The histone demethylase KDM5C functions as a tumor suppressor in AML by repression of bivalently marked immature genes.

Epigenetic regulators are frequently mutated in hematological malignancies including acute myeloid leukemia (AML). Thus, the identification and characterization of novel epigenetic drivers affecting AML biology holds potential to improve our basic understanding of AML and to uncover novel options for therapeutic intervention. To identify novel tumor suppressive epigenetic regulators in AML, we performed an in vivo short hairpin RNA (shRNA) screen in the context of CEBPA mutant AML. This identified the Histone 3 Lysine 4 (H3K4) demethylase KDM5C as a tumor suppressor, and we show that reduced Kdm5c/KDM5C expression results in accelerated growth both in human and murine AML cell lines, as well as in vivo in Cebpa mutant and inv(16) AML mouse models. Mechanistically, we show that KDM5C act as a transcriptional repressor through its demethylase activity at promoters. Specifically, KDM5C knockdown results in globally increased H3K4me3 levels associated with up-regulation of bivalently marked immature genes. This is accompanied by a de-differentiation phenotype that could be reversed by modulating levels of several direct and indirect downstream mediators. Finally, the association of KDM5C levels with long-term disease-free survival of female AML patients emphasizes the clinical relevance of our findings and identifies KDM5C as a novel female-biased tumor suppressor in AML.

The chromatin-binding protein Phf6 restricts the self-renewal of hematopoietic stem cells.

Recurrent inactivating mutations have been identified in the X-linked plant homeodomain finger protein 6 (PHF6) gene, encoding a chromatin-binding transcriptional regulator protein, in various hematological malignancies. However, the role of PHF6 in normal hematopoiesis and its tumor-suppressor function remain largely unknown. We herein generated mice carrying a floxed Phf6 allele and inactivated Phf6 in hematopoietic cells at various developmental stages. The Phf6 deletion in embryos augmented the capacity of hematopoietic stem cells (HSCs) to proliferate in cultures and reconstitute hematopoiesis in recipient mice. The Phf6 deletion in neonates and adults revealed that cycling HSCs readily acquired an advantage in competitive repopulation upon the Phf6 deletion, whereas dormant HSCs only did so after serial transplantations. Phf6-deficient HSCs maintained an enhanced repopulating capacity during serial transplantations; however, they did not induce any hematological malignancies. Mechanistically, Phf6 directly and indirectly activated downstream effectors in tumor necrosis factor α (TNFα) signaling. The Phf6 deletion repressed the expression of a set of genes associated with TNFα signaling, thereby conferring resistance against the TNFα-mediated growth inhibition on HSCs. Collectively, these results not only define Phf6 as a novel negative regulator of HSC self-renewal, implicating inactivating PHF6 mutations in the pathogenesis of hematological malignancies, but also indicate that a Phf6 deficiency alone is not sufficient to induce hematopoietic transformation.

A novel prospective isolation of murine fetal liver progenitors to study in utero hematopoietic defects.

In recent years, highly detailed characterization of adult bone marrow (BM) myeloid progenitors has been achieved and, as a result, the impact of somatic defects on different hematopoietic lineage fate decisions can be precisely determined. Fetal liver (FL) hematopoietic progenitor cells (HPCs) are poorly characterized in comparison, potentially hindering the study of the impact of genetic alterations on midgestation hematopoiesis. Numerous disorders, for example infant acute leukemias, have in utero origins and their study would therefore benefit from the ability to isolate highly purified progenitor subsets. We previously demonstrated that a Runx1 distal promoter (P1)-GFP::proximal promoter (P2)-hCD4 dual-reporter mouse (Mus musculus) model can be used to identify adult BM progenitor subsets with distinct lineage preferences. In this study, we undertook the characterization of the expression of Runx1-P1-GFP and P2-hCD4 in FL. Expression of P2-hCD4 in the FL immunophenotypic Megakaryocyte-Erythroid Progenitor (MEP) and Common Myeloid Progenitor (CMP) compartments corresponded to increased granulocytic/monocytic/megakaryocytic and decreased erythroid specification. Moreover, Runx1-P2-hCD4 expression correlated with several endogenous cell surface markers' expression, including CD31 and CD45, providing a new strategy for prospective identification of highly purified fetal myeloid progenitors in transgenic mouse models. We utilized this methodology to compare the impact of the deletion of either total RUNX1 or RUNX1C alone and to determine the fetal HPCs lineages most substantially affected. This new prospective identification of FL progenitors therefore raises the prospect of identifying the underlying gene networks responsible with greater precision than previously possible.

