Call number: LD2668 .T4 1961 S23

Anemia occurs in 60% of patients with Shwachman Diamond Syndrome (SDS). Although bi-allelic mutations in SBDS cause SDS, it is unclear whether SBDS is critical for erythropoiesis and what the pathogenesis of anemia is in SDS. I hypothesize that SBDS protects early erythroid progenitors from p53 family member mediated apoptosis by promoting ribosome biosynthesis and translation. SBDS deficiency by vector-based shRNA led to impaired cell expansion of differentiating K562 cells due to accelerated apoptosis and reduced proliferation. Furthermore, the cells showed general reduction of 40S, 60S, 80S ribosomal subunits, loss of polysomes and impaired global translation during differentiation. An upregulation of the pro-apoptotic p53 family member, TAp73, was found in resting SBDS deficient cells; however, not in differentiating cells. These results demonstrate SBDS plays a critical role in erythroid expansion by promoting survival of early erythroid progenitors and in maintaining ribosome biogenesis during erythroid maturation independently of p53 family members.

Anemia occurs in 60% of patients with Shwachman Diamond Syndrome (SDS). Although bi-allelic mutations in SBDS cause SDS, it is unclear whether SBDS is critical for erythropoiesis and what the pathogenesis of anemia is in SDS. I hypothesize that SBDS protects early erythroid progenitors from p53 family member mediated apoptosis by promoting ribosome biosynthesis and translation. SBDS deficiency by vector-based shRNA led to impaired cell expansion of differentiating K562 cells due to accelerated apoptosis and reduced proliferation. Furthermore, the cells showed general reduction of 40S, 60S, 80S ribosomal subunits, loss of polysomes and impaired global translation during differentiation. An upregulation of the pro-apoptotic p53 family member, TAp73, was found in resting SBDS deficient cells; however, not in differentiating cells. These results demonstrate SBDS plays a critical role in erythroid expansion by promoting survival of early erythroid progenitors and in maintaining ribosome biogenesis during erythroid maturation independently of p53 family members.

Polycomb proteins are epigenetic regulators that are critical in mediating gene repression at critical stages during development. Core and accessory proteins make up the Polycomb Repressive Complex 2 (PRC2), which is responsible for trimethylation of lysine 27 on histone 3 (H3K27me3), leading to maintenance of chromatin compaction and sustained gene repression. Classically, Polycomb accessory proteins are often thought of as having minor roles in fine-tuning the repressive action of PRC2. Their actions have often been attributed to chromatin recognition, targeting to specific loci and enhancing methyltransferase activity. In our previous work in mouse embryonic stem cells (ESCs), we showed that Polycomb-like 2 (Mtf2/Pcl2) is critical for PRC2-mediated regulation of stem cell self-renewal through feed-forward control of the pluripotency network. In moving beyond the ESC model system, we sought to interrogate the role of Mtf2 in vivo by creating a gene-targeted knockout mouse model. Surprisingly, we discovered a tissue-specific role for Mtf2 in controlling erythroid maturation and hematopoietic stem cell self-renewal. Via its regulation of other PRC2 members, Mtf2 is critical for global H3K27me3 methylation at promoter-proximal sites in developing erythroblasts. Thus, Mtf2 is required for proper maturation of erythroblasts. Loss of Mtf2 also reduces HSC self-renewal leading to stem cell pool exhaustion. Additionally, misregulation of Mtf2 in leukemia models contributes to massive leukemic blast expansion at the expense of leukemic stem cell self-renewal. In the developing hematopoietic system, Mtf2 functions as a core complex member, controlling epigenetic regulation of self-renewal and maturation of both stem and committed cells.

No description available.

