like in these mice led the authors catalyzes the dephosphorylation of phosphorylated serine–threonine residues in , and the only phosphatase that is regulated by Ca2+. Calcineurin is to conclude that reduced NFAT activity might be impli- involved in the regulation of many cellular processes, including cardiac cated in the appearance of cardiovascular, neurological, hypertrophy, skeletal-muscle development, synaptic plasticity and T-cell skeletal and gastrointestinal defects in DS individuals. activation. In this regard, calcineurin is the main target of the immunosup- pressive drugs cyclosporine A and FK506. The dysregulation of the NFAT pathway in DS had DSCR1: it belongs to a family of small and evolutionary conserved proteins that already been hinted at by the lower-than-normal calci- share the ability of binding to and inhibit calcineurin. In humans, the family neurin activity that has been detected in DS fetal brains consists of three members, DSCR1, DSCR1L1 and DSCR1L2, the expression of which is induced by different stimuli, including calcineurin activation in [9] and by the decreased activity of an NFAT-dependent the case of DSCR1. It has been proposed that the members of this family alter reporter in the heart and brain of the DS mouse the threshold for calcineurin signaling. For DSCR1, a cardioprotective role in model Ts16 [14]. In agreement with this, Arron et al. the heart has been reported. DYRK1A: it is a kinase that phosphorylates serine–threonine residues in [4] reported an increase in NFAT hyperphosphorylated proteins. DYRK1A belongs to the DYRK family, an evolutionarily conserved species in DS-tissue samples. Mechanistically, this is family of protein kinases, with members identified from yeast to humans, that explained by a cooperative effect of DSCR1 and DYRK1A participate in various cellular processes. The DYRK family is characterized by its activation mechanism that involves the autophosphorylation on a tyrosine on NFAT activity that, by negatively operating on residue in the activation loop. DYRK1A phosphorylates several cytosolic different steps of the signaling cascade, would amplify proteins and some splicing and transcription factors, including members of the NFAT family. the modest interference provoked by each protein alone Nuclear factor of activated T cells (NFAT): it is a family of transcription factors (Figure 1). with five members in humans. Their activity is mainly regulated by their However, some notes of caution need to be borne in subcellular localization, which depends on the phosphorylation status. They are well-known substrates of calcineurin, and one of the best examples to mind. show how Ca2+ signaling can control . , through the  In the study by Arron et al. [4] DSCR1 and DYRK1A induction of the expression of specific genes, are key regulators of the cooperation in vivo is supported by the defects detected differentiation of various cell types (e.g. T cells, neurons, myoblasts, endothelial cells and adipocytes), and they thus participate in the appropriate in double transgenic embryos in which expression is development of several organs and systems. driven by the b-actin promoter. Overexpression from a heterologous promoter might not recapitulate the endogenous gene-expression pattern and, therefore, products:ADAPT78,MCIP1,RCN1,CBP1andcalcipres- coincidence in cell type and/or time period cannot be sin1.AHumanGenomeOrganization(HUGO)initiative assumed with certainty. to rename DSCR1 as RCAN1 (regulator of calcineurin 1)  One of the isoforms of DSCR1 is itself under NFAT is underway. Dscr1 knockout mice are viable and present control. Thus, a negative effect of DYRK1A overexpres- phenotypes that link the gene to the regulation of sion on NFAT signaling might give rise to lowered calcineurin–NFAT-dependent transcription programs DSCR1 expression in a Ca2+-stimulated situation and [8]. However, no specific phenotypes associated to result in attenuation. DSCR1 overexpression and relevant for DS have been  It has been reported that, when DSCR1 trisomy is reported, except for the Drosophila DSCR1 ortholog reversed to disomy in the partial trisomic model Ts16, nebula.Indeed,nebula transgenic flies exhibit learning no significant rescue of the cardiac defects present in defects [9]. these mice is observed [14]. Similarly, having either Two separate publications have simultaneously shown DSCR1 or DYRK1A in two copies in a trisomic that a second HSA21 gene, dual-specificity tyrosine-(Y)- background for the other gene does not restore a normal phosphorylation regulated kinase 1A (DYRK1A), influ- craniofacial phenotype [14,15]. Thus, either cooperation encestheNFATpathwaythroughthekinaseactivityof between these two genes is not required for these DS its encoded protein on NFAT family members [4,10]. phenotypes or there are additional factors, including When DYRK1A was first identified in 