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Unexpected role for p19INK4d in posttranscriptional regulation of GATA1 and modulation of human terminal erythropoiesis

Xu Han, Jieying Zhang, Yuanliang Peng, Minyuan Peng, Xiao Chen, Huiyong Chen, Jianhui Song, Xiao Hu, Mao Ye, Jianglin Li, Vijay G. Sankaran, Christopher D. Hillyer, Narla Mohandas, Xiuli An and Jing Liu

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    CDKI expression in human terminal erythroid differentiation. (A) RNA-seq data showing CDKI members’ expression (fragments per kilobase of transcript per million) at each distinct stage of human terminal erythroid differentiation. Baso-E, basophilic erythroblast; ortho-E, orthochromatic erythroblast; poly-E, polychromatic erythroblast; pro-E, proerythroblast. (B) Representative image of western blotting of p19INK4d, p18INK4c, and p27KIP1 expression in whole-cell lysates prepared from erythroblasts cultured for different times (left). Quantitative analysis of data from 3 independent experiments of protein expression levels (right). GAPDH was used as a loading control. (C) qRT-PCR results showing p19INK4d expression in erythroblasts infected with lentivirus containing control (Lucif shRNA) or p19INK4d shRNA on day (D) 7, D 11, and D 15 of culture. The results were normalized to GAPDH mRNA. (D) Representative images of western blotting showing p19INK4d expression levels in erythroblasts infected with Lucif shRNA or p19INK4d shRNA (left). Quantitative analysis of protein expression data from 3 independent experiments (right). The results were normalized to GAPDH protein. Statistical analysis of data of 3 independent experiments and bar plot represents mean ± SD of triplicate samples. ***P < .001.

  • Figure 2.

    Effects of p19INK4don human terminal erythroid differentiation. (A) Representative images of flow cytometry analysis of GPA expression in erythroblasts infected with Lucif shRNA or p19INK4d shRNA on day 7; red numbers show statistical analysis of the GPA-positive rate from 3 independent experiments. (B) Representative images of flow cytometry analysis of band 3 and α4-integrin expression on D 7 and D 13 of GPA-positive erythroblasts infected with Lucif shRNA or p19INK4d shRNA. (C) Erythroid cell growth curves determined by manual cell counting of erythroblasts infected with Lucif shRNA or p19INK4d shRNA. (D) The cell-cycle distribution results of BrdU assay from erythroblasts infected with Lucif shRNA or p19INK4d shRNA. (E) Representative images of flow cytometry analysis of apoptosis by annexin V/7AAD staining in erythroblasts infected with Lucif shRNA or p19INK4d shRNA (top). The red numbers indicate percentage. Quantitative analysis from 3 independent experiments is shown (bottom). Statistical analysis of the data from 3 independent experiments and bar plot represents mean ± SD of triplicate samples. *P < .05.

  • Figure 3.

    p19INK4dknockdown decreased GATA1 protein expression and ectopic GATA1 expression rescues the differentiation delay. (A) Representative images of western blotting analysis showing GATA1, 4.1R, tropomodulin (Tmod), HBG, p18INK4c, KLF1, and CD44 expression in erythroblasts infected with Lucif shRNA or p19INK4d shRNA (day 11 cells) (left). Quantitative analysis of protein expression data from 3 independent experiments (right). GAPDH was used as a loading control and the results were normalized to GAPDH protein expression. (B) qRT-PCR analysis of GATA1 mRNA levels in erythroblasts infected with Lucif shRNA or p19INK4d shRNA. The results were normalized to GAPDH mRNA. (C) Representative data of flow cytometry analysis of erythroblasts infected with Lucif shRNA, p19INK4d shRNA, and p19INK4d shRNA combined with GATA1 overexpression. GPA expression was monitored on D 9 cells (left) and expression of band 3 and α4-integrin on D 15 cells (right). The red numbers indicate statistical analysis of GPA-positive rate from 3 independent experiments. (D) Representative images of western blotting showing GATA1 levels in whole cell lysates prepared from erythroblasts (left). Quantitative analysis of expression data from 3 independent experiments (right). GAPDH was used as a loading control. Statistical analysis of data from 3 independent experiments and bar plot represents mean ± SD of triplicate samples. *P < .05, **P < .01, ***P < .001.

