Control of human hemoglobin switching by LIN28B-mediated regulation of BCL11A translation

Abstract

Increased production of fetal hemoglobin (HbF) can ameliorate the severity of sickle cell disease and β-thalassemia1. BCL11A represses the genes encoding HbF and regulates human hemoglobin switching through variation in its expression during development2,3,4,5,6,7. However, the mechanisms underlying the developmental expression of BCL11A remain mysterious. Here we show that BCL11A is regulated at the level of messenger RNA (mRNA) translation during human hematopoietic development. Despite decreased BCL11A protein synthesis earlier in development, BCL11A mRNA continues to be associated with ribosomes. Through unbiased genomic and proteomic analyses, we demonstrate that the RNA-binding protein LIN28B, which is developmentally expressed in a pattern reciprocal to that of BCL11A, directly interacts with ribosomes and BCL11A mRNA. Furthermore, we show that BCL11A mRNA translation is suppressed by LIN28B through direct interactions, independently of its role in regulating let-7 microRNAs, and that BCL11A is the major target of LIN28B-mediated HbF induction. Our results reveal a previously unappreciated mechanism underlying human hemoglobin switching that illuminates new therapeutic opportunities.

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Fig. 1: Developmental expression of BCL11A in erythroid cells is regulated by altered protein synthesis.
Fig. 2: RNA-binding protein LIN28B associates with ribosomes in erythroid cells and is developmentally regulated.
Fig. 3: LIN28B alters BCL11A mRNA translation independently of the canonical let-7 microRNA pathway.
Fig. 4: BCL11A mRNA is directly bound by LIN28B and is the major target for fetal hemoglobin induction.

Data availability

The massively parallel sequencing data associated with this manuscript are available in the Gene Expression Omnibus database under accession code GSE118359.

The original mass spectra and the protein sequence database used for searches have been deposited in the public proteomics repository MassIVE (http://massive.ucsd.edu) and are accessible at accession MSV000084443. Source data for Figs. 14 and Extended Data Figs. 1, 35 and 10 are available online.

Code availability

Custom computer code for reproduction of sequencing-based analyses is available at https://github.com/sankaranlab/translation-regulation-bcl11a.

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Acknowledgements

We are grateful to D. Nathan, G. Daley, L. Zon and members of the Sankaran laboratory for valuable guidance and suggestions. We are grateful to D. Tenen for assistance with RNA immunoprecipitation and H. Keshishian for assistance with mass spectra data access. This work was supported by National Institutes of Health grants nos. U01 HL117720, R01 DK103794 and R33 HL120791 (to V.G.S.) and P01 DK32094 (to N.M.), a gift from the Lodish Family to Boston Children’s Hospital (to V.G.S.) and the New York Stem Cell Foundation (to V.G.S.). V.G.S. is a NYSCF–Robertson Investigator.

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Authors

Contributions

A.B. and V.G.S. conceived and designed the study. A.B., M.M. and K.E.M. performed experiments and analyzed data. C.A.L. and J.C.U. performed analyses. C.R.H., M.S., J.L., Y.W., Y.H., X.W., L.G., C.M.R., X.A., H.A.C., N.M., S.A.C., J.-J.C., S.H.O. and E.S.L. provided experimental assistance, reagents and advice. V.G.S. supervised all experimental and analytic aspects of this work. N.M. and V.G.S. acquired funding. A.B., M.M. and V.G.S. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Anindita Basak or Vijay G. Sankaran.

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Extended data

Extended Data Fig. 1 BCL11A protein and mRNA expression in fetal, newborn and adult erythroid cells.

a, Representative flow cytometry plots showing CD71 and CD235a surface expression in newborn (left) and adult (right) at differentiation day 7. Mean ± s.d shown. (n = 3; 3 biologically independent experiments). b, Representative westerns showing BCL11A expression, with GAPDH as control, in newborn (left) and adult (right) at differentiation day 7 (5 independent experiments). c, BCL11A mRNA expression (normalized to GAPDH), in newborn and adult at differentiation day 7 (n = 3; 3 biologically independent experiments). Mean ± s.d shown. Two-sided Student t-test used. N.S., not significant; P = 0.7153. d, Representative westerns showing BCL11A expression, with GAPDH used as control, at differentiation days 4, 7, & 10 (5 independent experiments). e, BCL11A mRNA expression (normalized to GAPDH) in newborn and adult (n = 3; 3 independent experiments) at differentiation days 4, 7, and 10. Mean ± s.d shown. Two-sided Student t-test used. N.S., not significant; P = 0.4395 (d4), P = 0.3051 (d7), P = 0.3672 (d10). f, XL isoform of BCL11A mRNA with 4 exons and qRT–PCR primers. FP, forward primer; RP, reverse primer. g, BCL11A mRNA expression (normalized to GAPDH), in newborn and adult (n = 3; 3 independent experiments) with 2 independent primer sets at differentiation day 7. Mean ± s.d shown. Two-sided Student t-test used. N.S., not significant, P = 0.2365 (pair 1), P = 0.4099 (pair 2). h, Stacked bar graphs showing fetal (HbF, red) and adult (HbA, grey) hemoglobin abundance (by HPLC) in newborn and adult on differentiation day 16. i, Representative westerns showing BCL11A expression with GAPDH as control at differentiation days 4, 7, 10, and 12 in fetal and adult (3 independent experiments). Blots have been cropped and the corresponding full blots are available in the Source Data files. Source data

