Characterization of Genome-Wide Binding of NUCLEAR-FACTOR I-X in Hematopoietic Progenitor Cells

We report that ectopic expression of Nfix in primary mouse HSPC extended their ex vivo culture from 20 to 40 days. HSPC overexpressing Nfix displayed hypersensitivity to supportive cytokines and reduced apoptosis when subjected to cytokine deprivation compared to controls. Ectopic Nfix resulted in elevated levels of c-Mpl transcripts and cell surface protein on primary murine HSPC as well as increased phosphorylation of STAT5, which is known to be activated down-stream of c-MPL signaling. Blocking c-MPL signaling by removal of its ligand, thrombopoietin (TPO), or addition of a c-MPL neutralizing antibody negated the anti-apoptotic effect of Nfix overexpression on cultured HSPC. Furthermore, NFIX-FLAG was capable of binding to and transcriptionally activating a proximal c-Mpl promoter fragment. In sum, these data suggest that NFIX-mediated up-regulation of c-Mpl transcription can protect primitive hematopoietic cells from stress ex vivo. Understanding the direct transcriptional targets or co-binding partners of NFIX would provide further insight into the mechanisms HSPC employ to maintain steady-state hematopoiesis or overcome stress hematopoiesis. To this end, we combined global transcriptional profiling and genome-wide binding to identify direct transcriptional targets of NFIX in Nfix+/+ and Nfix-/- HPC5 cells, a primitive multi-potent hematopoietic cell line. We find that NFIX preferentially binds enhancer and promoter genomic regions. Integrative analysis revealed >500 differentially expressed genes of which 58% were direct NFIX targets. Many of these genes were downregulated in the absence of NFIX, indicating that NFIX functions primarily as a transcriptional activator in this context. PANTHER pathway analysis implicated NFIX in the regulation of apoptosis, myeloid cell differentiation and cell-cell adhesion. Using archived ChIP-seq data, we revealed significant co-localization of NFIX with other well-known hematopoietic transcription factors, including pSTAT1, RUNX1, RAD21, STAT3, ETO2, FLI1, GATA2, LYL1, LDB1 and PU.1. We showed NFIX and PU.1 together target genes regulating hematopoietic cell adhesion, cell death and differentiation in hematopoietic cells. Our data support a model in which NFIX collaborates with PU.1 to regulate differentiation and apoptosis in hematopoietic cells. In summary, we identified direct transcription targets and putative co-regulatory partners of NFIX. In sum, the work here further characterizes the complex role of the NFI family member, NFIX. We show minor perturbations in PB lineages when transplanting NfixΔ/Δ HSPC. During secondary transplants, NfixΔ/Δ donor chimerism was similar to controls. Also, during steady-state hematopoiesis, we did not observe any overt phenotypes in the NfixΔ/Δ mice. We discovered that overexpressing Nfix ex vivo imparts cells with hypersensitivity to cytokines and resistance to apoptosis. These characteristics were due to an increase in c-MPL signaling and could be abolished if this signaling was blocked. We identified c-Mpl as the first transcriptional target of NFIX in a hematopoietic context. Finally, we have rigorously characterized the genome-wide binding of NFIX in a hematopoietic cell line as well as identified 291 putative transcriptional targets. This work provides more data towards illuminating the role of an NFI family member during hematopoiesis.

Phosphorylation of RUNX1 by Cyclin-Dependent Kinase Reduces Direct Interaction with HDAC1 and HDAC3 and Stimulates Marrow Progenitor Proliferation.

