Essential function for SAP family adaptors in the surveillance of hematopoietic cells by natural killer cells.

The adaptors SAP, EAT-2 and ERT are specific to cells of the immune system and belong to the SAP family. All three are expressed in natural killer (NK) cells. Here we examined the global function of the SAP family using mice lacking SAP, EAT-2 and ERT. These adaptors acted together in a mechanism that was essential for the elimination of hematopoietic but not nonhematopoietic cells by NK cells. This function was mediated by many receptors of the SLAM family on NK cells that were engaged by ligands found solely on hematopoietic cells. In the absence of SAP-related adaptors, SLAM receptors lost their activating function and became inhibitory receptors that repressed other activating receptors, such as NKG2D. Hence, the SAP family is essential for the elimination of unwanted hematopoietic cells by NK cells.

The human GFI136N variant induces epigenetic changes at the Hoxa9 locus and accelerates K-RAS driven myeloproliferative disorder in mice.

The coding single nucleotide polymorphism GFI136N in the human gene growth factor independence 1 (GFI1) is present in 3%-7% of whites and increases the risk for acute myeloid leukemia (AML) by 60%. We show here that GFI136N, in contrast to GFI136S, lacks the ability to bind to the Gfi1 target gene that encodes the leukemia-associated transcription factor Hoxa9 and fails to initiate histone modifications that regulate HoxA9 expression. Consistent with this, AML patients heterozygous for the GFI136N variant show increased HOXA9 expression compared with normal controls. Using ChipSeq, we demonstrate that GFI136N specific epigenetic changes are also present in other genes involved in the development of AML. Moreover, granulomonocytic progenitors, a bone marrow subset from which AML can arise in humans and mice, show a proliferative expansion in the presence of the GFI136N variant. In addition, granulomonocytic progenitors carrying the GFI136N variant allele have altered gene expression patterns and differ in their ability to grow after transplantation. Finally, GFI136N can accelerate a K-RAS driven fatal myeloproliferative disease in mice. Our data suggest that the presence of a GFI136N variant allele induces a preleukemic state in myeloid precursors by deregulating the expression of Hoxa9 and other AML-related genes.

The p150 subunit of the histone chaperone Caf-1 interacts with the transcriptional repressor Gfi1.

Modification of histones is critically involved in regulating chromatin structure and gene expression. The zinc finger protein Gfi1 silences transcription by recruiting a complex of histone modifying enzymes such as LSD-1/CoRest and HDAC-1 to target gene promoters. Here we present evidence that Gfi1 forms a complex with the p150 subunit of the histone chaperone chromatin assembly factor-1 (Caf-1). Gfi1 and p150 interact at endogenous expression levels and co-localize in distinct sub-nuclear structures. We show that p150 enhances Gfi1-mediated transcriptional repression and that it occupies Gfi1 target gene promoters in transfected cells and primary murine T cells only in the presence of Gfi1. Finally, size exclusion chromatography shows a fraction of p150 to coelute with Gfi1, LSD-1 and HDAC-1 and thus provides evidence that p150 is part of the Gfi1 repression complex. Since p150 binds directly to histones H3 and H4, our findings suggest that p150 may link the DNA-bound Gfi1 repressor complex to histones enabling modifications required for transcriptional silencing.

The X-Linked Helicase DDX3X Is Required for Lymphoid Differentiation and MYC-Driven Lymphomagenesis.

The X-linked gene DDX3X encodes an RNA helicase that is mutated at high frequencies in several types of human B-cell lymphoma. Females have two active DDX3X alleles and males carry a DDX3Y homolog on the Y chromosome. We show here that pan-hematopoietic, homozygous deletion of Ddx3x in female mice perturbs erythropoiesis, causing early developmental arrest. However, both hemizygous male and heterozygous female embryos develop normally, suggesting that one Ddx3x allele is sufficient for fetal hematopoietic development in females and that the Ddx3y allele can compensate for the loss of Ddx3x in males. In adult mice, DDX3X deficiency altered hematopoietic progenitors, early lymphoid development, marginal zone and germinal center B cells, and lymphomagenesis in a sex-dependent manner. Loss of both Ddx3x alleles abrogated MYC-driven lymphomagenesis in females, whereas Ddx3x deletion in males did not affect the formation of B-cell lymphoma in both mouse models. Moreover, tumors that appeared in male mice lacking DDX3X showed upregulated expression of DDX3Y, indicating a critical requirement for DDX3 activity for lymphomagenesis. These data reveal sex-specific roles of DDX3X in erythro- and lymphopoiesis as well as in MYC-driven lymphomagenesis. SIGNIFICANCE: The sex-dependent effects of DDX3X deficiency in malignant transformation of B cells and the compensatory role of DDX3Y support inhibition of DDX3 as a treatment strategy for MYC-driven B-cell lymphoma.

