4 research outputs found
The Notch Delta-4 ligand helps to maintain the quiescence and the short-term reconstitutive potential of Haematopoietic Progenitor Cells through activation of a key gene network: Delta-4/Notch pathway retains the HPCs potential
International audienceUnderstanding the role of Notch and its ligands within the different bone marrow niches could shed light on the mechanisms regulating haematopoietic progenitor cells (HPCs) maintenance and self renewal. Here, we report that murine bone marrow HPCs activation by the vascular Notch Delta4 ligand maintains a significant proportion of cells specifically in the G0 state. Furthermore, Delta4/Notch pathway limits significantly the loss of the in vivo short-term reconstitutive potential upon transplantation of Delta-4 activated HPCs into lethally irradiated recipient mice. Both effects are directly correlated with the decrease of cell cycle genes transcription such as CYCLIN-D1, â D2, and -D3, and the upregulation of stemness related genes transcription such as BMI1, GATA2, HOXB4 and C-MYC. In addition, the transcriptional screening also highlights new downstream post-transcriptional factors, named PUMILIO1 and â 2, as part of the stem signature associated with the Delta4/Notch signalling pathway
Etude des mĂ©canismes molĂ©culaires des protĂ©ines de liaison Ă lâARNm PUMILIO 1 et 2 dans la rĂ©gulation des cellules souches/progĂ©nitrices hĂ©matopoĂŻĂ©tiques normales et pathologiques
Les protĂ©ines de liaison Ă lâARN PUMILIO 1 et 2 (PUM1/2) exercent un rĂŽle central dans le maintien des cellules souches chez les InvertĂ©brĂ©s en se fixant, en association avec des partenaires protĂ©iques, sur la rĂ©gion 3â UTR de certains ARNm, rĂ©gulant ainsi leur devenir. A ce jour, le rĂŽle de PUM1/2 dans les cellules souches/progĂ©nitrices hĂ©matopoĂŻĂ©tiques (CSPHs) a Ă©tĂ© peu Ă©tudiĂ©. La perte de la coordination entre auto-renouvellement et diffĂ©renciation des CSPHs peut aboutir Ă des hĂ©mopathies chez l'Homme, dâoĂč la nĂ©cessitĂ© de comprendre les mĂ©canismes sous-jacents. Notre Ă©quipe a mis en Ă©vidence, par une approche de shARN, que lâinvalidation des protĂ©ines PUM1/2 dans les CSHs humaines et murines conduit Ă une rĂ©duction de leur expansion, associĂ©e Ă une apoptose accrue et un arrĂȘt du cycle cellulaire en phase G0/G1, et aussi Ă une perte du potentiel clonogĂ©nique in vitro et du potentiel de reconstitution in vivo. Lâobjectif de notre travail a consistĂ© Ă : a/ Ă©valuer les effets de la surexpression de PUM1/2 dans les CSPHs, b/ dĂ©terminer lâimplication de PUM1/2 dans les processus leucĂ©miques, c/ Ă©tudier les mĂ©canismes molĂ©culaires responsables de lâactivitĂ© de PUM1/2 en identifiant les cibles et les partenaires protĂ©iques par une approche de protĂ©omique globale. Nos rĂ©sultats suggĂšrent quâune surexpression modĂ©rĂ©e de PUM1 (2/3 fois) dans les cellules CD34+ limite la perte du potentiel clonogĂ©nique alors quâune expression plus Ă©levĂ©e (5/10 fois et plus) est toxique. Lâanalyse de lâexpression de PUM1/2 par RT-qPCR dans les Ă©chantillons de LeucĂ©mies Aigue MyeloĂŻdes (LAM) (GOELAMSthĂšque) montre une augmentation significative dans les Ă©chantillons les plus immatures (LAM0-2) comparĂ©s aux contrĂŽles sains. La perte de PUM1/2 par shARN dans les cellules primaires de leucĂ©mies ainsi que dans des lignĂ©es issues de diffĂ©rents processus leucĂ©miques rĂ©duit fortement leur survie. La recherche des partenaires associĂ©s Ă PUM par spectromĂ©trie de masse a permis de dĂ©couvrir Argonaute2 et MOV10 (tous les 2 impliquĂ©s dans la machinerie des miRNA), ainsi que des protĂ©ines de liaison aux ARNs, ELAV1 dĂ©jĂ connue pour son implication dans le maintien des CSH murines et IMP3, impliquĂ© dans