Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profiling of HOXB4-transduced murine hematopoietic stem cells

ABSTRACT: Overexpression of HOXB4 in hematopoietic stem cells (HSCs) leads to increased self-renewal without causing hematopoietic malignancies in transplanted mice. The molecular basis of HOXB4-mediated benign HSC expansion in vivo is not well understood. To gain further insight into the molecular events underlying HOXB4-mediated HSC expansion, we analyzed gene expression changes at multiple time points in Lin-Sca1+c-kit+ (LSK) cells from mice transplanted with bone marrow (BM) cells transduced with a MSCV-HOXB4-ires-YFP vector. A distinct HOXB4 transcriptional program was reproducibly induced and stabilized by 12 weeks after transplant. Dynamic expression changes were observed in genes critical for HSC self-renewal as well as genes involved in myeloid and B cell differentiation. Prdm16, a transcription factor associated with human acute myeloid leukemia (AML), was markedly repressed by HOXB4 but upregulated by HOXA9 and HOXA10, suggesting that Prdm16 downregulation was involved in preventing leukemia in HOXB4 transplanted mice. Functional evidence to support this mechanism was obtained by enforcing co-expression of sPrdm16 and HOXB4, which led to enhanced self-renewal, myeloid expansion, and leukemia. Altogether, these studies define the transcriptional pathways involved in HOXB4 HSC expansion in vivo and identify repression of Prdm16 transcription as a mechanism by which expanding HSCs avoid leukemic transformation. 5-FU treated bone marrow cells from female C57Bl/6L mice were transduced with concentrated supernatant from GPE+86-derived producer cells containing MSCV-HOXB4-ires-YFP or MSCV-ires-GFP retroviral vectors. Transduced cells were transplanted into lethally irradiated C57Bl/6L mice. HOXB4-YFP or GFP expressing LSK cells were sorted from transplanted mice at 6, 12 and 24 weeks post transplantation, of which RNA was extracted for microarray.

ORGANISM(S): Mus musculus

SUBMITTER: Geoffrey Neale

PROVIDER: E-GEOD-59804 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Spinocerebellar Ataxia Type 2 (SCA2) is caused by expansion of a polyglutamine encoding triplet repeat in the human ATXN2 gene beyond (CAG)31. This is thought to mediate toxic gain-of-function by protein aggregation, and affect RNA processing, resulting in degenerative processes affecting preferentially cerebellar neurons. As a faithful animal model, we generated a knock-in mouse replacing the single CAG of murine Atxn2 with CAG42, a frequent patient genotype. This expansion size was inherited stably. The mice showed phenotypes with reduced weight and later motor incoordination. Although brain Atxn2 mRNA became elevated, soluble ATXN2 protein levels diminished over time, which might explain partial loss-of-function effects. Deficits in soluble ATXN2 protein correlated with the appearance of insoluble ATXN2, a progressive feature in cerebellum possibly reflecting toxic gains-of-function. Since in vitro ATXN2 overexpression was known to reduce levels of its protein interactor PABPC1, we studied expansion effects on PABPC1. In cortex, PABPC1 transcript, soluble and insoluble protein levels were increased. In more vulnerable cerebellum the progressive insolubility of PABPC1 was accompanied by decreased soluble protein levels, with PABPC1 mRNA showing no compensatory increase. The sequestration of PABPC1 into insolubility by ATXN2 function gains was validated in human cell culture. To understand consequences on mRNA processing, transcriptome profiles at medium and old age in three different tissues were studied and demonstrated selective induction of Fbxw8 in old cerebellum. Fbxw8 is encoded next to the Atxn2 locus and was shown in vitro to decrease the level of expanded insoluble ATXN2 protein. In conclusion, our data support the concept that expanded ATXN2 undergoes progressive insolubility and affects PABPC1 by a toxic gain-of-function mechanism with tissue-specific effects, which may be partially alleviated by the induction of FBXW8. Factorial design comparing Atxn2 knock-in mice with wild type littermates in three different tissues (brainstem, cerebellum, liver)

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Gene expression profiling of HOXB4-transduced murine hematopoietic stem cells

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