Identification of UPS genes that regulate erythropoiesis. (A) Experimental scheme. Cas9-expressing HUDEP-2 erythroblasts were transduced with a lentiviral vector library encoding sgRNAs targeting 776 UPS genes (supplemental Table 1) selected in puromycin, expanded, and cultured under conditions promoting expansion or terminal maturation. For the latter, mature (Band3⁺) and immature (Band3^–) cells were purified by fluorescence-activated cell sorting (FACS) and analyzed by deep sequencing to compare sgRNA representation (panels B-C). Expansion-related genes were identified by comparing sgRNA representation in cells grown in culture for 0 or 8 days in expansion medium (panel D). (B) Differential representation of sgRNAs in immature vs mature erythroblasts. Each dot indicates a single gene ranked according to the relative sgRNA abundance (log₂ fold change in Band3⁺ vs Band3^– was an average of 4 sgRNAs per gene in 3 biological replicate experiments). Dots indicate the top 10 genes corresponding to underrepresented (blue) or overrepresented (black) sgRNAs in Band3⁺ erythroblasts. The yellow dot represents the average value of 19 control nontargeting sgRNAs (see also supplemental Table 2). (C) Heat map of the most differentially represented sgRNAs in mature (Band3⁺) vs immature (Band3^–) erythroblasts (an average of 4 sgRNAs per gene in 3 biological replicate experiments). Genes corresponding to over- or underrepresented sgRNAs correspond to positive and negative regulators of erythroid maturation, respectively. (D) Heat map showing the differential representation of sgRNAs for genes that promote HUDEP-2 erythroblast expansion. The data represent the average of 4 different sgRNAs for each gene in 3 biological replicate experiments. Genes corresponding to the 20 most depleted sgRNA sets after 8 days of growth in expansion medium are shown. (E) Gene Ontology (GO) enrichment analysis of genes identified by CRISPR screening as positively regulating HUDEP-2 cell maturation or expansion. The enriched GO terms are listed on the left, with the corresponding −log₁₀ adjusted P values shown in the graph. (F) Overlap between genes predicted to promote HUDEP-2 cell expansion or maturation. (G-K) Immature Cas9-expressing HUDEP-2 erythroblasts were transduced with lentiviral vectors encoding FBXO11 sgRNAs (F11-sg) or control sgRNAs (Ctrl), selected with puromycin, and expanded for 5 to 9 days. (G) Western blot showing FBXO11 protein expression. Bands labeled i1 and i4 indicate isoforms FBXO11-4 and FBXO11-1, as described previously.²⁰  The asterisk indicates a nonspecific band. (H) Cell pellets at day 5 of induced erythroid maturation. (I) Flow cytometry plots showing Band3 and CD49d expression at the indicated time points after induced erythroid maturation. (J) Summary of Band3 expression in multiple experiments performed as in panel I. Bars indicate the mean ± standard error of the mean (SEM) from 4 biological replicate experiments. (K) May-Grünwald-Giemsa–stained cells at maturation day 7. Red arrows indicate immature erythroblasts. Scale bar, 10 μm. **P < .01 (unpaired Student t test); n.s., not significant.