de nombreux cancers et dans la rĂ©gulation du cycle cellulaire. Lâinvalidation de IMP3 ou ELAV1 dans les CSPHs conduisent, in vitro, aux mĂȘmes effets observĂ©s avec la perte du PUM 1/2, une diminution de lâexpansion avec une augmentation de lâapoptose, et la perte du potentiel clonogĂ©nique. Enfin, nous avons identifiĂ© FoxP1 (Forkhead box P1) comme nouvelle cible directe de PUM1/2, dont le rĂŽle est encore trĂšs peu dĂ©crit dans lâhĂ©matopoĂŻĂšse. LâĂ©tude fonctionnelle de FoxP1 sur les CSPHs par shARN mime les effets observĂ©s avec les facteurs PUM1/2. De plus, la surexpression de FoxP1 restaure partiellement les activitĂ©s antiprolifĂ©ratives et pro-apoptotiques gĂ©nĂ©rĂ©es par les shPUM1/2. Enfin, le profil dâexpression de FoxP1 dans les LAM corrĂšle avec le profil dâexpression de PUM1/2. Nos rĂ©sultats confirment le rĂŽle majeur jouĂ© par les protĂ©ines PUM1/2 en partie via la rĂ©gulation positive de FoxP1 qui contribue au maintien les CSPHs normales et pathologiques.Pumilio 1 and 2 (PUM1/2) RNA-binding proteins exert a central role in stem cell maintenance among Invertebrates by binding the 3'UTR of mRNA targets in association with protein partners, thus regulating mRNA stability/translation. Nothing is known regarding normal and pathologic hematopoietic stem and progenitor cells (HSPCs). Loss of coordination between self-renewal and differentiation of HSPCs can lead to leukemia in humans, hence the need to understand the mechanisms. Our team has highlighted the fundamental role played by the post-transcriptional regulators Pumilio (PUM) 1/2 on normal HSPC properties. By a shRNA approach, PUM 1/2 knockdown in human and murine HSPCs leads to: a/ a reduced expansion associated with an increased apoptosis and a cell cycle arrest in G0/G1 phase, b/ the loss of their clonogenic capacity and their in vivo reconstitution potential. The objective of our work is to: a/ evaluate the effects of PUM 1/2 overexpression in HSPC, b/ determine PUM1/2 involvement in leukemic processes; c/ investigate the molecular mechanisms responsible of PUM activity in HSPC by identifying protein targets and partners. Our results showed that a moderate overexpression of PUM1 (2 to 3 fold) in normal CD34+ HSPCs limits the loss of their clonogenic potential, while a higher expression (5 to 10 fold or more) is toxic. The expression analysis of PUM1/2 transcripts in Acute Myeloid Leukemia (AML) (GOELAMSthĂšque) showed a significant increase in the most immature samples (AML0-2) as compared to healthy controls. PUM1/2 knockdown by shRNA in AML cells significantly reduced their survival. The same effect was observed in cell lines from several leukemic processes. We identified various PUM-associated partners by mass spectrometry, Argonaute2 and MOV10 (involved in the miRNA machinery), and the RNA-binding proteins IMP3 (involved in several cancer and in cell cycle regulation) and HuR/ELAV1 (already known to be involved in murine HSPCs maintenance). IMP3 or ELAV1 knockdown in HSPCs in vitro lead to the same effect of a PUM1/2 invalidation, a decreased expansion with an increased apoptosis and the loss of clonogenic potential. Finally, we identify the forkhead box P1 (FOXP1) transcription factor as a new direct target up-regulated by PUM1 and PUM2. Functional study of FoxP1 knockdown by shRNA in HSPCs mimic PUM1/2 activities. Moreover, FOXP1 overexpression partially rescued shPUM antiproliferative and pro-apoptotic effects. Also, the PUM1/2 and FOXP1 expression levels in leukemic primary cells were measured by RT-qPCR and revealed a positive correlation. Our results reveal that PUM1/2 are direct positive regulators of FOXP1 which contributes to the maintenance of normal and leukemic HSPCs