AF1q potentiates Notch signaling. (A) Gene-expression profiling of CD34⁺CD45RA⁻Lin⁻ HPCs transduced with nuclear-sequestered AF1q (A2M). CD34⁺CD45⁺Lin⁻ HPCs were FACS sorted, exposed to A2M (A1-A2) or control (P1-P2) vectors, sorted based on GFP expression, and subjected to global gene-expression analysis 72 hours later. Results are from 2 independent experiments (Exp1: A1/P1; Exp2: A2/P2). Statistical analysis, performed with the R package “locfdr” (lfdr < 2%), showed that 44 probe sets were differentially expressed between A2M (A1-A2)- and empty vector (P1-P2)–transduced CD34⁺CD45RA⁻Lin⁻ HPCs. Results are presented as a heat map of the average expression levels (up: red; down: green). (See also supplemental Table 2.) (B) AF1q down-modulates SPEN transcript levels in CD34⁺CD45RA⁻Lin⁻ HPCs. qRT-PCR analysis of CD34⁺CD45RA⁻Lin⁻ cells transduced with AF1q, A2M, or the empty vector (Ctl). Expression levels (nr) are normalized relative to those in cells transduced with the empty vector. Means ± SD percentages of 3 independent experiments are shown. (C-E) AF1q induces histone H3 modifications at the Spen locus. (C) Diagram illustrating the genomic context of Spen promoter loci. Regions amplified by site-specific qPCR are indicated by bars. (D-E) CD34⁺CD45RA⁻Lin⁻ cells were transduced as above with A2M or the empty vector and cultured for 72 hours before ChIP analysis via anti-H3ac (D) and anti-H3K9me3 (E) antibodies. Results are means of 2 ChIP experiments analyzed in triplicate. (F-G) Forced AF1q expression broadens the repertoire of Notch-responsive genes in CD34⁺CD45RA⁻Lin⁻ HPCs. (F) CD34⁺CD45RA⁻Lin⁻ HPCs were transduced with AF1q, A2M, or the empty vector (Ctl), sorted based on GFP expression, and cultured for 3 more days onto graded doses of plastic-immobilized Delta1^ext-IgG before qRT-PCR analysis. Expression levels are normalized relative to those in cells transduced with the empty vector. Means ± SD of 3 independent samples are shown. (G) Venn diagram detailing shared and distinct gene expression between Notch-stimulated A2M- and empty vector–transduced cells. CD34⁺CD45RA⁻Lin⁻ HPCs were transduced with A2M or the empty vector (Ctl), and cultured as above before being subjected to global gene-expression analysis. Statistical analysis performed with the R package “locfdr” (lfdr < 5%) showed that, after Notch activation, 551 probe sets were up-regulated in A2M-transduced cells, relative to only 262 for their homologs transduced with the empty vector (see also supplemental Table 5A-D).