FANCF is an ICSBP-target-gene in differentiating myeloid cells. A, a schematic representation of consensus sequences for IRF-protein binding in the proximal FANCF 5′-flank is shown. The sequence of the proximal FANCF 5′-flank was analyzed for IRF DNA binding consensus sequences (underlined in red). The ICSBP binding cis element is indicated by an asterisk. The red arrow indicates the 5′ end of the sequence of the clone obtained by ICSBP co-immunoprecipitation. The black arrow indicates the transcription start site. TSS, transcription start site. B, ICSBP interacts with the proximal 5′-flank of the FANCF gene in vitro in IFNγ-differentiated, but not untreated, U937 myeloid cells. Chromatin co-immunoprecipitation (IP) was performed with U937 myeloid leukemia cells (with or without IFNγ-induced differentiation) and an antibody to ICSBP (or irrelevant control antibody). Co-precipitating chromatin was amplified with primers encompassing 0 to −1.5 kb, 0 to −0.5 bp, 0 to −150 bp, −0.5 bp to −1.5 kb, or −1.5 to −2.0 kb in the proximal FANCF 5′-flank or +1.5 to +2.0 kb (in exon I).

ICSBP binds to a sequence in the proximal FANCF promoter in vitro and in vivo. A, ICSBP interacts with the −20 to −30 bp sequence from the FANCF promoter in vitro in DNA affinity purification assays with nuclear proteins from differentiated U937 cells. Nuclear proteins were isolated from U937 cells with or without IFNγ-induced differentiation. DNA affinity purification assays were performed using biotin-labeled probes representing the proximal 20 or 30 bp from the FANCF 5′-flank. Western blots (WB) of co-precipitating proteins were probed with antibody to ICSBP. Control Western blots were performed with amounts of nuclear proteins used in the affinity purification experiments. These blots were sequentially probed with antibody to ICSBP and with laminin A (as a loading control for nuclear proteins). B, ICSBP interacts with the −20 to −30 bp sequence from the FANCF promoter in vitro in electrophoretic mobility shift assays with nuclear proteins from differentiated U937 cells. The nuclear proteins described above were also analyzed by electrophoretic mobility shift assays using a radiolabeled DNA probe representing the proximal 60 bp of FANCF 5′-flank. Some binding reactions were preincubated with double-stranded (ds) oligonucleotide competitors (200-fold molar excess) representing the −20 to −30 bp or −30 to −60 bp sequences from the FANCF promoter, the ICSBP binding cis element from the CYBB or NF1 genes, or an irrelevant oligonucleotide as indicated. Other binding reactions were preincubated with antibody to ICSBP or preimmune serum, as indicated. The shifted complex, which is cross-immunoreactive with ICSBP, is indicated by the upper arrow, and the free probe is indicated by the by the lower arrow. Shifted bands in the middle panel are less intense in comparison to the flanking panels because less nuclear protein was used (1 μg versus 2 μg), and the exposure was shorter. C, ICSBP interacts with the −20 to −30 bp sequence from the FANCF promoter in vivo in chromatin co-immunoprecipitation (IP) assays with differentiated U937 cells. Chromatin co-immunoprecipitation was performed with U937 cells (with or without IFNγ-induced differentiation) and an antibody to ICSBP (or irrelevant control antibody). Co-precipitating chromatin was amplified by real time PCR with primers flanking the −20 to −30-bp sequence in the FANCF promoter. Results were normalized to total input chromatin. Statistically significant differences in the amount of co-precipitating chromatin in undifferentiated versus differentiated U937 cells is indicated by an asterisk. NE, nuclear extract.

