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ZFP36L2 is required for self-renewal of early burst-forming unit erythroid progenitors

Abstract

Stem cells and progenitors in many lineages undergo self-renewing divisions, but the extracellular and intracellular proteins that regulate this process are largely unknown. Glucocorticoids stimulate red blood cell formation by promoting self-renewal of early burst-forming unit–erythroid (BFU–E) progenitors1,2,3,4. Here we show that the RNA-binding protein ZFP36L2 is a transcriptional target of the glucocorticoid receptor (GR) in BFU–Es and is required for BFU–E self-renewal. ZFP36L2 is normally downregulated during erythroid differentiation from the BFU–E stage, but its expression is maintained by all tested GR agonists that stimulate BFU–E self-renewal, and the GR binds to several potential enhancer regions of ZFP36L2. Knockdown of ZFP36L2 in cultured BFU–E cells did not affect the rate of cell division but disrupted glucocorticoid-induced BFU–E self-renewal, and knockdown of ZFP36L2 in transplanted erythroid progenitors prevented expansion of erythroid lineage progenitors normally seen following induction of anaemia by phenylhydrazine treatment. ZFP36L2 preferentially binds to messenger RNAs that are induced or maintained at high expression levels during terminal erythroid differentiation and negatively regulates their expression levels. ZFP36L2 therefore functions as part of a molecular switch promoting BFU–E self-renewal and a subsequent increase in the total numbers of colony-forming unit–erythroid (CFU–E) progenitors and erythroid cells that are generated.

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Figure 1: Normal downregulation of ZFP36L2 during erythroid differentiation from the BFU–E stage is reversed by functional GR agonists.
Figure 2: ZFP36L2 is specifically required for BFU–E self-renewal.
Figure 3: ZFP36L2 is required for erythroid lineage expansion during stress erythropoiesis in vivo.
Figure 4: ZFP36L2 delays erythroid differentiation and preferentially binds to several mRNAs that are induced or maintained at higher expression levels during terminal erythroid differentiation.

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Gene Expression Omnibus

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All microarray data are available from the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo) under accession code GSE46216.

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Acknowledgements

This work is supported by NIH grant P01 HL 32262 to H.F.L.; L.Z. is supported by a graduate fellowship from Singapore-Massachusetts Institute of Technology Alliance. We thank C. Sieff, J. Zhang, and P. Ji for providing reagents, T. Chavarria and F. Reinhardt for assisting with mouse transplantation experiments, J. Shih for helping with the making of constructs, M. Bousquet and C. Patterson for discussion and assistance with mouse transplantation experiment and antibody testing, S. Gupta, J. Kwon and I. Barrasa for processing raw RNA-seq data, processing microarray and discussion of bioinformatics analyses, and Sanofi Aventis for providing many GR agonists.

Author information

Authors and Affiliations

Authors

Contributions

L.Z. conceived the project, designed and performed the experiments and bioinformatics analysis, analysed the data, and wrote the paper. L.P. assisted with luciferase reporter assays and BFU–E isolation. V.R.-E. performed the GR Chip-seq experiment. P.T. provided training in bioinformatics analyses. J.F. performed the initial experiments with the GR partial agonists. B.L. supervised part of the research. H.F.L. supervised the research and edited the paper.

Corresponding author

Correspondence to Harvey F. Lodish.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-17, Supplementary Table 4 and an additional reference. (PDF 1589 kb)

Supplementary Table 1

This file contains Supplementary Table 1 with legend, it describes Zfp36l2 as the most abundant gene that is upregulated by all GR agonists that stimulate BFU-E self- renewal. BFU-Es were cultured in self-renewal medium with indicated GR agonists for 4 hours and the gene expression pattern was profiled by RNA-seq. The expression levels of several genes that are commonly induced by functional GR agonists are shown as RPKM values. (XLSX 11 kb)

Supplementary Table 2

This file contains Supplementary Table 2 with its legend, listing mRNAs preferentially bound to Zfp36l2 in BFU-E cells. RIP-chip was performed on freshly isolated BFU-Es, where Zfp36l2 antibody or control IgG was incubated with BFU-E cell lysis and microarrays were performed on immunoprecipitated mRNAs. The microarray probes with relative intensity, calculated as a ratio between its intensity in the Zfp36l2 immunoprecipitates relative to control IgG immunoprecipitates. Those with a ratio of greater than 1.5 in both biological repeats are listed. (XLSX 245 kb)

Supplementary Table 3

This file contains Supplementary Table 3 with its legend, listing Zfp36l2 functional target genes. Microarray gene expression profiling was carried out on day 3 of in vitro cultured BFU-E infected with control shRNA or Zfp36l2 shRNAs. A group of genes were identified with lower expression levels in BFU-Es cultured with DEX, compared with their counterparts in BFU-Es cultured without DEX. The repression of several of these DEX repressed genes is mediated by Zfp36l2, as knockdown of Zfp36l2 eliminates this effect. The potential Zfp36l2 functional target genes were then identified by intersecting these repressed genes with the group of genes identified by RIP-chip assay. (XLSX 11 kb)

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Zhang, L., Prak, L., Rayon-Estrada, V. et al. ZFP36L2 is required for self-renewal of early burst-forming unit erythroid progenitors. Nature 499, 92–96 (2013). https://doi.org/10.1038/nature12215

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