1995, it was named other genes in trisomy, that need to be taken into minibrain after its ortholog in Drosophila,whichhad account. been associated with defects in the development of the Despite these caveats, Arron et al. [4] have provided a nervous system [11]. Conservation of this function in fine example of how gene-dosage imbalance, involving mammals was later confirmed in mice that only have several HSA21 genes acting cooperatively, can enhance one copy of the Dyrk1A gene [12].Thesemicedevelop the destabilization of regulatory circuits underlying dis- smaller brains than their disomic littermates. Analysis of ease. The report also nicely illustrated how cooperative mouse models with varying gene copy number has efforts from different areas of scientific expertise can result revealed that DYRK1A is a dosage-sensitive gene in in synergistic progress. HSA21 [13]. Finally, it is important to remember that DS is surely more than a disturbance of NFAT signaling. For DSCR1 and DYRK1A cooperation on NFAT signaling example, DYRK1A phosphorylates the microtubule- pathways associated protein tau [16]. Tau is dephosphorylated The recent report by Arron et al. [4] has elegantly by calcineurin [17],whichisthetargetofDSCR1.The combined phenotypic analysis of mouse models, signal- overexpression of DYRK1A and DSCR1 in trisomy might ing-pathway biochemistryandmathematicstomodel therefore lead to an increase in the tau-phosphorylated perturbations of gene networks. These authors undertook species. It is well known that tau hyperphosphorylation a systematic analysis of DS-related phenotypes in mice is correlated with the formation of the neurofibrillary www.sciencedirect.com Update TRENDS in Molecular Medicine Vol.12 No.10 453

Figure 1. Cooperation of DYRK1A and DSCR1 in the downregulation of the NFAT signaling pathway. In resting conditions, most of the NFAT molecules are in the hyperphosphorylated state because of the actions of NFAT kinases (phosphorylated residues are indicated as small brown circles in the NFAT diagram) and, therefore, accumulated in the cytosol. As a result of a specific stimulus that increases intracellular Ca2+ concentration, the phosphatase calcineurin is activated and, consequently, NFAT is dephosphorylated (this has been represented as a change in color from dark to light brown in the NFAT diagram), and translocated into the nucleus. There, NFAT can activate specific transcription programs, identified by the expression of a defined set of genes (represented as genes A to X). When DSCR1 is overexpressed, its activity as a calcineurin inhibitor leads to a decrease in NFAT dephosphorylation and, consequently, to a reduction in nuclear NFAT. Therefore, a subset of NFAT-dependent genes will be downregulated or not expressed at all. A similar situation is observed when DYRK1A is overexpressed as a result of the kinase activity of DYRK1A on NFAT. Considering that DYRK1A can be localized in both the nucleus and the cytosol of cells, the specific cellular compartment where the phosphorylation takes place has to be defined yet. When both DYRK1A and DSCR1 are overexpressed, as it occurs in trisomy 21, the independent negative effects of each protein on NFAT translocation are added and the number of NFAT nuclear molecules greatly diminishes. As a consequence, NFAT-dependent patterns of expression is more severely affected and a stronger impact in the phenotypic outcomes depending on the expression of these genes will be expected. tangles that are associated with AD. Therefore, DYRK1A The long road towards Down-syndrome therapeutics and DSCR1 might contribute to the early onset of There is no doubt that the development of new therapies the pathological traits of AD that is present in people would greatly improve the quality of life of people with with DS. DS. However, the issue is far from simple. First, many

Box 1. A historical perspective of Down syndrome research

www.sciencedirect.com 454 Update TRENDS in Molecular Medicine Vol.12 No.10 of the pathological traits of DS are likely to be linked to the References alteration of pathways that are required for normal embryo- 1 Epstein, C.J. (2001) Down syndrome (Trisomy 21). In Metabolic and Molecular Bases of Inherited Diseases (Scriver, C.R., Beaudet, A.L., Sly, nic development, as would be the case for some of the W.S. and Valle, D., eds), pp. 1223–1256, McGraw-Hill phenotypes related to disturbances in NFAT signaling. 2 Antonarakis, S.E. et al. (2004) Chromosome 21 and Down syndrome: Therefore, restoration of normality would require a precise from genomics to pathophysiology. Nat. Rev. Genet. 5, 725–738 definition of the appropriate developmental window for 3 Potier, M-C. et al. (2006) Transcriptional disruptions in Down intervention. Second, it is important to consider that the syndrome: a case study in the Ts1Cje mouse cerebellum during post-natal development. J. Neurochem. 