ICSBP deficiency increases sensitivity to mitomycin C-induced DNA damage in primary murine bone marrow cells in a FancF-dependent manner. A, ICSBP deficiency increases sensitivity to mitomycin C-induced DNA breaks in myeloid progenitor cells during M-CSF-induced differentiation in a FancF-dependent manner. Bone marrow cells were harvested from WT or ICSBP^−/− mice and cultured in GM-CSF, IL3, and SCF for 48 h or in GM-CSF, IL3, and SCF for 24 h followed by 24 h in M-CSF. MMC was added during the last 24 h of culture. Chromosome spreads were analyzed microscopically for DNA breaks (indicated as the number of breaks per 500 chromosomes). Statistically significant difference in DNA breaks for WT versus ICSBP^−/− cells is indicated by an asterisk. Other ICSBP^−/− bone marrow cells were cultured in GM-CSF, IL3, and SCF and transduced with a retroviral vector to express FancF (FancF/MSCV) or empty vector control (MSCV). After 48 h of antibiotic selection, cells were differentiated with M-CSF and treated with MMC, and chromosome spreads were analyzed for DNA breaks. Statistically significant differences in DNA breaks with versus without FancF re-expression are indicated by double asterisks. B, ICSBP deficiency increases sensitivity to mitomycin C-induced DNA radial formation in myeloid progenitor cells during M-CSF-induced differentiation. The chromosomes described above were also analyzed microscopically for the presence of DNA-radial forms (indicated as the number of radials per 300 chromosomes). A statistically significant difference in DNA radials for WT versus ICSBP^−/− cells is indicated by an asterisk and for ICSBP^−/− cells with versus without FancF re-expression is indicated by double asterisks. C, chromatin spread from ICSBP^−/− bone marrow cells with DNA breaks and radials. A representative chromatin spread from MMC-treated, M-CSF-differentiated ICSBP^−/− bone marrow cells. DNA breaks are indicated by an asterisk, and DNA radials are indicated by an arrow.

ICSBP activates FANCF transcription in differentiating myeloid cells. A, ICSBP activates a cis element found between −20 and −30 bp in the proximal FANCF 5′-flank in differentiating U937 cells. U937 cells were transfected with a series of plasmid constructs containing sequences from the FANCF 5′-flank (1.6 kb, and 500, 200, 100, 50, 30, and 20 bp) linked to a reporter. Cells were co-transfected with a vector to overexpress ICSBP or empty vector control. Reporter gene assays were performed with or without IFNγ-induced differentiation of the transfectants. A statistically significant difference in reporter gene expression with versus without ICSBP overexpression in IFNγ-differentiated transfectants is indicated by an asterisk. Statistically significant differences in reporter gene expression in ICSBP overexpressing, differentiated transfectants with 100 bp of FANCF versus 50 bp is indicated by double asterisks (**) and for 50 bp and 20 bp is indicated by the number symbol (#). CAT, chloramphenicol acetyltransferase. B, ICSBP tyrosine phosphorylation is necessary for FANCF promoter activation. U937 cells were co-transfected with a reporter vector with the proximal 1.6 kb of FANCF 5′-flank (or empty control reporter vector) and a vector to overexpress ICSBP, a mutant form of ICSBP with all tyrosine residues mutated to phenylalanine (Y-mut ICSBP) or empty vector control. Reporter gene assays were performed after IFNγ-induced differentiation. Statistically significant difference in reporter activity in transfectants with WT versus Y-mut ICSBP overexpression is indicated by an asterisk.