Regulation of natural cytotoxicity by the adaptor SAP and the Src-related kinase Fyn.

SAP is an adaptor protein that is expressed in NK and T cells. It is mutated in humans who have X-linked lymphoproliferative (XLP) disease. By interacting with SLAM family receptors, SAP enables tyrosine phosphorylation signaling of these receptors by its ability to recruit the Src-related kinase, Fyn. Here, we analyzed the role of SAP in NK cell functions using the SAP-deficient mouse model. Our results showed that SAP was required for the ability of NK cells to eliminate tumor cells in vitro and in vivo. This effect strongly correlated with expression of CD48 on tumor cells, the ligand of 2B4, a SLAM-related receptor expressed in NK cells. In keeping with earlier reports that studied human NK cells, we showed that SAP was necessary for the ability of 2B4 to trigger cytotoxicity and IFN-gamma secretion. In the absence of SAP, 2B4 function was shifted toward inhibition of NK cell-mediated cytotoxicity. By analyzing mice lacking Fyn, we showed that similarly to SAP, Fyn was strictly required for 2B4 function. Taken together, these results provide evidence that the 2B4-SAP-Fyn cascade defines a potent activating pathway of natural cytotoxicity. They also could help to explain the high propensity of patients who have XLP disease to develop lymphoproliferative disorders.

Binding of SAP SH2 domain to FynT SH3 domain reveals a novel mechanism of receptor signalling in immune regulation.

SAP (or SH2D1A), an adaptor-like molecule expressed in immune cells, is composed almost exclusively of a Src homology 2 (SH2) domain. In humans, SAP is mutated and either absent or non-functional in X-linked lymphoproliferative (XLP) syndrome, a disease characterized by an inappropriate response to Epstein-Barr virus (EBV) infection. Through its SH2 domain, SAP associates with tyrosines in the cytoplasmic domain of the SLAM family of immune cell receptors, and is absolutely required for the function of these receptors. This property results from the ability of SAP to promote the selective recruitment and activation of FynT, a cytoplasmic Src-related protein tyrosine kinase (PTK). Here, we demonstrate that SAP operates in this pathway by binding to the SH3 domain of FynT, through a second region in the SAP SH2 domain distinct from the phosphotyrosine-binding motif. We demonstrate that this interaction is essential for SAP-mediated signalling in T cells, and for the capacity of SAP to modulate immune cell function. These observations characterize a biologically important signalling mechanism in which an adaptor molecule composed only of an SH2 domain links a receptor devoid of intrinsic catalytic activity to the kinase required for its function.

GFI1 tethers the NuRD complex to open and transcriptionally active chromatin in myeloid progenitors.

Growth factor indepdendent 1 (GFI1) is a SNAG-domain, DNA binding transcriptional repressor which controls myeloid differentiation through molecular mechanisms and co-factors that still remain to be clearly identified. Here we show that GFI1 associates with the chromodomain helicase DNA binding protein 4 (CHD4) and other components of the Nucleosome remodeling and deacetylase (NuRD) complex. In granulo-monocytic precursors, GFI1, CHD4 or GFI1/CHD4 complexes occupy sites enriched for histone marks associated with active transcription suggesting that GFI1 recruits the NuRD complex to target genes regulated by active or bivalent promoters and enhancers. GFI1 and GFI1/CHD4 complexes occupy promoters that are either enriched for IRF1 or SPI1 consensus binding sites, respectively. During neutrophil differentiation, chromatin closure and depletion of H3K4me2 occurs at different degrees depending on whether GFI1, CHD4 or both are present, indicating that GFI1 is more efficient in depleting of H3K4me2 and -me1 marks when associated with CHD4. Our data suggest that GFI1/CHD4 complexes regulate histone modifications differentially to enable regulation of target genes affecting immune response, nucleosome organization or cellular metabolic processes and that both the target gene specificity and the activity of GFI1 during myeloid differentiation depends on the presence of chromatin remodeling complexes.