97, 104–109 increases in gene expression that underlie the appearance of 4 Arron, J.R. et al. (2006) NFAT dysregulation by increased dosage of DS defects are modest. It is thus possible that tilting the DSCR1 and DYRK1A on chromosome 21. Nature 441, 595–600 balance towards modest decreases in expression or activity 5 Graef, I.A. et al. (2001) NFAT signaling in vertebrate development. might also lead to disease, a situation that is clearly Curr. Opin. Genet. Dev. 11, 505–512 illustrated by the HSA21 monosomies. Finally, another 6 Hogan, P.G. et al. (2003) Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev. 17, 2205–2232 element to be considered is that there are often two facets 7 Fuentes, J.J. et al. (2000) DSCR1, overexpressed in Down syndrome, is to gene overexpression: one harmful and one advantageous. an inhibitor of calcineurin-mediated signaling pathways. Hum. Mol. For example, DSCR1, when overexpressed in mice, acts as Genet. 9, 1681–1690 an anti-angiogenic factor, and the associated blockade of 8 Ryeom, S. et al. (2003) The threshold pattern of calcineurin-dependent gene expression is altered by loss of the endogenous inhibitor new blood-vessel formation reduces tumor growth [18]. . Nat. Immunol. 4, 874–881 This effect seems to be mediated by the inhibition of 9 Chang, K.T. et al. (2003) The Drosophila homolog of Down’s syndrome NFAT-dependent activities induced by vascular-endothelial critical region 1 regulates learning: implications for mental growth factor. Thus, from this perspective, NFAT inhibition retardation. Proc. Natl. Acad. Sci. U. S. A. 100, 15794–15799 might be interpreted as beneficial and it might be seen as 10 Gwack, Y. et al. (2006) A genome-wide Drosophila RNAi screen identifies DYRK-family kinases as regulators of NFAT. Nature 441, contributing to the reduced incidence of solid tumors in DS 646–650 individuals. 11 Guimera, J. et al. (1996) A human homologue of Drosophila minibrain (MNB) is expressed in the neuronal regions affected in Concluding remarks Down syndrome and maps to the critical region. Hum. Mol. Genet. Trisomy 21 is considered to be a model for human disorders 5, 1305–1310 12 Fotaki, V. et al. (2002) Dyrk1A haploinsufficiency affects viability and involving gene-dosage imbalance. In the 1990s, research on causes developmental delay and abnormal brain morphology in mice. HSA21 contributed greatly to the development of many Mol. Cell. Biol. 22, 6636–6647 technical tools used during the genomic era. Consequently, 13 Altafaj, X. et al. (2001) Neurodevelopmental delay, motor HSA21 was one of the first human chromosomes to be abnormalities and cognitive deficits in transgenic mice completely sequenced (Box 1). Over the past few years, overexpressing Dyrk1A (minibrain), a murine model of Down’s syndrome. Hum. Mol. Genet. 10, 1915–1923 DS research has moved into the transcriptomic era, and a 14 Lange, A.W. et al. (2005) Restoration of DSCR1 to disomy in the large amount of data on changes in RNA expression has trisomy 16 mouse model of Down syndrome does not correct cardiac being collected both for HSA21 genes and for the rest of the or craniofacial development anomalies. Dev. Dyn. 233, 954–963 genome. In the coming years, an increased knowledge of the 15 Olson, L.E. et al. (2004) A chromosome 21 critical region does not cause specific Down syndrome phenotypes. Science 306, 687–690 functional roles of HSA21 genes is expected. This should 16 Woods, Y.L. et al. (2001) The kinase DYRK phosphorylates protein- contribute to a better understanding of how gene overex- synthesis initiation factor eIF2Be at Ser539 and the microtubule- pression affects the biological processes linked to HSA21- associated protein tau at Thr212: potential role for DYRK as encoded protein products. Consequently, more functional a glycogen synthase kinase 3-priming kinase. Biochem. J. 355, interactions such as the one reported by Arron et al. [4] will 609–615 17 Garver, T.D. et al. (1999) Reduction of calcineurin activity in brain be discovered, and this will enable distinct HSA21 genes to by antisense oligonucleotides leads to persistent phosphorylation be linked to the same pathway. Hopefully, trisomy 21 will of tau protein at Thr181 and Thr231. Mol. Pharmacol. 55, 632– become an appealing model for those working with gene- 641 network disturbances. The system-biology age for DS is 18 Minami, T. et al. (2004) Vascular endothelial growth factor- and approaching. thrombin-induced termination factor, Down syndrome critical region-1, attenuates endothelial cell proliferation and angiogenesis. J. Biol. Chem. 279, 50537–50554 Acknowledgements S.L. acknowledges support from the Spanish MEC (BFU2004–01768). We 1471-4914/$ – see front matter ß 2006 Elsevier Ltd. All rights reserved. thank S. Bartlett for his linguistic help. doi:10.1016/j.molmed.2006.08.001

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