Zfp281 (ZBP-99) plays a functionally redundant role with Zfp148 (ZBP-89) during erythroid development

Abstract

Erythroid maturation requires the concerted action of a core set of transcription factors. We previously identified the Krüppel-type zinc finger transcription factor Zfp148 (also called ZBP-89) as an interacting partner of the master erythroid transcription factor GATA1. Here we report the conditional knockout of Zfp148 in mice. Global loss of Zfp148 results in perinatal lethality from nonhematologic causes. Selective Zfp148 loss within the hematopoietic system results in a mild microcytic and hypochromic anemia, mildly impaired erythroid maturation, and delayed recovery from phenylhydrazine-induced hemolysis. Based on the mild erythroid phenotype of these mice compared with GATA1-deficient mice, we hypothesized that additional factor(s) may complement Zfp148 function during erythropoiesis. We show that Zfp281 (also called ZBP-99), another member of the Zfp148 transcription factor family, is highly expressed in murine and human erythroid cells. Zfp281 knockdown by itself results in partial erythroid defects. However, combined deficiency of Zfp148 and Zfp281 causes a marked erythroid maturation block. Zfp281 physically associates with GATA1, occupies many common chromatin sites with GATA1 and Zfp148, and regulates a common set of genes required for erythroid cell differentiation. These findings uncover a previously unknown role for Zfp281 in erythroid development and suggest that it functionally overlaps with that of Zfp148 during erythropoiesis.

Graphical abstract

Figure 1.

Gene targeting of Zfp148 locus. (A) Schematic diagram of the Zfp148 locus and gene targeting strategy, showing the Zfp148 allele with short Flippase recognition target (frt, F) and Cre recombinase recognition target (LoxP, L). (B) Diagram of the Zfp148 protein in WT and null allele showing >80% deletion, including all 4 zinc finger domains. (C) PCR-based genotyping of WT, Zfp148^fl/+, Zfp148^fl/fl, and Zfp148^−/− from tail DNA. (D-E) Western blot analysis of protein extracts from e14.5 WT, Zfp148^+/−, and Zfp148^−/− whole embryos on mixed genetic background strains using 2 independent anti-Zfp148 antibodies from Woo et al in panel D and Taniuchi et al in panel E. The epitope used to generate the antibodies is indicated on the schematic diagrams. (F) Photographs of representative Zfp148 conventional KO e18.5 embryos (top) and p14.5 (bottom) neonates on mixed C57BL/6, CD-1, and 129/Sv genetic background showing the runted postnatal phenotype. Scale bars, 1 cm. (G) Survival curves of Zfp148 WT (n = 14), Zfp148^+/− (n = 21), and Zfp148^−/− (n = 11) mice over the first 6 weeks of life. A significant difference in survival curve is observed between WT and Zfp148^−/− (Mantel-Cox Log-rank P = .0319), whereas the WT and Zfp148^+/− are not significant (Log-rank P = ns). (H) Growth curves of male WT (n = 5), Zfp148^+/− (n = 7), and Zfp148^−/− (n = 3) (left) panel, and female WT (n = 8), Zfp148^+/− (n = 11), and Zfp148^−/− (n = 6) mice (right) over the first 6 weeks of life. Growth of both female and male Zfp148^−/− mice is significantly delayed compared with WT (2-tailed paired Student t test, P < .05). PEST, peptide domain rich in proline, glutamic acid, serine, and threonine.

Figure 2.

Defective erythropoiesis in mice with pan-hematopoietic Zfp148 deletion. (A-C) Peripheral blood counts and indices from Zfp148^fl/fl and Vav1-Cre; Zfp148^fl/fl mice at 15 to 26 weeks of age. (D) Representative flow cytometric analysis profiles of bone marrow for erythroid progenitor maturation using anti-CD71 and anti-Ter119 antibodies in Zfp148^fl/fl and Vav1-Cre; Zfp148^fl/fl mice. (E) Dot plots depicting the percentage of live BM cells in non- and early erythroid cells (CD71⁻Ter119⁻), late proerythroblasts (CD71⁺Ter119⁻), early erythroid cells (CD71⁺Ter119⁺) and RBCs (CD71⁻Ter119⁺) from the flow analysis in panel D (n = 5). (F-G) Representative flow cytometric analysis profile for spleen as in panels D and E. (H-I) Recovery from phenylhydrazine (PHZ)-induced hemolytic anemia in male (n = 8) and female (n = 12) mice. Spun hematocrit levels from zfp148^fl/fl, Vav1-Cre, and Zfp148^fl/fl control littermates preceding and following a 2-day course of phenylhydrazine intraperitoneal injection. A baseline spun hematocrit was obtained prior to phenylhydrazine injection and then repeated on days 3, 4, 5, 6, and/or 7 following initial injection. Hgb, hemoglobin; MCH, mean cell hemoglobin; MCV, mean cell volume; WBC, white blood cell.

Figure 3.

