Emilia Stellacci

Heterozygous Germline Mutations in the CBL Tumor-Suppressor Gene Cause a Noonan Syndrome-like Phenotype

RAS signaling plays a key role in controlling appropriate cell responses to extracellular stimuli and participates in early and late developmental processes. Although enhanced flow through this pathway has been established as a major contributor to oncogenesis, recent discoveries have revealed that aberrant RAS activation causes a group of clinically related developmental disorders characterized by facial dysmorphism, a wide spectrum of cardiac disease, reduced growth, variable cognitive deficits, ectodermal and musculoskeletal anomalies, and increased risk for certain malignancies. Here, we report that heterozygous germline mutations in CBL, a tumor-suppressor gene that is mutated in myeloid malignancies and encodes a multivalent adaptor protein with E3 ubiquitin ligase activity, can underlie a phenotype with clinical features fitting or partially overlapping Noonan syndrome (NS), the most common condition of this disease family. Independent CBL mutations were identified in two sporadic cases and two families from among 365 unrelated subjects who had NS or suggestive features and were negative for mutations in previously identified disease genes. Phenotypic heterogeneity and variable expressivity were documented. Mutations were missense changes altering evolutionarily conserved residues located in the RING finger domain or the linker connecting this domain to the N-terminal tyrosine kinase binding domain, a known mutational hot spot in myeloid malignancies. Mutations were shown to affect CBL-mediated receptor ubiquitylation and dysregulate signal flow through RAS. These findings document that germline mutations in CBL alter development to cause a clinically variable condition that resembles NS and that possibly predisposes to malignancies.

Activation and repression of the 2-5A synthetase and p21 gene promoters by IRF-1 and IRF-2

The Interferon Regulatory Factors-1 and -2 (IRF-1 and IRF-2) were originally identified as transcriptional regulators of the interferon (IFN) and IFN-stimulated genes. These factors also modulate immune response and play a role in cell growth regulation. In this study we analysed the effect of the ectopic expression of IRF-1 and IRF-2 on the regulation of two potential IRF target genes involved in cell growth regulation, 2-5A synthetase and p21 (WAF/CP1), both of which contain consensus binding sites for IRF family members within their promoters. Following ectopic expression, IRF-1 transactivated 2-5A synthetase and p21 genes, an effect that was counterbalanced by concomitant ectopic expression of IRF-2. These effects were mediated by direct binding of IRF to the gene promoters. A construct expressing an IRF-2 antisense (FRI-2) was able to revert the inhibitory effect of IRF-2 on the IRF-1 transactivation. IRF-1 also induced expression of its homologous repressor IRF-2 as indicated by EMSA analysis using an IRF-E probe from the IRF-2 promoter; and by cotransfection of IRF-1 together with an IRF-2 promoter CAT construct. Therefore, the induction of IRF-1 by IFNs or other stimuli acts as a transactivator of genes involved in cell growth regulation, as well as of its own repressor IRF-2, thus providing autoinhibitory regulation of IRF-1 activated genes.

IRF-1 Is Required for Full NF-κB Transcriptional Activity at the Human Immunodeficiency Virus Type 1 Long Terminal Repeat Enhancer

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) gene expression is controlled by a complex interplay between viral and host factors. We have previously shown that interferon-regulatory factor 1 (IRF-1) is stimulated early after HIV-1 infection and regulates promoter transcriptional activity even in the absence of the viral transactivator Tat. In this work we demonstrate that IRF-1 is also required for full NF-κB transcriptional activity. We provide evidence that IRF-1 and NF-κB form a functional complex at the long terminal repeat (LTR) κB sites, which is abolished by specific mutations in the two adjacent κB sites in the enhancer region. Silencing IRF-1 with small interfering RNA resulted in impaired NF-κB-mediated transcriptional activity and in repressed HIV-1 transcription early in de novo-infected T cells. These data indicate that in early phases of HIV-1 infection or during virus reactivation from latency, when the viral transactivator is absent or present at very low levels, IRF-1 is an additional component of the p50/p65 heterodimer binding the LTR enhancer, absolutely required for efficient HIV-1 replication.