La biogenèse du ribosome est un processus indispensable à la prolifération cellulaire car elle permet la synthèse protéique assurant la croissance avant la division cellulaire. Les ribosomopathies telles que le syndrome myélodysplasique 5q- et l’anémie de Blackfan-Diamond sont dues respectivement à une mutation d’un gène codant une protéine ribosomique (RP) et à l’haploinsuffisance en RPS14, RP de la petite sous-unité du ribosome. Les patients atteints de l’une de ces ribosomopathies présentent un défaut de l’érythropoïèse suggérant que celle-ci est particulièrement dépendante du ribosome. L’érythropoïèse est le processus qui permet la formation de globules rouges à partir de cellules souches hématopoïétiques et consiste en différents stades de différenciation appelés érythroblastes. C’est dans ce contexte que je me suis intéressée au ribosome au cours de l’érythropoïèse. Dans un premier temps, nous avons caractérisé la biogenèse du ribosome dans des cellules érythroïdes primaires humaines et murines. Pour cela nous avons adapté une technique de SILAC pulsé et mis au point la ribomique, technique de protéomique permettant l’analyse de la biogenèse du ribosome dans des échantillons de cellules primaires basée sur l’identification presque exhaustive des protéines ribosomiques. À l’aide de la ribomique et par d’autres techniques, nous avons mis en évidence une diminution de la biogenèse du ribosome après le stade érythroblaste basophile. Nous avons également montré que cette biogenèse du ribosome est en partie sous le contrôle de la voie mTORC1 régulée par les deux cytokines fondamentales de l’érythropoïèse : le Stem Cell Factor (SCF) et l’érythropoïétine (EPO). L’expression par l’érythroblaste des récepteurs des deux cytokines permet une biogenèse du ribosome optimale. L’inhibition de la biogenèse du ribosome par le CX-5461, inhibiteur spécifique de l’ARN polymérase I, ou par la rapamycine, inhibiteur de mTORC1, entraîne une accélération de la différenciation érythroïde soulignant un rôle de la biogenèse du ribosome au cours de l’érythropoïèse. L’inhibition de la voie mTORC1 modifie l’ordre de clivage de l’ARNr, reflet d’une modification de sa maturation. Les expériences de ribomique dans les érythroblastes humains ont également permis de mettre en évidence la présence de paralogues de RP et la sous-représentation de certaines RPs au sein des ribosomes suggérant une hétérogénéité des ribosomes dans les érythroblastes humains. Parallèlement, un modèle mimant le syndrome 5q- a été développé par une approche shRPS14 dans une lignée humaine érythroleucémique dépendante de l’EPO. L’inhibition de RPS14 entraîne un défaut de biogenèse de la sous-unité 40S du ribosome aboutissant à une diminution des ribosomes entiers formés et une diminution de la traduction globale. Cependant une traduction est maintenue. Le défaut de biogenèse de la sous-unité 40S entraîne une augmentation de la quantité de c-KIT, récepteur du SCF et une diminution de la quantité de GATA1, facteur de transcription spécifique de l’érythropoïèse. Nous avons mis en évidence que la diminution de GATA1 est due à une diminution de sa traduction tandis que la traduction d’autres protéines est conservée dans ce contexte d’altération de la biogenèse du ribosome. Nous avons ensuite réalisé une analyse des transcrits présents dans les fractions polysomales correspondants à la traduction la plus efficace. Nous avons montré grâce à ce traductome que les propriétés thermodynamiques des parties 5’ et 3’UTR des ARNm modulent leur traduction dans le contexte d’inhibition de RPS14. Ces données suggèrent que l’altération de la biogenèse du ribosome peut aboutir à une modification du programme traductionnel. Ce travail montre que la biogenèse du ribosome diminue au cours de l’érythropoïèse et participe à la différenciation érythroïde. La voie mTORC1 participe au contrôle de cette biogenèse. / Ribosome biogenesis is a key event allowing cell growth before division. Defective RB recognized in ribosomopathyinherited Diamond-Blackfan anemia and 5q- syndrom. In this study, we aimed at investigating the regulatory role of RB during the erythroid precursor maturation which is characterized by a cell size reduction during 2 to 3 rapid cell divisions. We used two in vitro systemsé of expansion and differentiation of erythroblasts (E.) derived of immature hematopoietic progenitors from human mobilized peripheral blood or mouse fetal liver. The expansion step is supported by the Stem Cell Factor (SCF) and the second step depends on erythropoietin (EPO). The structure of the nucleolus was studied by electron microscopy. Compared to immature proerythroblasts (proE), a dramatic size reduction and change in nucleolar structure (ie. the disappearance of fibrillar and dense fibrillar components) is observed at the stage of mature polychromatophilic E. suggesting a loss of functionality. RB was measured by a pulsed SILAC (Stable Isotopic Labeling by Amino acids in Culture cell) proteomic assay that quantified the incorporation of newly synthesized ribosomal proteins in the ribosome. Both in mouse and human models, immature proE expanded upon SCF and EPO demonstrate a maximal RB with a renewal rate of 60% and 50% every 14h and 24h, respectively. By contrast, RB rapidly interrupted with the disappearance of proE and basophilic E after the switch to EPO alone. Consistently, the quantities of ribosomal RNA (rRNA) 45S precursor estimated by qPCR are maximal in proE and almost null in orthochromatophilic E. Inhibition of RB at proE stage by RNApol I specific inhibitor (CX-5461) accelerates the onset of terminal erythroid differentiation suggesting that RB is a rate limiting factor for final maturation. We then hypothesize that degree of signaling intensity in response to SCF and EPO may control the level of RB. To address this question, we investigated the mTORC1 (mechanistic Target Of Rapamycin Complex 1) pathway which is directly involved in RB through its substrate p70S6Kinase. Activation of P-p70S6Kinase and P-Rps6, as well as ribosome renewal, are twice more elevated in response to SCF and EPO than to EPO alone. Furthermore, inhibition of mTORC1/p70S6K/Rps6 pathway by rapamycin disrupts RB and leads to an acceleration of terminal erythroid differentiation.This study demonstrates that the collapse of RB promotes erythroid cell terminal maturation and shows the regulatory role of mTORC1 pathway on RB during erythropoiesis.