Mouse RUNX1C regulates premegakaryocytic/erythroid output and maintains survival of megakaryocyte progenitors.

RUNX1 is crucial for the regulation of megakaryocyte specification, maturation, and thrombopoiesis. Runx1 possesses 2 promoters: the distal P1 and proximal P2 promoters. The major protein isoforms generated by P1 and P2 are RUNX1C and RUNX1B, respectively, which differ solely in their N-terminal amino acid sequences. RUNX1C is the most abundantly expressed isoform in adult hematopoiesis, present in all RUNX1-expressing populations, including the cKit+ hematopoietic stem and progenitor cells. RUNX1B expression is more restricted, being highly expressed in the megakaryocyte lineage but downregulated during erythropoiesis. We generated a Runx1 P1 knock-in of RUNX1B, termed P1-MRIPV This mouse line lacks RUNX1C expression but has normal total RUNX1 levels, solely comprising RUNX1B. Using this mouse line, we establish a specific requirement for the P1-RUNX1C isoform in megakaryopoiesis, which cannot be entirely compensated for by RUNX1B overexpression. P1 knock-in megakaryocyte progenitors have reduced proliferative capacity and undergo increased cell death, resulting in thrombocytopenia. P1 knock-in premegakaryocyte/erythroid progenitors demonstrate an erythroid-specification bias, evident from increased erythroid colony-forming ability and decreased megakaryocyte output. At a transcriptional level, multiple erythroid-specific genes are upregulated and megakaryocyte-specific transcripts are downregulated. In addition, proapoptotic pathways are activated in P1 knock-in premegakaryocyte/erythroid progenitors, presumably accounting for the increased cell death in the megakaryocyte progenitor compartment. Unlike in the conditional adult Runx1 null models, megakaryocytic maturation is not affected in the P1 knock-in mice, suggesting that RUNX1B can regulate endomitosis and thrombopoiesis. Therefore, despite the high degree of structural similarity, RUNX1B and RUNX1C isoforms have distinct and specific roles in adult megakaryopoiesis.

Correction: RUNX1B Expression Is Highly Heterogeneous and Distinguishes Megakaryocytic and Erythroid Lineage Fate in Adult Mouse Hematopoiesis.

[This corrects the article DOI: 10.1371/journal.pgen.1005814.].

RUNX1B Expression Is Highly Heterogeneous and Distinguishes Megakaryocytic and Erythroid Lineage Fate in Adult Mouse Hematopoiesis.

The Core Binding Factor (CBF) protein RUNX1 is a master regulator of definitive hematopoiesis, crucial for hematopoietic stem cell (HSC) emergence during ontogeny. RUNX1 also plays vital roles in adult mice, in regulating the correct specification of numerous blood lineages. Akin to the other mammalian Runx genes, Runx1 has two promoters P1 (distal) and P2 (proximal) which generate distinct protein isoforms. The activities and specific relevance of these two promoters in adult hematopoiesis remain to be fully elucidated. Utilizing a dual reporter mouse model we demonstrate that the distal P1 promoter is broadly active in adult hematopoietic stem and progenitor cell (HSPC) populations. By contrast the activity of the proximal P2 promoter is more restricted and its upregulation, in both the immature Lineage- Sca1high cKithigh (LSK) and bipotential Pre-Megakaryocytic/Erythroid Progenitor (PreMegE) populations, coincides with a loss of erythroid (Ery) specification. Accordingly the PreMegE population can be prospectively separated into "pro-erythroid" and "pro-megakaryocyte" populations based on Runx1 P2 activity. Comparative gene expression analyses between Runx1 P2+ and P2- populations indicated that levels of CD34 expression could substitute for P2 activity to distinguish these two cell populations in wild type (WT) bone marrow (BM). Prospective isolation of these two populations will enable the further investigation of molecular mechanisms involved in megakaryocytic/erythroid (Mk/Ery) cell fate decisions. Having characterized the extensive activity of P1, we utilized a P1-GFP homozygous mouse model to analyze the impact of the complete absence of Runx1 P1 expression in adult mice and observed strong defects in the T cell lineage. Finally, we investigated how the leukemic fusion protein AML1-ETO9a might influence Runx1 promoter usage. Short-term AML1-ETO9a induction in BM resulted in preferential P2 upregulation, suggesting its expression may be important to establish a pre-leukemic environment.