Extended Data Fig. 2 Assessment of BCL11A and other mRNAs expressed in newborn and adult erythroid cells using RNA sequencing.

a, Scatter plots of gene expression, determined from RNA-sequencing reads, expressed as log2 TPM (transcripts per million reads) in adult and newborn primary erythroid cells. ‘r’ represents the Pearson correlation coefficient. b, Expression of BCL11A, represented as log2 TPM, between newborn (n = 2) and adult (n = 2) erythroid cells. Error bars show s.d. c, BCL11A mRNA structure and splicing is comparable between developmental stages. Sashimi plots depicting exon-exon spanning reads are shown for annotated isoforms of BCL11A. d, Representation of known BCL11A isoforms. e, Relative abundances of BCL11A isoforms in newborn (n = 2) and adult (n = 2). Error bars show s.d. No transcript was differentially expressed at P < 0.01 between the newborn and adult cells.

Extended Data Fig. 3 BCL11A protein expression is regulated via translation by polysome-associated LIN28B.

a, Western blots for BCL11A in the input and flow-through (FT) fractions of the immunoprecipitate in newborn (left) and adult (right) erythroid cells (2 independent experiments). b, Western blots for BCL11A and GATA1 in the total immunoprecipitate (IP) in newborn (left) and adult (right) erythroid cells after L-azidohomoalanine (L-AHA) labeling for 6 hours at day 7 of differentiation, followed by immunoprecipitation with BCL11A and GATA1 antibodies. c, Quantification of adult-BCL11A (blue) and newborn-BCL11A (purple) mRNAs across the different sucrose gradient fractions are shown as a percentage of the gradient. Cells were differentiated until day 7. Blots have been cropped and the corresponding full blots are available in the Source Data files. Source data

Extended Data Fig. 4 LIN28B association with polysomes and expression in newborn and adult cells.

Representative LIN28B occupancy across polysome fractions in newborn erythroid cells at day 7 of differentiation. LIN28B abundance is probed by western blot, using RPL5 and RPS20 as controls. Please note two gaps in the western blot between sequential polysome fractions that were placed to avoid overloading of proteins (2 independent experiments). Blots have been cropped and the corresponding full blots are available in the Source Data files. Source data

Extended Data Fig. 5 LIN28B partially dissociates from polysomes after RNase A treatment.

a, LIN28B occupancy across polysome fractions in erythroid cells either untreated (blue) or treated with RNase A (red). Experiment repeated 3 times. b, LIN28B abundance probed by western blot, using RPL5 and RPS20 as controls in the untreated sucrose gradient fractions. c, LIN28B abundance in polysome fractions with lysates digested with RNase A. Blots have been cropped and the corresponding full blots are available in the Source Data files. Source data

Extended Data Fig. 6 LIN28B expression in newborn and adult cells.

a, Volcano plot of differentially expressed genes between adult (n = 2) and newborn (n = 2) erythroid cells. Each dot is a gene with the value of the β coefficient (x-axis) from the sleuth linear model and the corresponding measure of statistical significance (y-axis). LIN28B is the most over-expressed gene in newborn cells compared to adult. Statistical test: generalized linear model from sleuth. b, Expression of LIN28B, represented as log2 TPM, between newborn and adult erythroid cells. Error bars show s.d. ***P < 0.001. Statistical test: generalized linear model from sleuth.

Extended Data Fig. 7 Effects of high-level LIN28B expression in adult erythroid cells.

a, BCL11A mRNA levels (normalized to GAPDH expression), upon high-level LIN28B expression (GFP-high) in adult erythroid cells, assessed on differentiation day 7 (n = 3; 3 biologically independent experiments). Mean is plotted and error bars show s.d. Two-sided Student t-test used. **P < 0.01. b, Relative γ-globin expression as a percentage of total globins (γ- and β-globins), upon high-level LIN28B expression (GFP-high) in adult erythroid cells, on differentiation day 15 (n=3; 3 independent experiments). Error bars show s.d. ***P < 0.001. c, Representative flow cytometry plots showing CD71 and CD235a surface expression in control (left) and physiological level LIN28B expressing (right) adult erythroid cells at day 7 of differentiation. Data represents mean ± s.d., n = 3; 3 biologically independent experiments.