Negative regulation of cytokine signalling is essential for maintaining hematopoietic homeostasis. We investigated the role of SH2B1 and CBL in the negative regulation of EPO and GM-CSF signaling, respectively. Erythropoiesis is driven by the cytokine erythropoietin (EPO), which mediates its signal by binding to its cognate receptor, the erythropoietin receptor (EPO-R). Murine knock-in studies have demonstrated EPO-R Tyr343 to play an important role in EPO mediated signalling. We have utilized a Cloning of Ligand Target (COLT) screen to identify the adaptor protein SH2B1 as an interactor of EPO-R pTyr343. We have demonstrated that SH2B1 binds to EPO-R via two mechanisms. The amino-terminus of SH2B1 and the membrane proximal region of EPO-R mediate SH2B1 constitutive binding to EPO-R. SH2B1 binds to EPO-R pTyr343 and pTyr 401 in an SH2 domain-dependent manner. SH2B1 displayed dose- and time- dependent Serine/Threonine phosphorylation in response to EPO stimulation. Knockdown of SH2B1 resulted in enhanced activation of Jak2 and EPO-R. These studies demonstrate SH2B1 as a novel negative regulator of EPO signalling. Mutations in the linker region and the RING finger of CBL have been identified in a number of myeloid malignancies, including juvenile myelomonocytic leukemia. We investigated how linker region mutant, CBL-Y371H, and RING finger mutant, CBL-C384R lead to GM-CSF hypersensitivity. Expression of these CBL mutants in the human hematopoietic cell line, TF-1, showed enhanced stimulation induced phosphorylation of GM-CSFR βc. We also demonstrated that the loss of E3 ligase activity of these CBL mutants results in increased expression of JAK2 and LYN kinases. Assessment of the effects of CBL mutants on downstream signalling revealed enhanced phosphorylation of SHP2, CBL and S6. Dasatinib induced inhibition of SRC family kinases abolished the elevated phosphorylation of CBL mutants, and equalized the phosphorylation of GM-CSFR βc in the wild type and CBL mutant cells.