SWI/SNF Subunits SMARCA4, SMARCD2 and DPF2 Collaborate in MLL-Rearranged Leukaemia Maintenance.

Alterations in chromatin structure caused by deregulated epigenetic mechanisms collaborate with underlying genetic lesions to promote cancer. SMARCA4/BRG1, a core component of the SWI/SNF ATP-dependent chromatin-remodelling complex, has been implicated by its mutational spectrum as exerting a tumour-suppressor function in many solid tumours; recently however, it has been reported to sustain leukaemogenic transformation in MLL-rearranged leukaemia in mice. Here we further explore the role of SMARCA4 and the two SWI/SNF subunits SMARCD2/BAF60B and DPF2/BAF45D in leukaemia. We observed the selective requirement for these proteins for leukaemic cell expansion and self-renewal in-vitro as well as in leukaemia. Gene expression profiling in human cells of each of these three factors suggests that they have overlapping functions in leukaemia. The gene expression changes induced by loss of the three proteins demonstrate that they are required for the expression of haematopoietic stem cell associated genes but in contrast to previous results obtained in mouse cells, the three proteins are not required for the expression of c-MYC regulated genes.

The histone demethylase Jarid1b is required for hematopoietic stem cell self-renewal in mice.

Jarid1b/KDM5b is a histone demethylase that regulates self-renewal and differentiation in stem cells and cancer; however, its function in hematopoiesis is unclear. Here, we find that Jarid1b is highly expressed in primitive hematopoietic compartments and is overexpressed in acute myeloid leukemias. Constitutive genetic deletion of Jarid1b did not impact steady-state hematopoiesis. In contrast, acute deletion of Jarid1b from bone marrow increased peripheral blood T cells and, following secondary transplantation, resulted in loss of bone marrow reconstitution. Our results reveal that deletion of Jarid1b compromises hematopoietic stem cell (HSC) self-renewal capacity and suggest that Jarid1b is a positive regulator of HSC potential.

shRNA screening identifies JMJD1C as being required for leukemia maintenance.

Epigenetic regulatory mechanisms are implicated in the pathogenesis of acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). Recent progress suggests that proteins involved in epigenetic control are amenable to drug intervention, but little is known about the cancer-specific dependency on epigenetic regulators for cell survival and proliferation. We used a mouse model of human AML induced by the MLL-AF9 fusion oncogene and an epigenetic short hairpin RNA (shRNA) library to screen for novel potential drug targets. As a counter-screen for general toxicity of shRNAs, we used normal mouse bone marrow cells. One of the best candidate drug targets identified in these screens was Jmjd1c. Depletion of Jmjd1c impairs growth and colony formation of mouse MLL-AF9 cells in vitro as well as establishment of leukemia after transplantation. Depletion of JMJD1C impairs expansion and colony formation of human leukemic cell lines, with the strongest effect observed in the MLL-rearranged ALL cell line SEM. In both mouse and human leukemic cells, the growth defect upon JMJD1C depletion appears to be primarily due to increased apoptosis, which implicates JMJD1C as a potential therapeutic target in leukemia.

Hemogenic endothelium--ontogenesis and role in blood production.

Endothelial and hematopoietic lineages have long been thought to develop from a common ancestor, the hemangioblast. Alternatively, clusters of hematopoietic cells in the dorsal aorta were observed to form in a close association with endothelial wall of the aorta, leading to the hypothesis that a special subset of endothelial cells, called the hemogenic endothelium, generates hematopoietic cells. Recent advances in time-lapse imaging, conditional labeling of cells in vivo and embryonic stem cell differentiation provided new evidence for the existence of both, the hemangioblast and hemogenic endothelium. Importantly, these seemingly contradictory theories can be merged into one model of hematopoietic differentiation from mesoderm.

Blood cell generation from the hemangioblast.

