Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of hematopoietic stem cells in the preleukemic phase from wiltype mice and mice deficient in transcription factor PU.1 to identify transcriptional changes preceding malignant transformation

ABSTRACT: Knockdown of the transcription factor PU.1 (Spi1) leads to acute myeloid leukemia (AML) in mice. We examined the transcriptome of PU.1 knockdown hematopoietic stem cells (HSC) in the preleukemic phase by linear amplification and genome-wide array analysis to identify transcriptional changes preceding malignant transformation. Hierarchical cluster analysis and principal component analysis clearly distinguished PU.1 knockdown from wildtype HSC. Jun family transcription factors c-Jun and JunB were among the top downregulated targets. Retroviral restoration of c-Jun expression in bone marrow cells of preleukemic mice partially rescued the PU.1-initiated myelomonocytic differentiation block. Lentiviral restoration of JunB at the leukemic stage led to reduced clonogenic growth, loss of leukemic self-renewal capacity, and prevented leukemia in transplanted NOD-SCID mice. Examination of 305 AML patients confirmed the correlation between PU.1 and JunB downregulation and suggests its relevance in human disease. These results delineate a transcriptional pattern that precedes the leukemic transformation in PU.1 knockdown HSC and demonstrate that decreased levels of c-Jun and JunB contribute to the development of PU.1-induced AML by blocking differentiation (c-Jun) and increasing self-renewal (JunB). Therefore, examination of disturbed gene expression in HSC can identify genes whose dysregulation is essential for leukemic stem cell function and are targets for therapeutic interventions. Experiment Overall Design: Total RNA of HSC of PU.1 knockdown or wildtype animals (3 pools of 3 animals each) was amplified by two rounds of RT and T7 promoter-based in-vitro transcription and the resultant biotinylated cRNA was then hybridized to Affymetrix Mouse Genome 430 2.0 arrays.

ORGANISM(S): Mus musculus

SUBMITTER: Ulrich Steidl

PROVIDER: E-GEOD-5654 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Essential role of Jun family transcription factors in PU.1 knockdown-induced leukemic stem cells.

Steidl Ulrich U Rosenbauer Frank F Verhaak Roel G W RG Gu Xuesong X Ebralidze Alexander A Otu Hasan H HH Klippel Steffen S Steidl Christian C Bruns Ingmar I Costa Daniel B DB Wagner Katharina K Aivado Manuel M Kobbe Guido G Valk Peter J M PJ Passegué Emmanuelle E Libermann Towia A TA Delwel Ruud R Tenen Daniel G DG

Nature genetics 20061015 11

Knockdown of the transcription factor PU.1 (encoded by Sfpi1) leads to acute myeloid leukemia (AML) in mice. We examined the transcriptome of preleukemic hematopoietic stem cells (HSCs) in which PU.1 was knocked down (referred to as 'PU.1-knockdown HSCs') to identify transcriptional changes preceding malignant transformation. Transcription factors c-Jun and JunB were among the top-downregulated targets. Restoration of c-Jun expression in preleukemic cells rescued the PU.1 knockdown-initiated mye ...[more]

PMID: 17041602

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Project description:To guarantee blood supply throughout adult life hematopoietic stem cells (HSCs) need to carefully balance between self-renewing cell divisions and quiescence. Identification of genes controlling HSC self-renewal is of utmost importance given that HSCs are the only stem cells with broad clinical applications. Transcription factor PU.1 is one of the major regulators of myeloid and lymphoid development. Recent reports suggest that PU.1 mediates its functions via gradual expression level changes rather than binary on/off states. So far, this has not been considered in any study of HSCs and thus, PU.1’s role in HSC function has remained largely unclear. Here we demonstrate using hypomorphic mice with an engineered disruption of an autoregulatory feedback loop that decreased PU.1 levels resulted in loss of key HSC functions, all of which could be fully rescued by restoration of proper PU.1 levels via a human PU.1 transgene. Mechanistically, we found excessive HSC cell divisions and altered expression of cell cycle regulators whose promoter regions were bound by PU.1 in normal HSCs. Adequate PU.1 levels were maintained by a mechanism of direct autoregulation restricted to HSCs through a physical interaction of a -14kb enhancer with the proximal promoter. Our findings identify PU.1 as novel regulator controling the switch between cell division and quiescence in order to prevent exhaustion of HSCs. Given that even moderate level changes greatly impact stem cell function, our data suggest important therapeutic implications for leukemic patients with reduced PU.1 levels. Moreover, we provide first proof, that autoregulation of a transcription factor, PU.1, has a crucial function in vivo. We anticipate that our concept of how autoregulation forms an active chromosomal conformation will impact future research on transcription factor networks regulating stem cell fate. HSCs of Pu.1 knock-in (PU.1ki/ki) mice were used for RNA extraction and hybridization on Affymetrix microarrays. We compared these microarray samples with the corresponding wild type.

Dataset Information

Transcription profiling of hematopoietic stem cells in the preleukemic phase from wiltype mice and mice deficient in transcription factor PU.1 to identify transcriptional changes preceding malignant transformation

Publications

Essential role of Jun family transcription factors in PU.1 knockdown-induced leukemic stem cells.

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