Chinavenmeni S. Velu

Gfi1 regulates miR-21 and miR-196b to control myelopoiesis.

The zinc finger protein growth factor independent-1 (Gfi1) is a transcriptional repressor that is critically required for normal granulocytic differentiation. GFI1 loss-of-function mutations are found in some patients with severe congenital neutropenia (SCN). The SCN-associated GFI1-mutant proteins act as dominant negatives to block granulopoiesis through selective deregulation of a subset of GFI1 target genes. Here we show that Gfi1 is a master regulator of microRNAs, and that deregulated expression of these microRNAs recapitulates a Gfi1 loss-of-function block to granulocyte colony-stimulating factor (G-CSF)-stimulated granulopoiesis. Specifically, bone marrow cells from a GFI1-mutant SCN patient and Gfi1(-/-) mice display deregulated expression of miR-21 and miR-196B expression. Flow cytometric analysis and colony assays reveal that the overexpression or depletion of either miR induces changes in myeloid development. However, coexpression of miR-21 and miR-196b (as seen in Gfi1(-/-) mice and a GFI1N382S SCN patient) completely blocks G-CSF-induced granulopoiesis. Thus, our results not only identify microRNAs whose regulation is required during myelopoiesis, but also provide an example of synergy in microRNA biologic activity and illustrate potential mechanisms underlying SCN disease pathogenesis.

MIR-23A microRNA cluster inhibits B-cell development

OBJECTIVE The transcription factor PU.1 (encoded by Sfpi1) promotes myeloid differentiation, but it is unclear what downstream genes are involved. Micro RNAs (miRNAs) are a class of small RNAs that regulate many cellular pathways, including proliferation, survival, and differentiation. The objective of this study was to identify miRNAs downstream of PU.1 that regulate hematopoietic development. MATERIALS AND METHODS miRNAs that change expression in a PU.1-inducible cell line were identified with microarrays. The promoter for an miRNA cluster upregulated by PU.1 induction was analyzed for PU.1 binding by electrophoretic mobility shift and chromatin immunoprecipitation assays. Retroviral transduction of hematopoietic progenitors was performed to evaluate the effect of miRNA expression on hematopoietic development in vitro and in vivo. RESULTS We identified an miRNA cluster whose pri-transcript is regulated by PU.1. The pri-miRNA encodes three mature miRNAs: miR-23a, miR-27a, and miR-24-2. Each miRNA is more abundant in myeloid cells compared to lymphoid cells. When hematopoietic progenitors expressing the 23a cluster miRNAs were cultured in B-cell-promoting conditions, we observed a dramatic decrease in B lymphopoiesis and an increase in myelopoiesis compared to control cultures. In vivo, hematopoietic progenitors expressing the miR-23a cluster generate reduced numbers of B cells compared to control cells. CONCLUSIONS The miR-23a cluster is a downstream target of PU.1 involved in antagonizing lymphoid cell fate acquisition. Although miRNAs have been identified downstream of PU.1 in mediating development of monocytes and granulocytes, the 23a cluster is the first downstream miRNA target implicated in regulating development of myeloid vs lymphoid cells.

Mutations in Growth Factor Independent-1 Associated with Human Neutropenia Block Murine Granulopoiesis through Colony Stimulating Factor-1

Severe congenital neutropenia (SCN) is characterized by a deficiency of mature neutrophils, leading to recurrent bacterial and fungal infections. Although mutations in Elastase-2, neutrophil (ELA2) predominate in human SCN, mutation of Ela2 in mice does not recapitulate SCN. The growth factor independent-1 (GFI1) transcription factor regulates ELA2. Mutations in GFI1 are associated with human SCN, and genetic deletion of Gfi1 results in murine neutropenia. We examined whether human SCN-associated GFI1N382S mutant proteins are causal in SCN and found that GFI1 functions as a rate-limiting granulopoietic molecular switch. The N382S mutation inhibited GFI1 DNA binding and resulted in a dominant-negative block to murine granulopoiesis. Moreover, Gfi1N382S selectively derepressed the monopoietic cytokine CSF1 and its receptor. Gfi1N382S-expressing Csf1-/- cells formed neutrophils. These results reveal a common transcriptional program that underlies both human and murine myelopoiesis, and that is central to the pathogenesis of SCN associated with mutations in GFI1. This shared transcriptional pathway may provide new avenues for understanding SCN caused by mutations in other genes and for clinical intervention into human neutropenias.