  • Figure 4.

    p19INK4dknockdown causes abnormal nuclear morphology of erythroblasts and ectopic GATA1 expression can reverse the abnormal phenotype. (A) Representative images of Lucif shRNA or p19INK4d shRNA-infected erythroblasts (day 15 cells) and quantitative analysis of cells with abnormal nuclei. (B) Flow cytometry analysis of sorted annexin V erythroblasts (left). Representative images of sorted annexin V erythroblasts (right). (C) Representative images of sorted erythroblasts at distinct development stages after Lucif shRNA or p19INK4d shRNA infection (left). Quantitative analysis of abnormal nuclear morphology of sorted erythroblasts at the polychromatic and orthochromatic stages from 3 independent experiments (right). (D) Representative images of erythroblasts infected with Lucif shRNA, p19INK4d shRNA, and p19INK4d shRNA combined with GATA1 overexpression (day 15 cells) (left). Quantitative analysis of nuclear morphology data from 3 independent experiments (right). (E) Representative images of western blotting showing GATA1 expression in erythroblasts transfected with GATA1 siRNA or scramble siRNA (day 15 cells) (left). Quantitative analysis of expression data from 3 independent experiments (right). GAPDH was used as a loading control and the results were normalized to GAPDH protein. (F) Representative images of erythroblasts (day 15 cells) transfected with GATA1 siRNA or scramble siRNA (left). Quantitative analysis of nuclear morphology data from 3 independent experiments (right). The percentage of abnormally nucleated cells = counts of abnormally nucleated cells in 2000 cells/2000. Statistical analysis of data from 3 independent experiments and bar plot represents mean ± SD of triplicate samples. **P < .01, ***P < .001.

  • Figure 5.

    Effect of p19INK4dknockdown on HSP70 expression, localization, and ERK activity. (A) Representative images of western blotting showing HSP70 and HSP27 expression in erythroblasts infected with Lucif shRNA or p19INK4d shRNA (left). Quantitative analysis of protein expression data from 3 independent experiments (right). GAPDH was used as a loading control and the results were normalized to GAPDH protein. (B) Representative immunofluorescence images showing HSP70 localization in erythroblasts infected with Lucif shRNA or p19INK4d shRNA. DAPI was used to stain the nucleus. (C) Western blotting analysis of nuclear and cytoplasmic fractions of HSP70. RCC1 and α-tubulin were used as nuclear and cytoplasmic markers, respectively. (D) Representative images of western blotting for p-ERK, ERK, p-AKT, and AKT in erythroblasts infected with Lucif shRNA or p19INK4d shRNA (left). Quantitative analysis of western blotting data from 3 independent experiments is shown (right). GAPDH was used as a loading control and the results were normalized to GAPDH protein. (E) Representative images of western blotting showing p-ERK, HSP70, GATA1, and KLF1 expression level in erythroblasts treated with 0, 5, or 10 μM FR180204 (left). Quantitative analysis of western blotting data from 3 independent experiments (right). GAPDH was used as a loading control and the results were normalized to GAPDH protein. (F) Representative immunofluorescence images showing HSP70 localization in erythroblasts treated with 0 or 10 μM FR180204. DAPI was used to stain the nucleus. (G) Western blotting analysis of nuclear and cytoplasmic fractions of HSP70 in erythroblasts from untreated and treated with 10μM FR180204. RCC1 and α-tubulin were used as nuclear and cytoplasmic markers, respectively. Statistical analysis of data from 3 independent experiments and bar plot represents mean ± SD of triplicate samples. *P < .05, **P < .01, ***P < .001.

  • Figure 6.

    p19INK4dinteracts with and negatively regulates PEBP1 and PEBP1 links p19INK4dwith the ERK pathway. (A) Representative images of silver-stained gels of IP proteins using IgG and p19INK4d antibody. (Upper) Nonspecific bands; (lower) specific band is marked with a red arrow. (B) Representative electrospray ionization-MS and MS/MS profiling of a tryptic peptide that owns higher content. The top right corner is the amino acid sequence of this peptide and the specific amino acid sequence belongs to PEBP1 through identification of proteins from the protein database. (C) Representative immunofluorescence images showing p19INK4d and PEBP1 localization in erythroblasts. DAPI was used to stain the nucleus. (D) Representative images of co-IP experiments with a p19INK4d (left) or PEBP1 antibody (right). (E) Representative images of western blotting showing PEBP1 expression in erythroblasts infected with Lucif shRNA or p19INK4d shRNA (left). Quantitative analysis of western blotting data from 3 independent experiments (right). GAPDH was used as a loading control and the results were normalized to GAPDH protein. (F) Representative images of western blotting showing PEBP1, p-ERK, ERK, HSP70, and GATA1 levels in erythroblasts transfected with PEBP1 siRNA or scramble siRNA (left). Quantitative analysis of western blotting data from 3 independent experiments (right). GAPDH was used as a loading control and the results were normalized to GAPDH protein. Statistical analysis of data from 3 independent experiments and bar plot represents mean ± SD of triplicate samples. * P < .05, ** P < .01.

  • Figure 7.

    Working model of p19INK4dfunction during human terminal erythroid differentiation. The blue arrow denotes a “decreased” expression, whereas the red arrow denotes an “increased” expression. p19INK4d knockdown increases PEBP1 expression and impairs the p-ERK-HSP70-GATA1 pathway, which delays human terminal erythroid differentiation and leads to generation of abnormal nucleus.