Association between SAP and FynT: Inducible SH3 domain-mediated interaction controlled by engagement of the SLAM receptor.

SAP is an intracellular adaptor molecule composed almost exclusively of an SH2 domain. It is mutated in patients with X-linked lymphoproliferative disease, a human immunodeficiency. Several immune abnormalities were also identified in SAP-deficient mice. By way of its SH2 domain, SAP interacts with tyrosine-based motifs in the cytoplasmic domain of SLAM family receptors. SAP promotes SLAM family receptor-induced protein tyrosine phosphorylation, due to its capacity to recruit the Src-related kinase FynT. This unusual property relies on the existence of a second binding surface in the SAP SH2 domain, centered on arginine 78 of SAP, that binds directly to the FynT SH3 domain. Herein, we wanted to further understand the mechanisms controlling the interaction between SLAM-SAP and FynT. Our experiments showed that, unlike conventional associations mediated by SH3 domains, the interaction of the FynT SH3 domain with SLAM-SAP was strictly inducible. It was absolutely dependent on engagement of SLAM by extracellular ligands. We obtained evidence that this inducibility was not due to increased binding of SLAM to SAP following SLAM engagement. Furthermore, it could occur independently of any appreciable SLAM-dependent biochemical signal. In fact, our data indicated that the induced association of the FynT SH3 domain with SLAM-SAP was triggered by a change in the conformation of SLAM-associated SAP caused by SLAM engagement. Together, these data elucidate further the events initiating SLAM-SAP signaling in immune cells. Moreover, they identify a strictly inducible interaction mediated by an SH3 domain.

A novel regulatory circuit between p53 and GFI1 controls induction of apoptosis in T cells.

Here we demonstrate a mode of reciprocal regulation between GFI1 and p53 that controls the induction of apoptosis in T cells. We show that GFI1 prevents induction of p53 dependent apoptosis by recruiting LSD1 to p53, which leads to the demethylation of its C-terminal domain. This is accompanied by a decrease of the acetylation of lysine 117 within the core domain of the murine p53 protein, which is required for transcriptional induction of apoptosis. Our results support a model in which the effect of GFI1's regulation of methylation at the c-terminus of p53 is ultimately mediated through control of acetylation at lysine 117 of p53. We propose that GFI1 acts prior to the occurrence of DNA damage to affect the post-translational modification state and limit the subsequent activation of p53. Once activated, p53 then transcriptionally activates GFI1, presumably in order to re-establish the homeostatic balance of p53 activity. These findings have implications for the activity level of p53 in various disease contexts where levels of GFI1 are either increased or decreased.

Reduced expression but not deficiency of GFI1 causes a fatal myeloproliferative disease in mice.

Growth factor independent 1 (Gfi1) controls myeloid differentiation by regulating gene expression and limits the activation of p53 by facilitating its de-methylation at Lysine 372. In human myeloid leukemia, low GFI1 levels correlate with an inferior prognosis. Here, we show that knockdown (KD) of Gfi1 in mice causes a fatal myeloproliferative disease (MPN) that could progress to leukemia after additional mutations. Both KO and KD mice accumulate myeloid cells that show signs of metabolic stress and high levels of reactive oxygen species. However, only KO cells have elevated levels of Lysine 372 methylated p53. This suggests that in contrast to absence of GFI1, KD of GFI1 leads to the accumulation of myeloid cells because sufficient amount of GFI1 is present to impede p53-mediated cell death, leading to a fatal MPN. The combination of myeloid accumulation and the ability to counteract p53 activity under metabolic stress could explain the role of reduced GF1 expression in human myeloid leukemia.

Gfi1b regulates the level of Wnt/ß-catenin signaling in hematopoietic stem cells and megakaryocytes.

