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HOXC4 homeoprotein efficiently expands human hematopoietic stem cells and triggers similar molecular alterations as HOXB4.

ABSTRACT: BACKGROUND: Expansion of hematopoietic stem cells represents an important objective for improving cell and gene therapy protocols. Retroviral transduction of the HoxB4 homeogene in mouse and human hematopoietic stem cells and hematopoietic progenitors is known to promote the cells' expansion. A safer approach consists in transferring homeobox proteins into hematopoietic stem cells taking advantage of the natural ability of homeoproteins to cross cell membranes. Thus, HOXB4 protein transfer is operative for expanding human hematopoietic cells, but such expansion needs to be improved. DESIGN AND METHODS: To that aim, we evaluated the effects of HOXC4, a protein encoded by a HOXB4 paralog gene, by co-culturing HOXC4-producing stromal cells with human CD34(+) hematopoietic cells. Numbers of progenitors and stem cells were assessed by in vitro cloning assays and injection into immuno-deficient mice, respectively. We also looked for activation or inhibition of target downstream gene expression. RESULTS: We show that the HOXC4 homeoprotein expands human hematopoietic immature cells by 3 to 6 times ex vivo and significantly improves the level of in vivo engraftment. Comparative transcriptome analysis of CD34(+) cells subjected or not to HOXB4 or HOXC4 demonstrated that both homeoproteins regulate the same set of genes, some of which encode key hematopoietic factors and signaling molecules. Certain molecules identified herein are factors reported to be involved in stem cell fate or expansion in other models, such as MEF2C, EZH2, DBF4, DHX9, YPEL5 and Pumilio. CONCLUSIONS: The present study may help to identify new HOX downstream key factors potentially involved in hematopoietic stem cell expansion or in leukemogenesis.

SUBMITTER: Auvray C

PROVIDER: S-EPMC3269473 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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HOXC4 homeoprotein efficiently expands human hematopoietic stem cells and triggers similar molecular alterations as HOXB4.

Auvray Céline C Delahaye Andrée A Pflumio Françoise F Haddad Rima R Amsellem Sophie S Miri-Nezhad Ayda A Broix Loïc L Yacia Azzedine A Bulle Frédérique F Fichelson Serge S Vigon Isabelle I

Haematologica 20120201 2

<h4>Background</h4>Expansion of hematopoietic stem cells represents an important objective for improving cell and gene therapy protocols. Retroviral transduction of the HoxB4 homeogene in mouse and human hematopoietic stem cells and hematopoietic progenitors is known to promote the cells' expansion. A safer approach consists in transferring homeobox proteins into hematopoietic stem cells taking advantage of the natural ability of homeoproteins to cross cell membranes. Thus, HOXB4 protein transfe ...[more]

PMID: 22298821

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Project description:Oncogenic Kras expression specifically in hematopoietic stem cells (HSCs) induces a rapidly fatal myeloproliferative neoplasm in mice, suggesting that Kras signaling plays a dominant role in normal hematopoiesis. However, such a conclusion is based on expression of an oncogenic version of Kras. Hence, we sought to determine the effect of simply increasing the amount of endogenous wild-type Kras on HSC fate. To this end, we utilized a codon-optimized version of the murine Kras gene (Krasex3op) that we developed, in which silent mutations in exon 3 render the encoded mRNA more efficiently translated, leading to increased protein expression without disruption to the normal gene architecture. We found that Kras protein levels were significantly increased in bone marrow (BM) HSCs in Krasex3op/ex3op mice, demonstrating that the translation of Kras in HSCs is normally constrained by rare codons. Krasex3op/ex3op mice displayed expansion of BM HSCs, progenitor cells, and B lymphocytes, but no evidence of myeloproliferative disease or leukemia in mice followed for 12 months. BM HSCs from Krasex3op/ex3op mice demonstrated increased multilineage repopulating capacity in primary competitive transplantation assays, but secondary competitive transplants revealed exhaustion of long-term HSCs. Following total body irradiation, Krasex3op/ex3op mice displayed accelerated hematologic recovery and increased survival. Mechanistically, HSCs from Krasex3op/ex3op mice demonstrated increased proliferation at baseline, with a corresponding increase in Erk1/2 phosphorylation and cyclin-dependent kinase 4 and 6 (Cdk4/6) activation. Furthermore, both the enhanced colony-forming capacity and in vivo repopulating capacity of HSCs from Krasex3op/ex3op mice were dependent on Cdk4/6 activation. Finally, BM transplantation studies revealed that augmented Kras expression produced expansion of HSCs, progenitor cells, and B cells in a hematopoietic cell-autonomous manner, independent from effects on the BM microenvironment. This study provides fundamental demonstration of codon usage in a mammal having a biological consequence, which may speak to the importance of codon usage in mammalian biology.