Zfp281 expression and physical interaction with GATA1 in erythroid cells. (A) qRT-PCR analysis showing Zfp148 and Zfp281 mRNA levels relative to Gapdh (glyceraldehyde-3-phosphate dehydrogenase) in C57BL/6 mouse tissues. (B) Heat map of Zfp148 and Zfp281 mRNA levels in flow cytometric sorted erythroid progenitor cells from ex vivo differentiated PBMC (GSE22552) (left) and CD34⁺ cord blood cells (GSE53983) (right). (C) Heat map showing mean copy number of Zfp148 and Zfp281 protein per erythroid progenitor cell, determined by mass spectrometry–based absolute quantification approach (PXD004313, PXD004314, PXD004315, and PXD004316). Following the expansion of cord blood CD34⁺ cells, CD36⁺ progenitors were flow sorted and differentiated ex vivo under erythroid conditions. Erythroid Prog1 and 2 equates to burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) cells, respectably. (D) Western blot analysis of Zfp281 protein in major hematopoietic organs, spleen (Sp), bone marrow (BM), and thymus (Th) in mice. (E) Western blot showing Zfp281 protein levels during erythroid ex vivo differentiation of hCD34⁺ cells. (F) Western blot analysis following streptavidin affinity purification (SA-IP) from nuclear extracts of MEL cells stably expressing Bir A alone or Bir A and recombinant GATA1 containing an amino-terminal FLAG and BirA recognition motif (FB-GATA1). Two percent of the input material is shown. (G) Western blot analysis following SA-IP from nuclear extracts of K562 cells stably expressing Bir A alone or Bir A and recombinant FB-Zfp148. Two percent of the input material is shown. (H) Western blot analysis following SA-IP from nuclear extracts of K562 cells stably expressing Bir A or Bir A and recombinant FB-Zfp281. Two percent of the input material is shown. Lg, large; Sm, small.

Figure 4.

Common gene targets of Zfp148 and Zfp281 in erythroid cells. (A) Venn diagram showing the overlap of Zfp148 and Zfp281 chromatin occupancy peaks (left). MEME-ChIP motif analysis result showing 3 representative motifs for unique and common peaks (right). (B) Pie graphs showing the distribution of chromatin occupancy peak location. UTRs, untranslated regions. (C) Venn diagram showing the number of genes commonly occupied by Zfp148, Zfp281, and/or GATA1. (D) GO term analysis of the 658 target genes common to Zfp148 and Zfp281, but devoid of GATA1. (E) GO term analysis of the 245 target genes common to GATA1 and Zfp148 and/or Zfp281. (F) GO term analysis of the 534 genes unique to GATA1. GO term analysis of unique Zfp148 and Zfp281 target genes are shown in supplemental Figure 8. (G) Distribution of Zfp148/Zfp281 peaks with respect to GATA1 peaks at the 245 commonly occupied genes. reg., regulation.

Figure 5.

Redundant roles of Zfp281 and Zfp148 in γ-globin gene expression in K562 cells. (A) Western blot showing knockout (KO) of Zfp148 in K562 cells by CRISPR/Cas9. (B) Western blot showing knockdown of Zfp281 protein levels in K562 cells using lentiviral transduction of shRNA constructs c#1 and c#2. A lentiviral shRNA against luciferase (shLuc) is shown as a control. (C) Western blot showing Zfp148 and Zfp281 protein levels in Zfp148 KO cells or control cells transduced with shZfp281c#1 or shLuc. (D) Representative BioChIP-seq signals at the γ-globin locus in hemin-induced K562 cells expressing Bir A alone or Bir A and ^BioZfp148 or ^BioZfp281. Peak calls for H3K27ac, H3K27me3, H3K4me1, H3K4me2, H3K4me3, and H3K9ac from ENCODE are indicated. (E) Quantitative RT-PCR analysis of γ-globin mRNA transcripts from cells in panel C. The levels were normalized to GAPDH mRNA transcript levels and are shown relative to the Zfp148 WT and shLuc control. HBG, hemoglobin subunit gamma.

Figure 6.

Redundant roles of Zfp148 and Zfp281 in primary murine fetal liver erythroid cells. (A) Western blot showing knockdown of murine Zfp281 protein levels in MEL cells transduced with lentiviral shRNA constructs shZfp281c#3, zhZfp281c#4, and shLuc. (B) Schematic diagram showing an experimental timeline of ex vivo culture, lentiviral transduction, and puromycin selection of fetal liver cells. (C) Representative benzidine stains with May-Grünwald counterstain of cytospun cells from murine e12.5-e13.5 d.p.c. Zfp148^−/− or WT (littermates) fetal liver cells transduced with the indicated shRNA lentiviruses, selected with puromycin, and ex vivo differentiated into erythroid cells (8 days in expansion medium followed by 48 hours in differentiation medium; see “Methods”). Magnification ×600. (D) Bar graph shows blinded benzidine counts from 5 biologic replicates of cultures in panel C. (E-F) Representative CD71/Ter119 flow cytometric plots and quantitation of the cultures in panel C. Each plot shows the percentage of R1 to R5 populations in 5 biological replicates. *P < .05; **P < .01.