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Hhex regulates HSC self-renewal and stress hematopoiesis via repression of Cdkn2a

ABSTRACT: The Hematopoietically-expressed homeobox transcription factor (Hhex) is important for the maturation of definitive hematopoietic progenitors and B-cells during development. We have recently shown that in adult hematopoiesis, Hhex is dispensable for maintenance of hematopoietic stem cells (HSCs) and myeloid lineages but essential for the commitment of Common Lymphoid Progenitors (CLPs) to lymphoid lineages. However, whether Hhex plays a role in HSC self-renewal and myeloid expansion during hematopoietic stress is unknown. Here we show that during serial bone marrow transplantation, Hhex-deleted HSCs are progressively lost, revealing an intrinsic defect in HSC self-renewal. Moreover, Hhex-deleted mice show markedly impaired hematopoietic recovery following myeloablation. In vitro, Hhex-null blast colonies were incapable of replating, implying a specific requirement for Hhex in immature hematopoietic progenitors. Transcriptome analysis of Hhex-null Lin-Sca+Kit+ (LSK) cells showed that Hhex deletion leads to the deregulation of Polycomb Repressive Complex 2 (PRC2) target genes, including an upregulation of Cdkn2a locus, encoding the cell cycle repressors p16Ink4a and p19Arf. Indeed, loss of Cdkn2a restored Hhex-null blast colony replating in vitro, as well as hematopoietic reconstitution following myeloablation in vivo. Thus, HSCs require Hhex to repress Cdkn2a to enable continued self-renewal and response to hematopoietic stress.

ORGANISM(S): Mus musculus

PROVIDER: GSE86209 | GEO | 2017/05/22

SECONDARY ACCESSION(S): PRJNA340892

REPOSITORIES: GEO

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Project description:MiRNAs have the potential to regulate cellular differentiation programs. However, miRNA-deficiency in primary hematopoietic stem cells (HSCs) results in HSC depletion in mice, leaving the question of whether miRNAs play a role in early-lineage decisions unanswered. To address this issue, we deleted Dicer1, which encodes an essential RNaseIII enzyme for miRNA biogenesis, in murine CCAAT/enhancer-binding protein alpha (C/EBPA)-positive myeloid-committed progenitors in vivo. In contrast to the results in HSCs, we found that miRNA depletion affected neither the number of myeloid progenitors nor the percentage of C/EBPA-positive progenitor cells. Analysis of gene-expression profiles from wild type and Dicer1-deficient granulocyte-macrophage progenitors (GMPs) revealed that 20 miRNA families were active in GMPs. Of the derepressed miRNA targets in Dicer1-null GMPs, 27% are normally exclusively expressed in HSCs or are specific for multi-potent progenitors and erythropoiesis, indicating an altered gene-expression landscape. Dicer1-deficient GMPs were defective in myeloid development in vitro and exhibited an increased replating capacity, indicating a regained self-renewal potential of these cells. In mice, Dicer1 deletion blocked monocytic differentiation, depleted macrophages and caused myeloid dysplasia with morphological features of Pelger-Huët anomaly. These results provide evidence for a miRNA-controlled switch for a cellular program of self-renewal and expansion towards myeloid differentiation in GMPs. To generate Cebpa-Cre;R26-LSL-Eyfp;Dicer1wt/fl/Dicer1fl/fl mice, we crossed mice that contain floxed Dicer1 alleles (Dicer1fl) with Cebpa-Cre;R26-LSL-Eyfp reporter mice 2. Fetal livers were obtained on embryonic day (E) 13.5. Routine genotyping of Dicer1; Cebpa-Cre;R26-LSL-Eyfp mice was performed by PCR assays of DNA from tail or toe biopsies. For transplantation, 6 to 8-week-old recipient mice (C57Bl/6, Jackson Laboratories) were irradiated (8.5 Gy) and tail-vein injected with fetal liver single-cell suspensions. Typically, cells from each fetal liver were transplanted into two recipient mice. Hematopoietic tissues were analyzed 6-10 weeks post transplantation. EYFP positive GMPs from the bone marrow of Dicer wt control (n=3), Dicer -/wt (n=3 and Dicer fl/fl (n=3) were sorted and analyzed for gene expression profiles.

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Hhex regulates HSC self-renewal and stress hematopoiesis via repression of Cdkn2a

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