Understanding how blood cells are generated is important from a biological perspective but also has potential implications in the treatment of blood diseases. Such knowledge could potentially lead to defining new conditions to amplify hematopoietic stem cells (HSCs) or could translate into new methods to produce HSCs, or other types of blood cells, from human embryonic stem cells or induced pluripotent stem cells. Additionally, as most key transcription factors regulating early hematopoietic development have also been implicated in various types of leukemia, understanding their function during normal development could result in a better comprehension of their roles during abnormal hematopoiesis in leukemia. In this review, we discuss our current understanding of the molecular and cellular mechanisms of blood development from the earliest hematopoietic precursor, the hemangioblast, a precursor for both endothelial and hematopoietic cell lineages.

The differential activities of Runx1 promoters define milestones during embryonic hematopoiesis.

The transcription factor RUNX1/AML1 is a master regulator of hematopoietic development. Its spatiotemporal expression is tightly regulated during embryonic development and is under the control of 2 alternative promoters, distal and proximal. Despite the functional significance of Runx1, the relative and specific activities of these 2 promoters remain largely uncharacterized. To investigate these activities, we introduced 2 reporter genes under the control of the proximal and distal promoters in embryonic stem cell and transgenic mouse lines. Our study reveals that both in vitro and in vivo the proximal Runx1 isoform marks a hemogenic endothelium cell population, whereas the subsequent expression of distal Runx1 defines fully committed definitive hematopoietic progenitors. Interestingly, hematopoietic commitment in distal Runx1 knockout embryos appears normal. Altogether, our data demonstrate that the differential activities of the 2 Runx1 promoters define milestones of hematopoietic development and suggest that the proximal isoform plays a critical role in the generation of hematopoietic cells from hemogenic endothelium. Identification and access to the discrete stages of hematopoietic development defined by the activities of the Runx1 promoters will provide the opportunity to further explore the cellular and molecular mechanisms of hematopoietic development.

Heme oxygenase-1 accelerates cutaneous wound healing in mice.

Heme oxygenase-1 (HO-1), a cytoprotective, pro-angiogenic and anti-inflammatory enzyme, is strongly induced in injured tissues. Our aim was to clarify its role in cutaneous wound healing. In wild type mice, maximal expression of HO-1 in the skin was observed on the 2(nd) and 3(rd) days after wounding. Inhibition of HO-1 by tin protoporphyrin-IX resulted in retardation of wound closure. Healing was also delayed in HO-1 deficient mice, where lack of HO-1 could lead to complete suppression of reepithelialization and to formation of extensive skin lesions, accompanied by impaired neovascularization. Experiments performed in transgenic mice bearing HO-1 under control of keratin 14 promoter showed that increased level of HO-1 in keratinocytes is enough to improve the neovascularization and hasten the closure of wounds. Importantly, induction of HO-1 in wounded skin was relatively weak and delayed in diabetic (db/db) mice, in which also angiogenesis and wound closure were impaired. In such animals local delivery of HO-1 transgene using adenoviral vectors accelerated the wound healing and increased the vascularization. In summary, induction of HO-1 is necessary for efficient wound closure and neovascularization. Impaired wound healing in diabetic mice may be associated with delayed HO-1 upregulation and can be improved by HO-1 gene transfer.

In vitro differentiation of mouse embryonic stem cells as a model of early hematopoietic development.

Embryonic Stem (ES) are pluripotent cells derived from the inner cell mass of blastocysts. ES cells differentiate in vitro into all kind of cells and the development of endothelial and hematopoietic cells from mouse ES cells has been especially established. As such, the in vitro differentiation of ES cells provides a powerful experimental model to study and determine the role of specific genes in the development of the hematopoietic system. Using this approach we have demonstrated the critical function of the transcription factor AML1/Runx1 at the onset of hematopoietic development (Blood 100:458-466, 2002; Blood 103:886-889, 2004). In this chapter, we will describe our protocols and methods for the culture of healthy ES cells, their effective differentiation toward hematopoiesis, and the quantitative analysis of their hematopoietic potential by replating or gene expression analyses.

The haemangioblast generates haematopoietic cells through a haemogenic endothelium stage.