Activating mutations in RRAS underlie a phenotype within the RASopathy spectrum and contribute to leukaemogenesis

RASopathies, a family of disorders characterized by cardiac defects, defective growth, facial dysmorphism, variable cognitive deficits and predisposition to certain malignancies, are caused by constitutional dysregulation of RAS signalling predominantly through the RAF/MEK/ERK (MAPK) cascade. We report on two germline mutations (p.Gly39dup and p.Val55Met) in RRAS, a gene encoding a small monomeric GTPase controlling cell adhesion, spreading and migration, underlying a rare (2 subjects among 504 individuals analysed) and variable phenotype with features partially overlapping Noonan syndrome, the most common RASopathy. We also identified somatic RRAS mutations (p.Gly39dup and p.Gln87Leu) in 2 of 110 cases of non-syndromic juvenile myelomonocytic leukaemia, a childhood myeloproliferative/myelodysplastic disease caused by upregulated RAS signalling, defining an atypical form of this haematological disorder rapidly progressing to acute myeloid leukaemia. Two of the three identified mutations affected known oncogenic hotspots of RAS genes and conferred variably enhanced RRAS function and stimulus-dependent MAPK activation. Expression of an RRAS mutant homolog in Caenorhabditis elegans enhanced RAS signalling and engendered protruding vulva, a phenotype previously linked to the RASopathy-causing SHOC2S2G mutant. Overall, these findings provide evidence of a functional link between RRAS and MAPK signalling and reveal an unpredicted role of enhanced RRAS function in human disease.

IFN Regulatory Factor-1 Negatively Regulates CD4 + CD25 + Regulatory T Cell Differentiation by Repressing Foxp3 Expression

Regulatory T (Treg) cells are critical in inducing and maintaining tolerance. Despite progress in understanding the basis of immune tolerance, mechanisms and molecules involved in the generation of Treg cells remain poorly understood. IFN regulatory factor (IRF)-1 is a pleiotropic transcription factor implicated in the regulation of various immune processes. In this study, we report that IRF-1 negatively regulates CD4+CD25+ Treg cell development and function by specifically repressing Foxp3 expression. IRF-1-deficient (IRF-1−/−) mice showed a selective and marked increase of highly activated and differentiated CD4+CD25+Foxp3+ Treg cells in thymus and in all peripheral lymphoid organs. Furthermore, IRF-1−/− CD4+CD25− T cells showed extremely high bent to differentiate into CD4+CD25+Foxp3+ Treg cells, whereas restoring IRF-1 expression in IRF-1−/− CD4+CD25− T cells impaired their differentiation into CD25+Foxp3+ cells. Functionally, both isolated and TGF-β-induced CD4+CD25+ Treg cells from IRF-1−/− mice exhibited more increased suppressive activity than wild-type Treg cells. Such phenotype and functional characteristics were explained at a mechanistic level by the finding that IRF-1 binds a highly conserved IRF consensus element sequence (IRF-E) in the foxp3 gene promoter in vivo and negatively regulates its transcriptional activity. We conclude that IRF-1 is a key negative regulator of CD4+CD25+ Treg cells through direct repression of Foxp3 expression.

Repression of Interferon Regulatory Factor 1 by Hepatitis C Virus Core Protein Results in Inhibition of Antiviral and Immunomodulatory Genes

ABSTRACT Hepatitis C virus (HCV) proteins are known to interfere at several levels with both innate and adaptive responses of the host. A key target in these effects is the interferon (IFN) signaling pathway. While the effects of nonstructural proteins are well established, the role of structural proteins remains controversial. We investigated the effect of HCV structural proteins on the expression of interferon regulatory factor 1 (IRF-1), a secondary transcription factor of the IFN system responsible for inducing several key antiviral and immunomodulatory genes. We found substantial inhibition of IRF-1 expression in cells expressing the entire HCV replicon. Suppression of IRF-1 synthesis was mainly mediated by the core structural protein and occurred at the transcriptional level. The core protein in turn exerted a transcriptional repression of several interferon-stimulated genes, targets of IRF-1, including interleukin-15 (IL-15), IL-12, and low-molecular-mass polypeptide 2. These data recapitulate in a unifying mechanism, i.e., repression of IRF-1 expression, many previously described pathogenetic effects of HCV core protein and suggest that HCV core-induced IRF-1 repression may play a pivotal role in establishing persistent infection by dampening an effective immune response.