Extended Data Fig. 8 LIN28B associates with BCL11A mRNA.

a, RNA-immunoprecipitation (RNA-IP) in newborn erythroid cells at day 7 of differentiation, with antibodies against LIN28B (4196, Cell Signaling) or control IgG. Detection of BCL11A, HMGA1, GATA1, ALAS2, LDB1, KLF1, and LMO2 mRNAs (n = 3; 3 independent experiments). Mean is plotted and error bars show s.d. Two-sided Student t-test used. **P < 0.01; N.S., not significant, P = 0.6395 (GATA1), P = 0.8782 (ALAS2), P = 0.8999 (LDB1), P = 0.9571 (KLF1), P = 0.9550 (LMO2). b, Detection of BCL11A, HMGA1, GATA1, ALAS2, LDB1, KLF1, and LMO2 mRNAs (n = 3; 2 independent experiments) in the input for LIN28B antibody (4196, Cell Signaling). Mean is plotted and error bars show s.d. Two-sided Student t-test used. N.S., not significant, P = 0.3867 (BCL11A), P = 0.8050 (HMGA1), P = 0.6420 (GATA1), P = 0.8656 (ALAS2), P = 0.6413 (LDB1), P = 0.3257 (KLF1), P = 0.7152 (LMO2). c, RNA-IP in newborn erythroid cells with antibodies against LIN28B (A303-588A, Bethyl Labs) or control rabbit IgG. Detection of BCL11A (left), HMGA1 (center), and GATA1 (right) mRNAs (n = 3; 2 independent experiments). Mean is plotted and error bars show s.d. Two-sided Student t-test used. *P < 0.05; ***P < 0.001; N.S., not significant, P = 0.5393. d, Detection of BCL11A (left), HMGA1 (center) and GATA1 (right) mRNAs (n = 3; 3 independent experiments) in the input for LIN28B antibody (A303-588A, Bethyl Labs). Mean is plotted and error bars show s.d. Two-sided Student t-test used. N.S., not significant, P = 0.5261 (BCL11A), P = 0.4871 (HMGA1), P = 0.9464 (GATA1).

Extended Data Fig. 9 CLIP-seq of LIN28B in newborn erythroid cells identifies genome-wide binding peaks.

a, Genomic annotation of LIN28B binding sites. Peaks passing a 1% IDR for the consensus between replicates and a 5% false-discovery rate for each replicate are shown. b, 4-mer motifs associated with LIN28B binding sites. The dashed lines represent a threshold of 60%. 17 4-mers are present at >60% in both exons and UTRs, including GGAG, GAAG, and AAGA. c, Fold enrichment of genomic distance covered by LIN28B binding peaks compared to the genome-wide proportions. d, Rank-order enrichment of LIN28B binding peaks genome-wide. Each peak’s enrichment, measured by its -log10 q-value is plotted against its rank genome-wide for both replicates (n = 2). Statistical test: macs2 peak calling algorithm. e, Total coverage and mutation proportion for each replicate of the LIN28B/BCL11A binding site. f, Volcano plots showing the log-fold change with Benjamini-Hochberg adjusted P-values for LIN28B target genes compared between newborn (n = 2) and adult (n = 2) proerythroblasts. Statistical test: generalized linear model from sleuth.

Extended Data Fig. 10 Suppression of γ-globin by BCL11A expression in newborn erythroid cells.

a, γ-globin levels upon BCL11A expression in newborn erythroid cells on differentiation day 12 (n = 3; 3 biologically independent experiments) in control and BCL11A expressing cells. Mean is plotted and error bars show s.d. Two-sided student t-test used. ****P < 0.0001. b, Representative western blots showing BCL11A expression from lentiviral construct in newborn erythroid cells at day 12 of differentiation. GAPDH is used as a loading control (3 independent experiments). c, Representative flow cytometry plots showing CD71 and CD235a surface expression in control (left) and BCL11A expressing (right) newborn erythroid cells at days 8, 10 and 12 of differentiation (3 biologically independent experiments). Blots have been cropped and the corresponding full blots are available in the Source Data files. Source data

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Basak, A., Munschauer, M., Lareau, C.A. et al. Control of human hemoglobin switching by LIN28B-mediated regulation of BCL11A translation. Nat Genet 52, 138–145 (2020). https://doi.org/10.1038/s41588-019-0568-7

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