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HB9 induces senescence
confirming the HB9-dependent erythroid-biased differen- tiation observed in vivo. HBZ is an embryonic α-like globin gene, which is expressed in primitive erythroid cells. As hemoglobin is a tetramer of 2 α-like globin polypeptide chains and 2 β-like globin chains, also the embryonic β-like globin gene HBE1 was significantly up-regulated (4.6-fold; P=0.019).
SLC4A1 is part of the anion exchanger family and is expressed in the erythrocyte plasma membrane, where it functions as a chloride/bicarbonate exchanger.
Thus, HB9 expression in CD34+ HSPCs initiates de novo expression of genes related to erythropoiesis, which is in line with the differentiation bias towards the megakary- ocytic/erythroid cell lineage observed in vivo.
The computational gene set enrichment analysis soft- ware was used to statistically determine altered pathways related to HB9 expression associated with biological process-related gene ontology terms. Among the positive enriched biological processes, mitosis-related processes showed the strongest enrichment, while, in contrast, cytoskeleton organization-related processes showed the strongest negative enrichment (Online Supplementary Table S2 and Online Supplementary Figure S14). This phenotype
resembles our observations made in vitro, where HB9 led to cell cycle arrest and multinuclearity as a consequence of senescence-response.
With regard to clonogenic potential, HB9-transduced CD34+ HSPCs showed a decreased clonogenic capacity compared to GFP-transduced cells (Online Supplementary Figure S15), corresponding to reduced cellularity in bone marrow (Online Supplementary Figure S6A) and peripheral blood (Figure 5C) of B6[HB9] mice.
Discussion
The homeobox gene HB9 is expressed during early embryonic development, being essential for pancreatic as well as motor neuronal differentiation.7-11 Aberrant HB9 expression has been detected in distinct tumor entities; especially in translocation t(7;12) AML it is assumed to be a key factor in leukemic transformation.15,18 However, up to now only poor functional studies exist addressing its oncogenic potential or its influence on hematopoiesis. We evaluated the oncogenic potential of HB9 regarding its influence on proliferation and cell cycle using a model of
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Figure 7. De novo gene expression in human CD34+ HSPCs upon HB9 transduction. (A) qRT-PCR analysis of HB9 expression, normalized to β-Actin, in cord blood derived CD34+ cells, transduced with GFP (CD34[GFP]) or HB9-GFP (CD34[HB9]). (B) Gel electrophoretic analysis of the qRT-PCR products. (C) De novo expressed genes in CD34[HB9] compared to CD34[GFP]; normalized read counts of three independent RNA-Seq experiments are depicted.
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