Fig 2: SETD1A Suppresses Fork Degradation after Replication Stress(A–F) U-2-OS cells were transfected with control siRNA or those targeting: SETD1A and SETD1B (A); BOD1L and SETD1A (B); SETD1A and SMARCAL1 (C); SETD1A and BRCA2 (D); SETD1A, BRCA2, and either KMT2C (E), or KMT2D (F). 72 hr post transfection, cells were pulsed for 20 min each with CldU and IdU and exposed to 4 mM HU for 5 hr (as in the schematic). DNA was visualized with antibodies to CldU and IdU, and tract lengths were calculated. Plots denote average ratios of IdU:CldU label length from 3 independent experiments; arrows indicate mean ratios. Plots in (E) and (F) amalgamate data from the same experiments. See also Table S1.

Fig 3: The conditional knockout (cKO) of Setd1b in the adult mouse is lethal.(A) Setd1b knockout strategy. The conditional allele (FD) carries loxP sites (red triangles) flanking exon 5 (E5) for Cre-mediated recombination. Excision of E5 leads to a frameshift and a premature stop codon in exon 6 (E6) (asterisk). The resulting frameshifted FDC allele corresponds to the null allele. Coding exons of the Setd1b wild type (WT) allele are depicted in black, non-coding exons in white. Single FRT and rox sites are the remnants of previous Flp and Dre recombination. (B) Breeding strategy and experimental setup. Female Setd1bFD/FD mice were mated with Setd1bFD/+ males homozygous for the Rosa26-Cre-ERT2 allele (RC/RC). The resulting offspring was heterozygous for Rosa26-Cre-ERT2 (RC/+) and either heterozygous (FD/+) or homozygous (FD/FD) for the conditional Setd1b allele. To induce Cre recombination, each mouse received five doses of 4.5 mg tamoxifen via gavage over a period of 8 days according to the depicted scheme. (C) qRT-PCR analysis in kidney, spleen, and liver showed absence of Setd1b expression in a homozygous conditional knockout (cKO) mouse compared to a heterozygous control (Ctrl). (D) The loss of Setd1b protein (215 kDa) was validated by western blot analysis in cKO liver. (E) Kaplan-Meier survival curve. While the majority of controls (n = 18) survived, all cKO mice (n = 44) died within 30 weeks after induction with a median survival of 7–8 weeks (dashed line). (F) Male cKO mice between 12 and 26 weeks after induction (Ctrl n = 9, cKO n = 10) had significantly reduced body weights compared to control littermates (p**=0.0043). The graph depicts the mean ± SEM (data derived from five independent tamoxifen inductions).

Fig 4: H3K4 methylation activities of human SET1A, SET1B, and MLL2 complexes. (A and B) SDS-PAGE/Coomassie blue staining (A) and immunoblot analyses (B) of SET1A, SET1B, and MLL2 complexes purified from HEK293E cells transfected with combinations of plasmids expressing subunits that constitute each complex. MLL2N and MLL2C, N-terminal and C-terminal parts of MLL2, respectively, resulting from Taspase 1 cleavage (54). (C and D) Differential H3K4 methylation activities of SET1A, SET1B, and MLL2 complexes. H3 (C) and unmodified or H2Bub chromatin substrates (D) were subjected to in vitro HMT assays with the indicated complexes (5 nM).

Fig 5: Hematopoietic-specific deletion of Setd1b using Vav-Cre is lethal and impedes production of early progenitors.(A) Breeding strategy. Mice heterozygous for Vav-Cre (VC/+) and either heterozygous (Ctrl) or homozygous (vKO) for the conditional Setd1b allele were used for analysis. (B) Kaplan-Meier survival curve. All vKO mice died with a median survival of 25 weeks (dashed line). (C) Peripheral blood counts of mice aged between 10 and 32 weeks (Ctrl n = 30, vKO n = 35). Both lymphocyte and thrombocyte counts were severely diminished in vKO blood (p***<0.0001). Furthermore, mice showed mild but significant anemia (p***=0.0002) (data representative of >20 measurements). (D) Wright-Giemsa stained blood smears. Immature myeloid forms, including myeloblasts and myelocytes, were detected in vKO blood. (E) The majority of vKO mice (n = 11) aged between 5 and 31 weeks developed mild splenomegaly as compared to controls (n = 14) (p**=0.004). (F) FACS analysis of splenic cell compartments (Ctrl n = 6, vKO n = 7). In spleens from vKO mice, lymphocytes were highly reduced with respect to spleen weight (mg) (p**<0.008), while CD11b+ Gr-1high (p*=0.035) as well as CD11b+ Gr-1low granulocytes (p**=0.001) were increased. (G) FACS analysis of HSPCs in BM. In vKO mice (n = 7), LMPPs were almost undetectable (p**=0.003) and GMPs were more than twofold decreased (p**=0.005) compared to control littermates (n = 6). The data in (F) and (G) are representative of four independent experiments. The graphs in (C), (F), and (G) depict the mean ± SD; in (E) the mean ± SEM is illustrated.10.7554/eLife.27157.017Figure 6—source data 1.Numerical data used to generate Figure 6G (HSPCs BM VC).