It has been proposed that during embryonic development haematopoietic cells arise from a mesodermal progenitor with both endothelial and haematopoietic potential called the haemangioblast. A conflicting theory instead associates the first haematopoietic cells with a phenotypically differentiated endothelial cell that has haematopoietic potential (that is, a haemogenic endothelium). Support for the haemangioblast concept was initially provided by the identification during mouse embryonic stem cell differentiation of a clonal precursor, the blast colony-forming cell (BL-CFC), which gives rise to blast colonies with both endothelial and haematopoietic components. Although recent studies have now provided evidence for the presence of this bipotential precursor in vivo, the precise mechanism for generation of haematopoietic cells from the haemangioblast still remains completely unknown. Here we demonstrate that the haemangioblast generates haematopoietic cells through the formation of a haemogenic endothelium intermediate, providing the first direct link between these two precursor populations. The cell population containing the haemogenic endothelium is transiently generated during BL-CFC development. This cell population is also present in gastrulating mouse embryos and generates haematopoietic cells on further culture. At the molecular level, we demonstrate that the transcription factor Tal1 (also known as Scl; ref. 10) is indispensable for the establishment of this haemogenic endothelium population whereas the core binding factor Runx1 (also known as AML1; ref. 11) is critical for generation of definitive haematopoietic cells from haemogenic endothelium. Together our results merge the two a priori conflicting theories on the origin of haematopoietic development into a single linear developmental process.

The stepwise specification of embryonic stem cells to hematopoietic fate is driven by sequential exposure to Bmp4, activin A, bFGF and VEGF.

The differentiation of embryonic stem (ES) cells offers a powerful approach to study mechanisms implicated in cell fate decision. A major hurdle, however, is to promote the directed and efficient differentiation of ES cells toward a specific lineage. Here, we define in serum-free media the minimal factor requirement controlling each step of the differentiation process, resulting in the production of highly enriched hematopoietic progenitors. Four factors - Bmp4, activin A, bFGF (Fgf2) and VEGF (VegfA) - are sufficient to drive the selective and efficient differentiation of mouse ES cells to hematopoiesis. Each of these factors appears to regulate a step of the process: Bmp4 promotes the very efficient formation of mesoderm; bFGF and activin A induce the differentiation of these mesodermal precursors to the hemangioblast fate; and VEGF is required for the production of fully committed hematopoietic progenitors. The stimulation of mesodermal precursors by bFGF and activin A switches on very rapidly the hematopoietic program, allowing us to dissect the molecular events leading to the formation of the hemangioblast. Runx1, Scl (Tal1) and Hhex expression is upregulated within 3 hours of stimulation, whereas upregulation of Lmo2 and Fli1 is observed later. Interestingly, increased expression levels of genes such as cMyb, Pu.1 (Sfpi1), Gata1 and Gata2 are not observed at the onset of hemangioblast commitment. This stepwise control of differentiation is extremely efficient, giving rise to a very high frequency of hematopoietic precursors, and provides an optimal system for understanding the molecular machineries involved in blood progenitor commitment.

Effect of heme and heme oxygenase-1 on vascular endothelial growth factor synthesis and angiogenic potency of human keratinocytes.

BACKGROUND: Skin injury leads to the release of heme, a potent prooxidant which is degraded by heme oxygenase-1 (HO-1) to carbon monoxide, iron, and biliverdin, subsequently reduced to bilirubin. Recently the involvement of HO-1 in angiogenesis has been shown; however, the role of heme and HO-1 in wound healing angiogenesis has not been yet investigated. RESULTS: Treatment of HaCaT keratinocytes with hemin (heme chloride) induced HO-1 expression and activity. The effect of heme on vascular endothelial growth factor (VEGF) synthesis is variable: induction is significant after a short, 6 h treatment with heme, while longer stimulation may attenuate its production. The involvement of HO-1 in VEGF synthesis was confirmed by inhibition of VEGF expression by SnPPIX, a blocker of HO activity and by attenuation of HO-1 mRNA expression with specific siRNA. Importantly, induction of HO-1 by hemin was able to overcome the inhibitory effect of high glucose on VEGF synthesis. Moreover, HO-1 expression was also induced in keratinocytes cultured in hypoxia, with concomitant augmentation of VEGF production, which was further potentiated by hemin stimulation. Accordingly, conditioned media from keratinocytes overexpressing HO-1 enhanced endothelial cell proliferation and augmented formation of capillaries in angiogenic assay in vitro. CONCLUSIONS: HO-1 is involved in hemin-induced VEGF expression in HaCaT and may play a role in hypoxic regulation of this protein. HO-1 overexpression may be beneficial in restoring the proper synthesis of VEGF disturbed in diabetic conditions.