Gfi1b is a transcriptional repressor expressed in hematopoietic stem cells (HSCs) and megakaryocytes (MKs). Gfi1b deficiency leads to expansion of both cell types and abrogates the ability of MKs to respond to integrin. Here we show that Gfi1b forms complexes with ß-catenin, its co-factors Pontin52, CHD8, TLE3 and CtBP1 and regulates Wnt/ß-catenin-dependent gene expression. In reporter assays, Gfi1b can activate TCF-dependent transcription and Wnt3a treatment enhances this activation. This requires interaction between Gfi1b and LSD1 and suggests that a tripartite ß-catenin/Gfi1b/LSD1 complex exists, which regulates Wnt/ß-catenin target genes. Consistently, numerous canonical Wnt/ß-catenin target genes, co-occupied by Gfi1b, ß-catenin and LSD1, have their expression deregulated in Gfi1b-deficient cells. When Gfi1b-deficient cells are treated with Wnt3a, their normal cellularity is restored and Gfi1b-deficient MKs regained their ability to spread on integrin substrates. This indicates that Gfi1b controls both the cellularity and functional integrity of HSCs and MKs by regulating Wnt/ß-catenin signaling pathway.

GFI1 facilitates efficient DNA repair by regulating PRMT1 dependent methylation of MRE11 and 53BP1.

GFI1 is a transcriptional regulator expressed in lymphoid cells, and an "oncorequisite" factor required for development and maintenance of T-lymphoid leukemia. GFI1 deletion causes hypersensitivity to ionizing radiation, for which the molecular mechanism remains unknown. Here, we demonstrate that GFI1 is required in T cells for the regulation of key DNA damage signaling and repair proteins. Specifically, GFI1 interacts with the arginine methyltransferase PRMT1 and its substrates MRE11 and 53BP1. We demonstrate that GFI1 enables PRMT1 to bind and methylate MRE11 and 53BP1, which is necessary for their function in the DNA damage response. Thus, our results provide evidence that GFI1 can adopt non-transcriptional roles, mediating the post-translational modification of proteins involved in DNA repair. These findings have direct implications for treatment responses in tumors overexpressing GFI1 and suggest that GFI1's activity may be a therapeutic target in these malignancies.

Molecular dissection of 2B4 signaling: implications for signal transduction by SLAM-related receptors.

2B4 is a SLAM-related receptor expressed on natural killer (NK) cells and cytotoxic T cells. It can regulate killing and gamma interferon secretion by NK cells, as well as T-cell-mediated cytotoxicity. There are conflicting data regarding the mechanism of action of 2B4. In these studies, we attempted to understand better the nature and basis of 2B4 signaling. Our studies showed that engagement of 2B4 on NK cells triggered a tyrosine phosphorylation signal implicating 2B4, Vav-1, and, to a lesser extent, SHIP-1 and c-Cbl. Structure-function analyses demonstrated that this response was defined by a series of tyrosine-based motifs in the cytoplasmic region of 2B4 and was not influenced by the extracellular or transmembrane segment of 2B4. In addition, the 2B4-induced signal was absolutely dependent on coexpression of SAP, a Src homology 2 (SH2) domain-containing adaptor associating with SLAM-related receptors and mutated in X-linked lymphoproliferative disease. It was also observed that 2B4 was detectably associated with the Src-related protein tyrosine kinase FynT in an immortalized NK cell line. Mutation of arginine 78 of SAP, a residue critical for binding of SAP to FynT, eliminated 2B4-mediated protein tyrosine phosphorylation, implying that SAP promotes 2B4 signaling most probably by recruiting FynT. Finally, despite the similarities in the signaling modalities of 2B4 and its relative SLAM, the natures of the tyrosine phosphorylation signals induced by these two receptors were found to be different. These differences were not caused by variations in the extent of binding to SAP but rather were dictated by the tyrosine-based sequences in the cytoplasmic domain of the receptors. Taken together, these data lead to a better understanding of 2B4 signaling. Furthermore, they provide firm evidence that the signals transduced by the various SLAM-related receptors are unique and that the specificity of these signals is defined by the distinctive arrays of intracytoplasmic tyrosines in the receptors.

Threshold Levels of Gfi1 Maintain E2A Activity for B Cell Commitment via Repression of Id1.

