Fig 2: GFI1’s role in DNA Repair is mediated through PRMT1 activity. a Nuclear extracts were prepared from MEFs expressing an R/K mutant form of MRE11 and wild-type control cells. Extracts were immunoprecipitated for MRE11 and blotted for ADMA. b MEFs expressing an R/K mutant form of MRE11 and wild-type control cells were exposed to 5 Gy IR and allowed to recover for the indicated time. Cells were then lysed and analyzed by alkaline Comet assay. Comet tail moment averages are shown. One of three replicate experiments is shown. Error bars represent s.d. *p < 0.05, **p < 0.01, ***p < 0.001 on a Welch corrected T-test. c GFI1 KO Jurkat cells and parental control cells were treated for 48 h with 500 nM MS023 inhibitor. Nuclear extracts were then immunoprecipitated for MRE11 and blotted for ADMA. d GFI1 KO Jurkat cells and parental control cells (left) and SupT1 overexpressing GFI1 and vector control cells (right) were pre-treated with 500 nM of MS023 inhibitor or vehicle were seeded at 1 million cells per ml and exposed to 5 Gy IR. Cells were counted each following day. Dashed lines show average cell numbers and individual data points of a triplicate experiment are shown. e Cells as in d were exposed to 5 Gy IR and allowed to recover for the indicated time. Cells were then lysed and analyzed by Comet assay as in b. f SupT1 overexpressing GFI1 and vector control cells were electroporated with an siRNA against PRMT1. Electroporated cells were FACS sorted 24 h later. 24 additional hours later, cells were exposed to 5 Gy IR, allowed to recover for the indicated time, then lysed and analyzed by alkaline Comet assay as in b. g GFI1 KO Jurkat cells and parental control cells were treated as in f and analyzed by alkaline Comet assay as in b. h GFI1 KO Jurkat cells and parental control cells were electroporated with a pcDNA3.1 plasmid expressing PRMT1 or a vector control. Electroporated cells were FACS sorted 24 h later. 24 additional hours later, cells were exposed to 5 Gy IR, allowed to recover for the indicated time, then lysed and analyzed by alkaline Comet assay as in b

Fig 3: GFI1 is a negative feedback regulator of p53 (A) Thymocytes were extracted from p53 WT or KO mice. Cells were exposed to 5Gy IR or left untreated. mRNA was extracted and the levels of Gfi1 were measured at the indicated time points by qPCR relative to Gapdh. (B) Thymocytes were extracted from GFI1 WT and KO mice. Cells were exposed to 5Gy IR or left untreated and fixed with formaldehyde after 2 hours. Chromatin immunoprecipitation was performed to isolate p53-bound DNA fragments. Fold enrichment amplification at the Gfi1 promoter relative to a non-specific IgG control is shown. (C) mRNA was extracted from thymocytes as in (B). The levels of KO mutant mRNA were measured at the indicated time points after IR by qPCR relative to Gapdh. (D) Diagram representing the regulatory circuit between GFI1 and p53.

Fig 4: H3K4 methylation and chromatin status at sites occupied by GFI1, CHD4, or both CHD4 and GFI1 during neutrophil differentiation.a Heatmap of data from ChIP-Seq and ATAC-Seq experiments and to determine the occupation of GFI1, CHD4, and H3K4 di-methylation patterns during neutrophil differentiation from GMPs via preNeu (preneutrophils) to matNeu (mature neutrophils). Data were filtered for promoter regions, ordered according to CHD4 occupation, and were separated into three groups: occupation by CHD4 alone, by GFI1/CHD4 and by GFI1 alone. b Heatmap of data from ChIP-Seq and ATAC-Seq experiments as in (a), but for enhancer regions. c ATAC-Seq and ChIP-seq read on loci covering the myeloid-specific enhancers at the 3' end of the Plbd1 gene and at downstream regions for sites occupied by GFI1, CHD4, or both, CEBPa and carrying the indicated histone marks. Annotations represent locations on the mouse genome version GRCm38 (mm10). d Metagene analysis for ATAC-seq reads and H3K4me2 levels (e) in RPM values for GMPs, preNeu, and matNeu cells at promoters occupied by CHD4 alone, by GFI1/CHD4, and by GFI1 alone. Shown are regions 2 kb 5' and 3' of the TSS. e Metagene analysis for ATAC-seq reads and for H3K4me2 levels in mean RKPB for GMPs, preNeu and matNeu cells at enhancers occupied by CHD4 alone, by GFI1/CHD4, and by GFI1 alone. Shown are regions 2 kb 5' and 3' of the site of the enhancer (Ctr).

Fig 5: De novo motif analysis and histone methylation aggregation plots for sites occupied by GFI1, CHD4, and CEBPa in GMPs.a Comparison of ChIP-seq data for the occupation of GFI1, CHD4, and H3K4 methylation at promoter regions in GMPs with an analogous ChIP-seq data set for the transcription factor CEBPa. Data are ordered according to GFI1 occupation and are separated into four groups: occupation by GFI1 alone, by GFI1/CEBPa, by GFI1/CEBPa/CHD4, and by GFI1/CHD4. b De novo motif analysis of consensus DNA binding sites at promoter regions by GFI1 alone, by GFI1/CEBPa, by GFI1/CEBPa/CHD4, and by GFI1/CHD4. c) Aggregation plots of histone H3K4 methylation at promoters as defined in (a) for regions 2 kb 5' or 3, of the TSS (transcription start site).