Influence of GFI1^36N allele on progression. (A) Prognostic impact of GFI1^36N on disease progression of AML patients. GFI1^36S (homozygous for GFI1^36S) and GFI1^36N (heterozygous for GFI1^36N or homozygous for GFI1^36N; 1 patient only) de novo AML patients, recruited from the DSIL, had the same prognosis for a 5-year overall survival rate of approximately 27% (GFI1^36S) and 34% (GFI1^36N). Also, the 5-year relapse-free survival was not different between both groups (data not shown). (B) Genome-wide mRNA expression pattern of patients heterozygous for the GFI1^36N allele was not overall different from GFI1^36S-homozygous patients. Genome-wide RNA expression data were obtained by gene array analysis (Affymetrix) from a patient cohort from the HOVON study group analyzed in The Netherlands (Rotterdam; n = 350).

GFI1^36S and GFI1^36N proteins show different subnuclear localization. (A) NIH-3T3 cells were transfected with GFI1^36S expression plasmid. GFI1 appears green; DNA, blue. The right column represents the merging of both staining. The numbers of cells analyzed for GFI1^36S are indicated. GFI1^36S is mainly localized in a dotted pattern. (B) NIH-3T3 cells were transfected with a GFI1^36N expression plasmid. The numbers of cells analyzed for GFI1^36N are indicated. GFI1^36N is localized at the nuclear border. (C) NIH-3T3 cells were transfected with either a GFI1^36S or GFI1^36N expression plasmid. Cell lysates were fractionated. Both protein variants are localized in the nuclear cell fraction and cannot be found in the cytosolic fraction. (D) Analysis of a bone marrow sample from the only available homozygous for GFI1^36N. As in transfected cells, GFI1^36N was mainly located at the nuclear/cytoplasmic border. As a control, cells from the Kasumi 1 t(8;21)-positive AML cell line were used. This cell line was originally derived from a patient with French-American-British M2 AML and is homozygous for GFI1^36S. (E) Nuclear matrix preparation of NIH-3T3 cells transiently transfected with expression vectors for GFI1^36S or GFI1^36N. Both variants are still attached to nuclear matrix components, although the GFI1^36N variant appears to be more concentrated at the nuclear membrane, consistent with our observation in regular cell transfection assays.

AML1/ETO colocalizes with GFI1^36S but not with the variant GFI1^36N. (A) NIH 3T3 cells were transfected with expression vectors for AML1/ETO and GFI1^36S (original magnification ×100). (B) NIH 3T3 cells were transfected with expression vectors for AML1/ETO and the GFI1^36N variant form. Expression of GFI1 proteins was revealed by immune-fluorescence with an α-GFI1 antibody and a secondary FITC labeled antibody. The presence of AML1/ETO was revealed by staining with the α-ETO antibody and a secondary rhodamine-labeled antibody. Nuclei were visualized by treatment with the DNA dye TO-PRO-3 (blue). The staining for each protein and DNA is represented in a separate column. Examples of 2 different cells are given for each setting in 2 different rows. The merging of all 3 fluorescence signals is depicted in the last column at the right side and results in a white staining, which can be detected when the signals corresponding to the GFI1^36S protein and AML1/ETO are merged. White areas or white spots in the nuclei of transfected cells suggest a colocalization of AML1/ETO and the GFI1^36S protein (A). In contrast, no such white areas can be detected between GFI1^36N and AML1/ETO (B). The green signal representing the GFI1^36N protein remained at the nuclear/cytoplasmic border, well separated from the red signal in the nucleus that represents AML1/ETO (B), clearly indicating a lack of colocalization between both. The number of cells analyzed for the subnuclear localization and a colocalization with AML1/ETO are indicated (original magnification ×100).

GFI1^36S and GFI1^36N maintain similar repressor activity but are differentially regulated by AML1/ETO. (A) Transient transfection of HeLa cells with expression plasmids for GFI1^36S, GFI1^36N, AML1/ETO fusion protein, and with a luciferase reporter gene containing Gfi1 binding sequences. Both GFI1^36S and GFI1^36N were able to repress the Gfi1 promoter in a concentration-dependent manner (lanes 1–2 and 3–4, respectively), whereas AML1/ETO alone did not influence reporter gene activity (lane 8). In the presence of AML1/ETO, the GFI1^36S protein lost its repression capacity, but GFI1^36N retained its repressor activity (lanes 5–6; P < .001). Error bars represent SEM. (B) Same as panel A but with different amounts of expression plasmid transfected as indicated. (C) GFI1^36S and GFI1^36N both coprecipitate in transfected cells with AML1/ETO. Cos7 cells were transfected with GFI1^36S, GFI1^36N, or AML1/ETO expression plasmids. Immunoprecipitations (IP) were performed with α-GFI1 antibody. AML1/ETO immunoprecipitates in the lysates with GFI1^36S or the variant GFI1^36N (lanes 2 and 3, respectively). A control IP was performed with a goat α-CD2 antibody (lane 1). An input control demonstrated the presence of both GFI1 and the AML1/ETO protein. (D) GFI1^36S coprecipitates with AML1/ETO in extracts from Kasumi1 cells, which is a cell line derived from a t(8;21) AML patient. Nuclear and cytosolic extracts of Kasumi1 cells were prepared and incubated either with the α-Gfi1 (N20) antibody or with a nonspecific α-actin antibody. The cell extracts were immunoblotted and developed with an α-ETO antibody. As a positive control, AML1/ETO transfected Cos7 cells were used.

Expression, genomic representation, and LD of GFI1^36N. (A) Chromatogram of the GFI1^36S/36N cDNA sequence of one of the patients. Change of G to A at position c107 (c107G>A) results in the replacement of serine by asparagine. The neighboring amino acid sequences of the more common human and mouse sequences are shown. (B) Location of the GFI1^36N variant on genomic and protein level. The SNP is located in exon 2 and replaces a serine by an asparagine at amino acid position 36. Green indicates N-terminal Snail/Growth factor independence 1 repressor domain of GFI1; blue, 6 C₂H₂ zinc finger domains. (C) GFI1 mRNA expression in different patients. GFI1 expression in different AML patients was semiquantitatively assayed by reverse transcriptase PCR. Lanes 1 to 9 represent bone marrow and peripheral blood samples of AML patients at diagnosis. Lane 10 shows cell from the t(8;21)-positive Kasumi 1 AML cell line. Lane 11 shows the control without reverse transcriptase and lane 12 a peripheral blood aphaeresis sample from an AML patient. Lane 13 shows HeLa cells (human cervical cancer) and lane 14 cells originating from a patient with a chronic lymphocytic leukemia (CLL). (D) Results of LD analysis in the genomic region encompassing the GFI1 and EVI5 loci and neighboring regions. LD block 3 spans part of GFI1 and EVI5. (E) LD as determined after genotyping of 39 GFI1^36N heterozygous AML patients from Essen, Marburg, and the DSIL study group. The genotypes of 5 SNPs in the proximity of the GFI1^36N SNP were determined. The results show that GFI1^36N is not within the LD block that spans part of EVI5. (F) Results of LD of a group consisting of the aforementioned 39 GFI1^36N heterozygous AML patients and 26 healthy persons homozygous for GFI1^36S from Essen. Similar to the analysis described previously, GFI1^36N is not within the LD block that spans part of EVI5.