Project description:At the molecular level, the mechanisms of action of Hox genes during hematopoiesis remain elusive. Although reports have described the implication of Hox genes in some molecular pathways in mice, the targets of HOX proteins in human hematopoietic cells are mostly unidentified. Since HOXB4 and HOXC4 are transcription factors encoded by paralogous genes that display similar effects on HSCs and progenitors, we presumed that HOXB4 and HOXC4 regulate the same target genes during human HSC expansion. This hypothesis was supported by the fact that HOXB4 and HOXC4 have similar molecular structure and similar roles and expression patterns during development. To identify early factors and signaling pathways involved in hematopoietic cell expansion consecutive to the action of HOXB4 or HOXC4, we performed a differential transcriptome analysis of RNAs from cord blood-derived CD34+ cells cultured for 24 hours in the presence of MS-5 cells engineered to secrete HOXB4 or HOXC4 proteins. MS-5/GFP cells were used as control in separate co-cultures to exclude nonspecific changes in gene expression that may be associated with the presence of lentivirus-transduced MS-5 cells. Human CD34+ cord blood (4.106) cells were co-cultured for 24 hours with irradiated EGFP-, HOXB4-, or HOXC4-transduced MS-5 cells. 12 arrays were analysed. Three competitive hybridizations to the arrays were performed in duplicate using each time two dye-swap following this experimental design: AVZ056 : CY5 (CD34+/MS5+HOXB4) / CY3 (CD34+/MS5+GFP) AVZ059 : CY5 (CD34+/MS5+GFP) / CY3 (CD34+/MS5+HOXB4) AVZ064 : CY5 (CD34+/MS5+GFP) / CY3 (CD34+/MS5+HOXB4) AVZ067 : CY5 (CD34+/MS5+HOXB4) / CY3 (CD34+/MS5+GFP) AVZ057 : CY5 (CD34+/MS5+GFP) / CY3 (CD34+/MS5+HOXC4) AVZ058 : CY5 (CD34+/MS5+HOXC4) / CY3 (CD34+/MS5+GFP) AVZ060 : CY5 (CD34+/MS5+HOXC4) / CY3 (CD34+/MS5+GFP) AVZ068 : CY5 (CD34+/MS5+GFP) / CY3 (CD34+/MS5+HOXC4) AVZ061 : CY5 (CD34+/MS5+HOXB4) / CY3 (CD34+/MS5+HOXC4) AVZ062 : CY5 (CD34+/MS5+HOXC4) / CY3 (CD34+/MS5+HOXB4) AVZ065 : CY5 (CD34+/MS5+HOXC4) / CY3 (CD34+/MS5+HOXB4) AVZ066 : CY5 (CD34+/MS5+HOXB4) / CY3 (CD34+/MS5+HOXC4)

Dataset Information

HOXC4 homeoprotein efficiently expands human hematopoietic stem cells and triggers similar molecular alterations as HOXB4.

Publications

HOXC4 homeoprotein efficiently expands human hematopoietic stem cells and triggers similar molecular alterations as HOXB4.

Similar Datasets

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