Abstract 2508 Poster Board II-485 RUNX1 helps direct the formation of definitive HSC during embryogenesis and the subsequent maturation of the lymphoid, myeloid, and megakaryocytic lineages in adults. RUNX1 regulates both lineage-specific genes and G1 to S cell cycle progression, the latter in part via induction of cyclin D3 and cdk4 transcription. Recent studies demonstrate that RUNX1 homologs in sea urchin blastocyts or in C. elegans seam cells, a stem cell subset, also stimulate cell proliferation. Of note, RUNX1 activities are commonly perturbed in human AML or ALL cases. RUNX1 residues S48, S303, and S424 fit the consensus, (S/T)PX(R/K), for phosphorylation by cyclin dependent kinases (cdk). Mutation of these residues to the phosphomimetic aspartic acid increases RUNX1 trans-activation, and mutation to alanine reduces trans-activation, without perturbing p300 interaction (Zhang et al 2008). We now demonstrate that cdk phosphorylation of RUNX1 reduces its interaction with HDAC1 and HDAC3. First, co-immunoprecipitation of FLAG-tagged HDAC1 or HDAC3 with myc-tagged RUNX1, RUNX1(tripleA), with S48, S303, and S424 mutated to alanine, or RUNX1(tripleD), with these three residues mutated to aspartic acid, was assessed using extracts from transiently transfected 293T cells. Both HDAC1 and HDAC3 bound RUNX1(tripleD) with greatly reduced affinity compared to RUNX1 or its tripleA variant. Consistent with previous mapping of HDAC1 and HDAC3 interaction to the C-terminal residues 380–432 of RUNX1, when this same assay was repeated using RUNX1(S48D), RUNX1(S303D), or RUNX1(S424D), carrying single residue mutations to aspartic acid, only S424D reduced HDAC1 binding, and S424D had a greater effect than S48D or S303D on HDAC3 binding. Second, when GST-RUNX1, GST-RUNX1(424A), or GST-RUNX1(424D) fusion proteins isolated from bacteria were incubated with in vitro translated, S35-methionine labeled HDAC1 or HDAC3, reduced interaction was again seen to the 424D variant. Finally, incubation of wild-type GST-RUNX1 with cdk1/cyclinB led to reduced interaction with radio-labeled HDAC1 or HDAC3, indicating that cdk phosphorylation and not just change of cdk target residues to aspartic acid reduces HDAC binding to RUNX1. In addition, endogenous HDAC1 in the Ba/F3 hematopoietic cell line co-immunoprecipitated with exogenous RUNX1, and we are attempting to optimize methods to detect interaction between the endogenous proteins. We previously provided data suggesting that RUNX1 cdk phosphorylation stimulates proliferation of the Ba/F3 hematopoietic cell line (Zhang et al 2008). We now find that RUNX1 or RUNX1(tripleD) reproducibly stimulate proliferation of transduced, lineage-negative murine marrow progenitors more potently than RUNX1(tripleA). Together our findings indicate that stimulation of RUNX1 trans-activation activity by cdk-mediated reduction in direct HDAC1 and HDAC3 interaction facilitates hematopoietic progenitor cell proliferation. Future studies will endeavor to determine whether activation of cdk in HSC or myeloid progenitors, for example by Notch or Wnt signaling, stimulates RUNX1 cdk phosphorylation to facilitate induction of cell cycle regulatory genes and cell proliferation. Conversely, reduced RUNX1 cdk phosphorylation may facilitate HSC quiescence. Disclosures: No relevant conflicts of interest to declare.

Penumbra encodes a novel tetraspanin that is highly expressed in erythroid progenitors and promotes effective erythropoiesis

In a search for new genes involved in the regulation of erythropoiesis, we identified murine Penumbra cDNA from a multipotent hematopoietic cell line based on its predominant expression in erythroblasts. Subsequently, we identified the human PENUMBRA from a bone marrow cDNA library. Penumbra is a new member of the tetraspanin superfamily of membrane proteins, many of which are thought to function as organizers of supramolecular signaling complexes. Human and murine Penumbras contain 283 amino acids and are 97% identical. The human PENUMBRA gene is mapped to chromosome 7q32, a hot spot for deletions in myelodysplastic syndromes and acute myelogenous leukemias. Penumbra is targeted to the cell surface and forms disulfide-bonded homodimers. To study the effects of Penumbra deletions, we created a knockout mouse model by gene targeting. Penumbra−/− mice develop massive splenomegaly, basophilic macrocytic red blood cells, and anemia as they age. A multipotent hematopoietic cell line, EMX, was established from the bone marrow of a Penumbra−/− mouse. EMX exhibits ineffective erythropoiesis in the presence of erythropoietin, a defect that is reversed by reexpression of Penumbra. These findings indicate that Penumbra has a positive function in erythropoiesis and its deletion or mutation may result in anemia.