Gfi1 integrates progenitor versus granulocytic transcriptional programming

In patients with severe congenital neutropenia (SCN) and mice with growth factor independent-1 (Gfi1) loss of function, arrested myeloid progenitors accumulate, whereas terminal granulopoiesis is blocked. One might assume that Gfi-null progenitors accumulate because they lack the ability to differentiate. Instead, our data indicate that Gfi1 loss of function deregulates 2 separable transcriptional programs, one of which controls the accumulation and lineage specification of myeloid progenitors, but not terminal granulopoiesis. We demonstrate that Gfi1 directly represses HoxA9, Pbx1, and Meis1 during normal myelopoiesis. Gfi1-/- progenitors exhibit elevated levels of HoxA9, Pbx1 and Meis1, exaggerated HoxA9-Pbx1-Meis1 activity, and progenitor transformation in collaboration with oncogenic K-Ras. Limiting HoxA9 alleles corrects, in a dose-dependent manner, in vivo and in vitro phenotypes observed with loss of Gfi1 in myeloid progenitor cells but did not rescue Gfi1-/- blocked granulopoiesis. Thus, Gfi1 integrates 2 events during normal myeloid differentiation; the suppression of a HoxA9-Pbx1-Meis1 progenitor program and the induction of a granulopoietic transcription program.

Gfi1 expressed in bone marrow stromal cells is a novel osteoblast suppressor in patients with multiple myeloma bone disease

Protracted inhibition of osteoblast (OB) differentiation characterizes multiple myeloma (MM) bone disease and persists even when patients are in long-term remission. However, the underlying pathophysiology for this prolonged OB suppression is unknown. Therefore, we developed a mouse MM model in which the bone marrow stromal cells (BMSCs) remained unresponsive to OB differentiation signals after removal of MM cells. We found that BMSCs from both MM-bearing mice and MM patients had increased levels of the transcriptional repressor Gfi1 compared with controls and that Gfi1 was a novel transcriptional repressor of the critical OB transcription factor Runx2. Trichostatin-A blocked the effects of Gfi1, suggesting that it induces epigenetic changes in the Runx2 promoter. MM-BMSC cell-cell contact was not required for MM cells to increase Gfi1 and repress Runx2 levels in MC-4 before OBs or naive primary BMSCs, and Gfi1 induction was blocked by anti-TNF-α and anti-IL-7 antibodies. Importantly, BMSCs isolated from Gfi1(-/-) mice were significantly resistant to MM-induced OB suppression. Strikingly, siRNA knockdown of Gfi1 in BMSCs from MM patients significantly restored expression of Runx2 and OB differentiation markers. Thus, Gfi1 may have an important role in prolonged MM-induced OB suppression and provide a new therapeutic target for MM bone disease.

The 3′ Region of the Chicken Hypersensitive Site-4 Insulator Has Properties Similar to Its Core and Is Required for Full Insulator Activity

Chromatin insulators separate active transcriptional domains and block the spread of heterochromatin in the genome. Studies on the chicken hypersensitive site-4 (cHS4) element, a prototypic insulator, have identified CTCF and USF-1/2 motifs in the proximal 250 bp of cHS4, termed the “core”, which provide enhancer blocking activity and reduce position effects. However, the core alone does not insulate viral vectors effectively. The full-length cHS4 has excellent insulating properties, but its large size severely compromises vector titers. We performed a structure-function analysis of cHS4 flanking lentivirus-vectors and analyzed transgene expression in the clonal progeny of hematopoietic stem cells and epigenetic changes in cHS4 and the transgene promoter. We found that the core only reduced the clonal variegation in expression. Unique insulator activity resided in the distal 400 bp cHS4 sequences, which when combined with the core, restored full insulator activity and open chromatin marks over the transgene promoter and the insulator. These data consolidate the known insulating activity of the canonical 5′ core with a novel 3′ 400 bp element with properties similar to the core. Together, they have excellent insulating properties and viral titers. Our data have important implications in understanding the molecular basis of insulator function and design of gene therapy vectors.