IRF-1 deficiency skews the differentiation of dendritic cells toward plasmacytoid and tolerogenic features

Members of the IFN regulatory factors (IRFs) family are transcriptional regulators that play essential roles in the homeostasis and function of the immune system. Recent studies indicate a direct involvement of some members of the family in the development of different subsets of dendritic cells (DC). Here, we report that IRF‐1 is a potent modulator of the development and functional maturation of DC. IRF‐1‐deficient mice (IRF‐1−/−) exhibited a predominance of plasmacytoid DC and a selective reduction of conventional DC, especially the CD8α+ subset. IRF‐1−/− splenic DC were markedly impaired in their ability to produce proinflammatory cytokines such as IL‐12. By contrast, they expressed high levels of IL‐10, TGF‐β, and the tolerogenic enzyme indoleamine 2,3 dioxygenase. As a consequence, IRF‐1−/− DC were unable to undergo full maturation and retained plasmacytoid and tolerogenic characteristics following virus infection ex vivo and in vivo. Accordingly, DC from IRF‐1−/− mice were less efficient in stimulating the proliferation of allogeneic T cells and instead, induced an IL‐10‐mediated, suppressive activity in allogeneic CD4+CD25+ regulatory T cells. Together, these results indicate that IRF‐1 is a key regulator of DC differentiation and maturation, exerting a variety of effects on the functional activation and tolerogenic potential of these cells.

Mutations Impairing GSK3-Mediated MAF Phosphorylation Cause Cataract, Deafness, Intellectual Disability, Seizures, and a Down Syndrome-like Facies

Transcription factors operate in developmental processes to mediate inductive events and cell competence, and perturbation of their function or regulation can dramatically affect morphogenesis, organogenesis, and growth. We report that a narrow spectrum of amino-acid substitutions within the transactivation domain of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (MAF), a leucine zipper-containing transcription factor of the AP1 superfamily, profoundly affect development. Seven different de novo missense mutations involving conserved residues of the four GSK3 phosphorylation motifs were identified in eight unrelated individuals. The distinctive clinical phenotype, for which we propose the eponym Aymé-Gripp syndrome, is not limited to lens and eye defects as previously reported for MAF/Maf loss of function but includes sensorineural deafness, intellectual disability, seizures, brachycephaly, distinctive flat facial appearance, skeletal anomalies, mammary gland hypoplasia, and reduced growth. Disease-causing mutations were demonstrated to impair proper MAF phosphorylation, ubiquitination and proteasomal degradation, perturbed gene expression in primary skin fibroblasts, and induced neurodevelopmental defects in an in vivo model. Our findings nosologically and clinically delineate a previously poorly understood recognizable multisystem disorder, provide evidence for MAF governing a wider range of developmental programs than previously appreciated, and describe a novel instance of protein dosage effect severely perturbing development.

Mutations in ZBTB20 cause Primrose syndrome

Primrose syndrome and 3q13.31 microdeletion syndrome are clinically related disorders characterized by tall stature, macrocephaly, intellectual disability, disturbed behavior and unusual facial features, with diabetes, deafness, progressive muscle wasting and ectopic calcifications specifically occurring in the former. We report that missense mutations in ZBTB20, residing within the 3q13.31 microdeletion syndrome critical region, underlie Primrose syndrome. This finding establishes a genetic link between these disorders and delineates the impact of ZBTB20 dysregulation on development, growth and metabolism.

Impaired myelopoiesis in mice devoid of interferon regulatory factor 1

Interferon regulatory factor (IRF)-1 is a transcription factor controlling the expression of several genes, which are differentially induced depending on the cell type and signal. IRF-1 modulates multiple functions, including regulation of immune responses and host defence, cell growth, cytokine signalling and hematopoietic development. Here, we investigated the role of IRF-1 in granulocytic differentiation in mice with a null mutation in the IRF-1 gene. We show that IRF-1−/− bone marrow cells exhibit an increased number of immature granulocytic precursors, associated with a decreased number of mature granulocytic elements as compared to normal mice, suggestive of a defective maturation process. Clonogenetic analyses revealed a reduced number of CFU-G, CFU-M and CFU-GM colonies in IRF-1−/− mice, while the number of BFU-E/CFU-E colonies was unchanged. At the molecular level, the expression of CAAT-enhancer-binding protein (C/EBP)-ɛ, -α and PU.1 was substantially lower in the CD11b+ cells from the bone marrow of IRF-1−/− mice as compared to cells from wild-type mice. These results, together with the fact that IRF-1 is markedly induced early during granulo-monocytic differentiation of CD34+ cells, highlight the pivotal role of IRF-1 in the early phases of myelopoiesis.