Negative regulation of cytokine signalling is essential for maintaining hematopoietic homeostasis. We investigated the role of SH2B1 and CBL in the negative regulation of EPO and GM-CSF signaling, respectively. Erythropoiesis is driven by the cytokine erythropoietin (EPO), which mediates its signal by binding to its cognate receptor, the erythropoietin receptor (EPO-R). Murine knock-in studies have demonstrated EPO-R Tyr343 to play an important role in EPO mediated signalling. We have utilized a Cloning of Ligand Target (COLT) screen to identify the adaptor protein SH2B1 as an interactor of EPO-R pTyr343. We have demonstrated that SH2B1 binds to EPO-R via two mechanisms. The amino-terminus of SH2B1 and the membrane proximal region of EPO-R mediate SH2B1 constitutive binding to EPO-R. SH2B1 binds to EPO-R pTyr343 and pTyr 401 in an SH2 domain-dependent manner. SH2B1 displayed dose- and time- dependent Serine/Threonine phosphorylation in response to EPO stimulation. Knockdown of SH2B1 resulted in enhanced activation of Jak2 and EPO-R. These studies demonstrate SH2B1 as a novel negative regulator of EPO signalling. Mutations in the linker region and the RING finger of CBL have been identified in a number of myeloid malignancies, including juvenile myelomonocytic leukemia. We investigated how linker region mutant, CBL-Y371H, and RING finger mutant, CBL-C384R lead to GM-CSF hypersensitivity. Expression of these CBL mutants in the human hematopoietic cell line, TF-1, showed enhanced stimulation induced phosphorylation of GM-CSFR βc. We also demonstrated that the loss of E3 ligase activity of these CBL mutants results in increased expression of JAK2 and LYN kinases. Assessment of the effects of CBL mutants on downstream signalling revealed enhanced phosphorylation of SHP2, CBL and S6. Dasatinib induced inhibition of SRC family kinases abolished the elevated phosphorylation of CBL mutants, and equalized the phosphorylation of GM-CSFR βc in the wild type and CBL mutant cells.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 4 uppsatser.

Although the key role played by stem cells in maintaining hemopoiesis is well recognized, mechanisms that determine stem cell behaviour (e.g. self-renewal) have remained poorly defined. Historical precedent has illustrated the usefulness of in viitro colony assays in facilitating the investigation of various primitive hemopoietic progenitor classes with restricted differentiation and proliferative potential. In particular, such assays have made possible the definition of the sequential nature of a series of events that early erythropoietic cells undergo and suggested properties that might be anticipated to characterize colonies derived from stem cells proliferating and differentiating in vitro. The present studies were undertaken to test the hypothesis that very large, late maturing erythroid colonies previously noted on occasion in routine erythroid colony assays were, in fact, derived from progenitors with the self-renewal and multiple myeloid differentiation properties associated with stem cells. Initial experiments showed that cells capable of yielding macroscopic sized erythroid colonies were present at low frequency in normal marrow but became the predominant erythropoietic cell type after 2 to 3 weeks in flask culture. Macroscopic erythroid colony formation in assays of cells from either source were shown to have: 1) identical very late maturation kinetics (onset of hemoglobinization after 1 week of initial colony growth), 2) the same high erythropoietin requirements, and 3) similar responsiveness to factors present in media conditioned by mitogen stimulated spleen cells. Optimization of these 2 classes of stimulant yielded an assay that was linear down to very low cell concentrations (less than 5 x 103 cells/ml). This made possible the cytological analysis of macroscopic erythroid colonies under conditions where overlap with other colony types was minimal. Such studies revealed that macroscopic colonies contained, in addition to erythroid cells, megakaryocytes ( > 90% of colonies) and granulocyte cells (30% of colonies) including cells of the eosinophil lineage. Such colonies were also found to contain cells capable of macroscopic spleen colony formation in irradiated mice, the conventional assay for mouse hemopoietic stem cells (on average 1 CFU-S per colony of flask culture origin, and 0.3 CFU-S per colony in assays of fresh marrow). Direct evidence for self-renewal was obtained from replating experiments using irradiated feeders to optimize plating efficiency. Mixed colonies of macroscopic size were regularly demonstrable in replating assays after 1, and even 2, generations of mixed colony formation indicating up to 6 self-renewal divisions during colony formation. By comparison to flask culture cells, the extent of self-renewal exhibited by cells in fresh marrow yielding macroscopic erythroid colonies was found to be 5-fold lower. Finally, the in vitro expression of stem cell self-renewal behaviour was investigated in individual colonies. The number of stem cells generated, when assessed either by CFU-S or replating assays was found to vary markedly. This variation was not accounted for by errors of detection or by variations in colony size. Such findings are similar to previous data on the CFU-S content of individual spleen colonies and provide the first evidence that the type of variation in stem cell self-renewal observed in vivo also occurs in vitro where microenvironmental factors are unlikely to be contributing factors. These results are consistent with a model of stem cell self-renewal in which intrinsic cellular factors play the key role in influencing the decision of individual cells to self-renew. / Medicine, Faculty of / Medical Genetics, Department of / Graduate