Rho GTPase Cdc42 is essential for B lymphocyte development and activation

Cdc42 is a member of the Rho GTPase family that has been implicated in several cell functions including proliferation and migration, but its physiologic role needs to be dissected in each cell type. We achieved B-cell and hematopoietic stem cell deletion of Cdc42 by conditional gene targeting in mice. Deletion of Cdc42 from proB/preB-cell stage significantly blocked B-cell development at T1 and later stages, resulting in reduced mature B-cell populations and reduced antigen-specific immunoglobulin M (IgM), IgG1, and IgG3 production. The Cdc42(-/-) B cells, themselves, were abnormal with impaired proliferation and survival. The mutant B cells were further characterized by a B-cell receptor (BCR) signaling defect with increased Erk and decreased Akt activation, as well as a defect in BCR-mediated B-cell-activating factor (BAFF) receptor up-regulation and subsequent BAFF receptor signaling in mature resting B cells. Surprisingly, Cdc42 was dispensable for stromal cell-derived factor-1alpha (SDF-1alpha)- or B-lymphocyte chemoattractant (BLC)-induced B-cell migration. Finally, loss of Cdc42 from hematopoietic stem cells did not alter common lymphoid progenitor production but severely reduced proB/preB- and immature B-cell populations, indicating that Cdc42 is also involved in B-cell precursor differentiation. These results reveal multifaceted roles of Cdc42 in B-cell development and activation.

Ajuba functions as a histone deacetylase-dependent co-repressor for autoregulation of the growth factor-independent-1 transcription factor.

Growth factor independent-1 (Gfi1) is a zinc finger protein with a SNAG-transcriptional repressor domain. Ajuba is a LIM domain protein that shuttles between the cytoplasm and the nucleus. Ajuba functions as a co-repressor for synthetic Gfi1 SNAG-repressor domain-containing constructs, but a role for Ajuba co-repression of the cognate DNA bound Gfi1 protein has not been defined. Co-immunoprecipitation of synthetic and endogenous proteins and co-elution with gel filtration suggest that an endogenous Ajuba·Gfi1·HDAC multiprotein complex is possible. Active histone deacetylase activity co-immunoprecipitates with Ajuba or Gfi1, and both proteins depend upon histone deacetylases for full transcriptional repression activity. Ajuba LIM domains directly bind to Gfi1, but the association is not SNAG domain-dependent. ChIP analysis and reciprocal knockdown experiments suggest that Ajuba selectively functions as a co-repressor for Gfi1 autoregulation. The data suggest that Ajuba is utilized as a corepressor selectively on Gfi1 target genes.

Therapeutic antagonists of microRNAs deplete leukemia-initiating cell activity

Acute myelogenous leukemia (AML) subtypes that result from oncogenic activation of homeobox (HOX) transcription factors are associated with poor prognosis. The HOXA9 transcription activator and growth factor independent 1 (GFI1) transcriptional repressor compete for occupancy at DNA-binding sites for the regulation of common target genes. We exploited this HOXA9 versus GFI1 antagonism to identify the genes encoding microRNA-21 and microRNA-196b as transcriptional targets of HOX-based leukemia oncoproteins. Therapeutic inhibition of microRNA-21 and microRNA-196b inhibited in vitro leukemic colony forming activity and depleted in vivo leukemia-initiating cell activity of HOX-based leukemias, which led to leukemia-free survival in a murine AML model and delayed disease onset in xenograft models. These data establish microRNA as functional effectors of endogenous HOXA9 and HOX-based leukemia oncoproteins, provide a concise in vivo platform to test RNA therapeutics, and suggest therapeutic value for microRNA antagonists in AML.

Coordination of IL-7 receptor and T-cell receptor signaling by cell-division cycle 42 in T-cell homeostasis

T-cell homeostasis is essential for normal functioning of the immune system. IL-7 receptor (IL-7R) and T-cell receptor (TCR) signaling are pivotal for T-cell homeostatic regulation. The detailed mechanisms regulating T-cell homeostasis and how IL-7R and TCR signaling are coordinated are largely unknown. Here we demonstrate that T cell-specific deletion of cell-division cycle 42 (Cdc42) GTPase causes a profound loss of mature T cells. Deletion of Cdc42 leads to a markedly increased expression of growth factor independence-1 (Gfi-1) and represses expression of IL-7Rα. In the absence of Cdc42, aberrant ERK1/2 MAP kinase activity results in enhanced, TCR-mediated T-cell proliferation. In vivo reconstitution of effector-binding–defective Cdc42 mutants and the effector p21 protein-activated kinase 1 (PAK1) into Cdc42-deficient T cells showed that PAK1 is both necessary and sufficient for Cdc42-regulated T-cell homeostasis. Thus, T-cell homeostasis is maintained through a concerted regulation of Gfi-1–IL-7R–controlled cytokine responsiveness and ERK-mediated TCR signaling strength by the Cdc42-PAK1 signaling axis.