The Interferon Consensus Sequence Binding Protein (ICSBP/IRF8) Activates Transcription of the FANCF Gene during Myeloid Differentiation

Abstract

The interferon consensus sequence binding protein (ICSBP) is an interferon regulatory transcription factor with leukemiasuppressor activity. ICSBP regulates genes that are involved in phagocyte function, proliferation, and apoptosis. In murine models ICSBP deficiency results in a myeloproliferative disorder (MPD) with increased mature neutrophils. Over time this MPD progresses to acute myeloid leukemia (AML), suggesting that ICSBP deficiency is adequate for MPD, but additional genetic lesions are required for AML. The hypothesis of these studies is that dysregulation of key target genes predisposes to disease progression under conditions of decreased ICSBP expression. To investigate this hypothesis, we used chromatin co-immunoprecipitation to identify genes involved the ICSBPleukemia suppressor effect. In the current studies, we identify the gene encoding Fanconi F (FANCF) as an ICSBP target gene. FancF participates in a repair of cross-linked DNA. We identify a FANCF promoter cis element, which is activated by ICSBP in differentiating myeloid cells. We also determine that DNA cross-link repair is impaired in ICSBP-deficient myeloid cells in a FancF-dependent manner. This effect is observed in differentiating cells, suggesting that ICSBP protects against the genotoxic stress of myelopoiesis. Decreased ICSBP expression is found in human AML and chronic myeloid leukemia during blast crisis (CML-BC). Our studies suggest that ICSBP deficiency may be functionally important for accumulation of chromosomal abnormalities during disease progression in these myeloid malignancies.The interferon consensus sequence binding protein (ICSBP) 2 is an interferon regulatory factor (also known as IRF8) that is expressed in CD34ϩ bone marrow progenitor cells and in differentiating and mature myeloid and B cells (1, 2). ICSBP functions as an activator or repressor of gene transcription, depending upon the sequence of the cis element and cellular context. For example, during myeloid differentiation, ICSBP activates transcription of the genes encoding the phagocyte oxidase proteins gp91PHOX and p67 PHOX , the Toll-like receptor 4, and interleukin-12 (3-6). Therefore, ICSBP contributes to functional differentiation of phagocytic cells.ICSBP also activates transcription of the genes encoding Neurofibromin (Nf1) and Ink4b in differentiating myeloid cells (7,8). ICSBP-induced expression of Nf1 and Ink4b decreases the proliferative effects of cytokines such as GM-CSF and SCF. In contrast, ICSBP represses transcription of the gene encoding Fas-associated phosphatase (Fap1; the PTPN13 gene) in myeloid progenitors (9). Because Fap1 antagonizes Fas-induced apoptosis, a decrease in ICSBP-induced repression of PTPN13 transcription during myelopoiesis increases the sensitivity of differentiating cells to Fas (10 -12). Therefore, ICSBP-deficient myeloid cells would be anticipated to exhibit apoptosis resistance, cytokine hypersensitivity, and phagocyte functional defects.Consistent with this hypothesis, studies in murine models identified a leukemi...

Increased Fanconi C expression contributes to the emergency granulopoiesis response

“…This suggested a possible IRF8-binding cis element between -48 and -56 bp. Consistent with this, the -50 to -56 bp Fancc sequence had homology with an IRF-binding consensus (5′-GAAAAT-3′ compared with the ISRE consensus [5′-GAAANN-3′] and the IRF8-binding Fancf cis element [5′-GAAAGC-3′]) (17,33). We mutated this sequence in the context of the -56-bp Fancc promoter construct (5′-GAAAAT-3′ to 5′-GAACAT-3′) and found that the mutant construct did not exhibit IRF8-induced promoter activity ( Figure 2B).…”

“…This set included genes that terminate cytokine-induced proliferation (NF1 and GAS2), increase sensitivity of phagocytes to Fas-induced apoptosis (PTPN13), and are involved in DNA repair (FANCF) (14)(15)(16)(17). We found that IRF8 activated FANCF (the gene encoding Fanconi F) in bone marrow progenitor cells that were stimulated with differentiating cytokines (17).…”

“…We previously determined that treatment of myeloid progenitor cells with differentiating cytokines increased expression of FANCF (17). In this study, we considered the possibility that these cytokines also induce expression of other Fanconi proteins.…”

“…Some binding assays were incubated with excess unlabeled homologous oligonucleotide, homologous oligonucleotide with a mutation of the ISRE-like sequence, or oligonucleotides with 2 irrelevant Fancc promoter sequences or the IRF8-binding FANCF cis element (17). Other binding assays were preincubated with IRF8 antibody (or control antibody).…”

“…In these studies, the reporter plasmid is treated with mitomycin C (MMC) to generate DNA crosslinks prior to transfection into U937 cells (a TK-luciferase plasmid for the current studies) (17). Decreased reporter activity from MMC-treated plasmid versus untreated plasmid reflects MMC-induced DNA damage in this assay (17). Therefore, conditions that increase reporter activity in cells transfected with MMC-treated plasmid (but not control plasmid) are presumed to increase the efficiency of DNA repair.…”