Nadia Danilova

Ribosomal protein S19 deficiency in zebrafish leads to developmental abnormalities and defective erythropoiesis through activation of p53 protein family

Mutations in several ribosomal proteins (RPs) lead to Diamond-Blackfan anemia (DBA), a syndrome characterized by defective erythropoiesis, congenital anomalies, and increased frequency of cancer. RPS19 is the most frequently mutated RP in DBA. RPS19 deficiency impairs ribosomal biogenesis, but how this leads to DBA or cancer remains unknown. We have found that rps19 deficiency in ze-brafish results in hematopoietic and developmental abnormalities resembling DBA. Our data suggest that the rps19-deficient phenotype is mediated by dysregulation of deltaNp63 and p53. During gastrulation, deltaNp63 is required for specification of nonneural ectoderm and its up-regulation suppresses neural differentiation, thus contributing to brain/craniofacial defects. In rps19-deficient embryos, deltaNp63 is induced in erythroid progenitors and may contribute to blood defects. We have shown that suppression of p53 and deltaNp63 alleviates the rps19-deficient phenotypes. Mutations in other ribosomal proteins, such as S8, S11, and S18, also lead to up-regulation of p53 pathway, suggesting it is a common response to ribosomal protein deficiency. Our finding provides new insights into pathogenesis of DBA. Ribosomal stress syndromes represent a broader spectrum of human congenital diseases caused by genotoxic stress; therefore, imbalance of p53 family members may become a new target for therapeutics.

Ribosomal protein L11 mutation in zebrafish leads to haematopoietic and metabolic defects

Mutations in ribosomal proteins are associated with a congenital syndrome, Diamond–Blackfan anaemia (DBA), manifested by red blood cell aplasia, developmental abnormalities and increased risk of malignancy. Recent studies suggest the involvement of p53 activation in DBA. However, which pathways are involved and how they contribute to the DBA phenotype remains unknown. Here we show that a zebrafish mutant for the rpl11 gene had defects both in the development of haematopoietic stem cells (HSCs) and maintenance of erythroid cells. The molecular signature of the mutant included upregulation of p53 target genes and global changes in metabolism. The changes in several pathways may affect haematopoiesis including upregulation of pro‐apoptotic and cell cycle arrest genes, suppression of glycolysis, downregulation of biosynthesis and dysregulation of cytoskeleton. Each of these pathways has been individually implicated in haematological diseases. Inhibition of p53 partially rescued haematopoiesis in the mutant. Altogether, we propose that the unique phenotype of DBA is a sum of several abnormally regulated molecular pathways, mediated by the p53 protein family and p53‐independent, which have synergistic impact on haematological and other cellular pathways affected in DBA. Our results provide new insights into the pathogenesis of DBA and point to the potential avenues for therapeutic intervention.

p53 family in development

The p53 family network is a unique cellular processor that integrates information from various pathways and determines cellular choices between proliferation, replication arrest/repair, differentiation, senescence, or apoptosis. The most studied role of the p53 family is the regulation of stress response and tumor suppression. By removing damaged cells from the proliferating pool, p53 family members preserve the integrity of the genome. In addition to this well recognized role, recent data implicate the p53 protein family in a broader role of controlling cell proliferation, differentiation and death. Members of the p53 protein family with opposing activity perform coordination of these processes. Imbalance of p53 protein family may contribute to a significant proportion of congenital developmental abnormalities in humans.

Zebrafish Models for Dyskeratosis Congenita Reveal Critical Roles of p53 Activation Contributing to Hematopoietic Defects through RNA Processing

Dyskeratosis congenita (DC) is a rare bone marrow failure syndrome in which hematopoietic defects are the main cause of mortality. The most studied gene responsible for DC pathogenesis is DKC1 while mutations in several other genes encoding components of the H/ACA RNP telomerase complex, which is involved in ribosomal RNA(rRNA) processing and telomere maintenance, have also been implicated. GAR1/nola1 is one of the four core proteins of the H/ACA RNP complex. Through comparative analysis of morpholino oligonucleotide induced knockdown of dkc1 and a retrovirus insertion induced mutation of GAR1/nola1 in zebrafish, we demonstrate that hematopoietic defects are specifically recapitulated in these models and that these defects are significantly reduced in a p53 null mutant background. We further show that changes in telomerase activity are undetectable at the early stages of DC pathogenesis but rRNA processing is clearly defective. Our data therefore support a model that deficiency in dkc1 and nola1 in the H/ACA RNP complex likely contributes to the hematopoietic phenotype through p53 activation associated with rRNA processing defects rather than telomerase deficiency during the initial stage of DC pathogenesis.

TNF-mediated inflammation represses GATA1 and activates p38 MAP kinase in RPS19-deficient hematopoietic progenitors

Diamond-Blackfan anemia (DBA) is an inherited disorder characterized by defects in erythropoiesis, congenital abnormalities, and predisposition to cancer. Approximately 25% of DBA patients have a mutation in RPS19, which encodes a component of the 40S ribosomal subunit. Upregulation of p53 contributes to the pathogenesis of DBA, but the link between ribosomal protein mutations and erythropoietic defects is not well understood. We found that RPS19 deficiency in hematopoietic progenitor cells leads to decreased GATA1 expression in the erythroid progenitor population and p53-dependent upregulation of tumor necrosis factor-α (TNF-α) in nonerythroid cells. The decrease in GATA1 expression was mediated, at least in part, by activation of p38 MAPK in erythroid cells and rescued by inhibition of TNF-α or p53. The anemia phenotype in rps19-deficient zebrafish was reversed by treatment with the TNF-α inhibitor etanercept. Our data reveal that RPS19 deficiency leads to inflammation, p53-dependent increase in TNF-α, activation of p38 MAPK, and decreased GATA1 expression, suggesting a novel mechanism for the erythroid defects observed in DBA.