FancF expression is differentiation stage-specific and ICSBP-dependent. A, FancF mRNA expression is differentiation stage-specific and ICSBP-dependent in primary murine myeloid progenitor cells. Bone marrow was harvested from WT or ICSBP^−/− mice and Sca1-separated and cultured in GM-CSF, IL3, and SCF (bi-potential myeloid progenitors). Some of these cells were differentiated to granulocytes with G-CSF or monocytes with M-CSF. RNA was isolated from the cells and analyzed by quantitative real time PCR for FancF expression. Results were normalized to 18 S. Statistically significant differences in FancF mRNA expression in WT myeloid progenitors in comparison to M-CSF- or G-CSF-differentiated cells are indicated by single and double asterisks, respectively. Statistically significant differences in FancF expression between WT versus ICSBP^−/− cells differentiated with M-CSF or G-CSF are indicated by single and double number symbols (# and ##), respectively. B, FancF mRNA expression increases during IFNγ-induced differentiation of U937 myeloid leukemia cells. U937 cells were treated with IFNγ for 0, 24, or 48 h. RNA was isolated and analyzed for FancF mRNA expression by quantitative real time PCR. Results were normalized to 18 S. Statistically significant differences in FancF expression at 24 or 48 h in comparison to untreated cells are indicated by single and double asterisks and, respectively. C, FancF protein expression increases during IFNγ-induced differentiation of U937 myeloid leukemia cells. Proteins isolated from the U937 cells, described above, were analyzed by Western blot for FancF expression. The blot was re-probed with antibody to tubulin as a loading control. D, FancF mRNA expression is increased by ICSBP overexpression and decreased by ICSBP knockdown in differentiating U937 myeloid leukemia cells. Stable transfectants of U937 cells were generated with various combinations of vectors to overexpress ICSBP (or empty pcDNA vector control) or an ICSBP-specific shRNA (or with scrambled shRNA control). RNA isolated from the stable transfectants (with or without IFNγ-induced differentiation) was analyzed by quantitative real time PCR for FancF mRNA expression. ICSBP mRNA expression was also determined as a control for overexpression or knockdown. Statistically significant ICSBP overexpression or knockdown is indicated by one or two asterisks, respectively. Statistically significant differences in ICSBP expression in cells with ICSBP knockdown with versus without ICSBP re-expression is indicated by three asterisks. A statistically significant increase in FancF expression in control vector-transduced cells during differentiation is indicated by the number symbol (#) or ampersand. Statistically significant differences in FancF expression in differentiated cells with ICSBP overexpression versus vector control is indicated by the double number symbol (#). Statistically significant differences in FancF expression in differentiated cells with ICSBP knockdown versus control shRNA is indicated by a double ampersand. Statistically significant differences in FancF expression in cells with ICSBP knockdown with versus without ICSBP re-expression is indicated by three ampersands. E, FancF protein expression is increased by ICSBP overexpression and decreased by ICSBP knockdown in differentiating U937 myeloid leukemia cells. Proteins isolated from the U937 cells, described above, were analyzed by Western blot. The blots were sequentially probed with antibodies to ICSBP, FancF, and tubulin (as a loading control).

ICSBP overexpression increases DNA-cross-linked repair in differentiating U937 cells in a FancF-dependent manner. A, ICSBP overexpression improves the repair of mitomycin C-treated plasmid in differentiating U937 myeloid leukemia cells in a FancF-dependent manner. U937 stable transfectants were generated with various combinations of vectors to overexpress ICSBP or FancF (or with empty vector control) or with vectors to express an ICSBP or FancF-specific shRNA (or scrambled shRNA control). These transfectants were co-transfected with MMC-treated or sham-treated reporter plasmid, and reporter assays were performed with or without IFNγ-induced differentiation. A statistically significant difference in reporter expression from MMC-treated plasmid in control cells with versus without differentiation is indicated by an asterisk. CAT, chloramphenicol acetyltransferase. Statistically significant difference in MMC-treated reporter gene activity in differentiated transfectants with vector control versus ICSBP overexpression is indicated by double asterisks. Statistically significant difference in reporter gene activity from MMC-treated plasmid in ICSBP-overexpressing, IFNγ-treated transfectants with versus without FancF-specific shRNA are indicated by three asterisks. Statistically significant difference in reporter gene activity from MMC-treated plasmid in IFNγ-treated transfectants with ICSBP-knock down with versus without re-expression of ICSBP is indicated by the number symbol (#). B, mitomycin C treatment results in DNA-cross-link generation in vitro. Reporter plasmid DNA (chloramphenicol acetyltransferase reporter with a cytomegalovirus promoter) was treated in vitro with MMC (versus sham-treated control). Plasmid DNA was analyzed by native agarose gel electrophoresis and ethidium bromide staining to verify the presence of cross-linked DNA. C, FancF expression in stable transfectants of U937 myeloid leukemia cell with FancF overexpression or knock down. U937 stable transfectants were generated with a vector to overexpress FancF (or empty vector control) or with a vector to express a FancF-specific shRNA (or scrambled control shRNA). Western blots of lysates from differentiated stable transfectants were serially probed with antibodies to FancF and tubulin (as a loading control).