A regulatory circuit that controls myeloid versus B lymphoid cell fate in hematopoietic progenitors has been proposed, in which a network of the transcription factors Egr1/2, Nab, Gfi1 and PU.1 forms the core element. Here we show that a direct link between Gfi1, the transcription factor E2A and its inhibitor Id1 is a critical element of this regulatory circuit. Our data suggest that a certain threshold of Gfi1 is required to gauge E2A activity by adjusting levels of Id1 in multipotent progenitors, which are the first bipotential myeloid/lymphoid-restricted progeny of hematopoietic stem cells. If Gfi1 levels are high, Id1 is repressed enabling E2A to activate a specific set of B lineage genes by binding to regulatory elements for example the IL7 receptor gene. If Gfi1 levels fall below a threshold, Id1 expression increases and renders E2A unable to function, which prevents hematopoietic progenitors from engaging along the B lymphoid lineage.

A monoclonal antibody to visualize PtdIns(3,4,5)P(3) in cells.

Phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] is a second messenger produced in response to agonist stimulation. Traditionally, visualization of phosphoinositide polyphosphates (PtdInsP(n)) in living cells is accomplished using chimeric green fluorescent protein (GFP)-pleckstrin homology (PH) domain proteins, while PtdInsP(n) quantitation is accomplished by extraction and separation of radiolabeled cellular PtdInsP(n)s. Here we describe preparation of a covalent protein-PtdIns(3,4,5)P(3) immunogen, characterization of binding selectivity of an anti-PtdIns(3,4,5)P(3) IgM, and immunodetection of PtdIns(3,4,5)P(3) in stimulated mammalian cells. This antibody has greater than three orders of magnitude selectivity for binding PtdIns(3,4,5)P(3) relative to its precursor, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), and is therefore optimal for studies of cell function. The immunodetection in platelet-derived growth factor (PDGF)-stimulated NIH 3T3 cells was benchmarked against HPLC analysis of [3H]-myo-inositol-labeled cellular PtdInsP(n)s. In addition, the changes in subcellular amounts and localizations of both PtdIns(3,4,5)P(3) and PtdIns(4,5)P(2) in stimulated NIH 3T3 fibroblasts and human neutrophils were observed by immunofluorescence. In insulin- or PDGF-stimulated fibroblasts, PtdIns(3,4,5)P(3) levels increased in the cytoplasm, peaking at 10 min. In contrast, increases in the PtdIns(4,5)P(2) levels were detected in nuclei, corresponding to the production of new substrate following depletion by phosphoinositide (PI) 3-kinase.

Growth factor independence 1 antagonizes a p53-induced DNA damage response pathway in lymphoblastic leukemia.

Most patients with acute lymphoblastic leukemia (ALL) fail current treatments highlighting the need for better therapies. Because oncogenic signaling activates a p53-dependent DNA damage response and apoptosis, leukemic cells must devise appropriate countermeasures. We show here that growth factor independence 1 (Gfi1) can serve such a function because Gfi1 ablation exacerbates p53 responses and lowers the threshold for p53-induced cell death. Specifically, Gfi1 restricts p53 activity and expression of proapoptotic p53 targets such as Bax, Noxa (Pmaip1), and Puma (Bbc3). Subsequently, Gfi1 ablation cures mice from leukemia and limits the expansion of primary human T-ALL xenografts in mice. This suggests that targeting Gfi1 could improve the prognosis of patients with T-ALL or other lymphoid leukemias.

Negative regulation of natural killer cell function by EAT-2, a SAP-related adaptor.

EAT-2 is an adaptor expressed in innate immune cells, including natural killer (NK) cells. It is closely related to the adaptor SAP, which regulates signaling lymphocyte activation molecule (SLAM)-related receptors by recruiting the kinase FynT to the receptors. Here we have studied the function of EAT-2 in NK cells by creating mice lacking or overexpressing EAT-2. Like SAP, EAT-2 was associated with the SLAM-related receptor 2B4 in NK cells. However, unlike SAP, EAT-2 was an inhibitor of NK cell function. EAT-2 repressed natural cytotoxicity and interferon-gamma secretion by a mechanism involving tyrosine phosphorylation of its C terminus. We have demonstrated a similar function for the adaptor ERT, a newly identified SAP family member expressed in mouse NK cells. These data identify a previously unknown mechanism of NK cell inhibition. Moreover, they indicate that EAT-2 and SAP have distinct and at times opposing functions in natural immunity.