The role of the DNA damage response in zebrafish and cellular models of Diamond Blackfan anemia.

Ribosomal biogenesis involves the processing of pre-ribosomal RNA. A deficiency of some ribosomal proteins (RPs) impairs processing and causes Diamond Blackfan anemia (DBA), which is associated with anemia, congenital malformations and cancer. p53 mediates many features of DBA, but the mechanism of p53 activation remains unclear. Another hallmark of DBA is the upregulation of adenosine deaminase (ADA), indicating changes in nucleotide metabolism. In RP-deficient zebrafish, we found activation of both nucleotide catabolism and biosynthesis, which is consistent with the need to break and replace the faulty ribosomal RNA. We also found upregulation of deoxynucleotide triphosphate (dNTP) synthesis – a typical response to replication stress and DNA damage. Both RP-deficient zebrafish and human hematopoietic cells showed activation of the ATR/ATM-CHK1/CHK2/p53 pathway. Other features of RP deficiency included an imbalanced dNTP pool, ATP depletion and AMPK activation. Replication stress and DNA damage in cultured cells in non-DBA models can be decreased by exogenous nucleosides. Therefore, we treated RP-deficient zebrafish embryos with exogenous nucleosides and observed decreased activation of p53 and AMPK, reduced apoptosis, and rescue of hematopoiesis. Our data suggest that the DNA damage response contributes to p53 activation in cellular and zebrafish models of DBA. Furthermore, the rescue of RP-deficient zebrafish with exogenous nucleosides suggests that nucleoside supplements could be beneficial in the treatment of DBA.

p53 Upregulation Is a Frequent Response to Deficiency of Cell-Essential Genes

Background The role of p53 in the prevention of development of embryos damaged by genotoxic factors is well recognized. However, whether p53 plays an analogous role in preventing birth defects from genetic mutations remains an unanswered question. Genetic screens for mutations affecting development show that only a fraction of developmentally lethal mutations leads to specific phenotypes while the majority results in similar recurrent phenotypes characterized by neuronal apoptosis and developmental delay. Mutations in cell-essential genes typically fall into this group. The observation that mutations in diverse housekeeping genes lead to a similar phenotype suggests a common mechanism underlying this phenotype. For some mutants, p53 inhibition was shown to attenuate the phenotype. Methodology/Principal Findings To find out how common p53 involvement is in this phenotype, we analyzed zebrafish mutants from various categories of cell essential genes. Several thousand zebrafish mutants have been identified; many of them are kept at stock centers and available for the research community. We selected mutants for genes functioning in DNA replication, transcription, telomere maintenance, ribosome biogenesis, splicing, chaperoning, endocytosis, and cellular transport. We found that mutants have similar phenotypes including neural apoptosis, failure to develop structures originated from the neural crest cells, and hematopoietic defects. All mutants share p53 upregulation and similar changes in several p53-dependent and independent molecular pathways. Conclusion/Significance Our results suggest that mutations in housekeeping genes often canalize on the p53-mediated phenotype. p53 prevents the development of embryos with defects in such genes. p53-mediated changes in gene expression may also contribute to many human congenital malformations.

Role of p53 family in birth defects: Lessons from zebrafish

p53 Protein family is an important teratologic suppressor, but in certain conditions it can cause congenital abnormalities. p53 Family performs this dual role in development by integrating information from cells interior with that from the environment to determine the choice between life and death. Understanding of p53 family developmental functions may lead to new therapeutic approaches for treatment and prevention of birth defects. Zebrafish is becoming the vertebrate system of choice for studying p53 family role in development.

Innate immune system activation in zebrafish and cellular models of Diamond Blackfan Anemia

Deficiency of ribosomal proteins (RPs) leads to Diamond Blackfan Anemia (DBA) associated with anemia, congenital defects, and cancer. While p53 activation is responsible for many features of DBA, the role of immune system is less defined. The Innate immune system can be activated by endogenous nucleic acids from non-processed pre-rRNAs, DNA damage, and apoptosis that occurs in DBA. Recognition by toll like receptors (TLRs) and Mda5-like sensors induces interferons (IFNs) and inflammation. Dying cells can also activate complement system. Therefore we analyzed the status of these pathways in RP-deficient zebrafish and found upregulation of interferon, inflammatory cytokines and mediators, and complement. We also found upregulation of receptors signaling to IFNs including Mda5, Tlr3, and Tlr9. TGFb family member activin was also upregulated in RP-deficient zebrafish and in RPS19-deficient human cells, which include a lymphoid cell line from a DBA patient, and fetal liver cells and K562 cells transduced with RPS19 shRNA. Treatment of RP-deficient zebrafish with a TLR3 inhibitor decreased IFNs activation, acute phase response, and apoptosis and improved their hematopoiesis and morphology. Inhibitors of complement and activin also had beneficial effects. Our studies suggest that innate immune system contributes to the phenotype of RPS19-deficient zebrafish and human cells.