Gene-centric functional dissection of human genetic variation uncovers regulators of hematopoiesis

  1. Satish K Nandakumar
  2. Sean K McFarland
  3. Laura M Mateyka
  4. Caleb A Lareau
  5. Jacob C Ulirsch
  6. Leif S Ludwig
  7. Gaurav Agarwal
  8. Jesse M Engreitz
  9. Bartlomiej Przychodzen
  10. Marie McConkey
  11. Glenn S Cowley
  12. John G Doench
  13. Jaroslaw P Maciejewski
  14. Benjamin L Ebert
  15. David E Root
  16. Vijay G Sankaran  Is a corresponding author
  1. Harvard Medical School, United States
  2. Broad Institute of MIT and Harvard, United States
  3. Ruprecht-Karls-University Heidelberg, Germany
  4. University of Oxford, United Kingdom
  5. Harvard Stem Cell Institute, United States
  6. Harvard University, United States
  7. Cleveland Clinic, United States
  8. Brigham and Women’s Hospital, United States
  9. Dana-Farber Cancer Institute, United States
  10. Howard Hughes Medical Institute, United States
6 figures, 1 table and 1 additional file

Figures

Figure 1 with 3 supplements
Design and Execution of an shRNA Screen Using Blood Cell Trait GWAS Hits to Identify Genetic Actors in Erythropoiesis.

(A) Overview of shRNA library design.75 loci associated with red blood cell traits (van der Harst et al., 2012) were used as the basis to calculate 75 genomic windows of LD 0.8 or greater from the …

https://doi.org/10.7554/eLife.44080.002
Figure 1—source data 1

Table containing annotations and information for the 75 SNPs used to seed the shRNA library.

https://doi.org/10.7554/eLife.44080.006
Figure 1—source data 2

Table containing annotations and information for all hairpins, as well as shRNA counts for each time point and replicate.

https://doi.org/10.7554/eLife.44080.007
Figure 1—figure supplement 1
Characteristics of GWAS Loci and Gene Selection for Pooled Screen.

(A) Counts of loci from among the original 75 annotated with linkage to each of the six RBC traits, hemoglobin (Hb), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration …

https://doi.org/10.7554/eLife.44080.003
Figure 1—figure supplement 2
Feasibility of Loss of Function Approaches to Perform Pooled Screens in Primary Hematopoietic Stem and Progenitor Cells (HSPCs).

(A) Schematic of the loss of function lentiviral constructs tested for pooled screens in primary CD34+ cells. (B) FACS plots showing the proportion of infected GFP+ cells 4 days after transduction …

https://doi.org/10.7554/eLife.44080.004
Figure 1—figure supplement 3
Pooled shRNA screen in primary HSPCs undergoing erythroid differentiation.

(A) Histogram showing distribution of number of independent hairpins included in the library to target each of the candidate’s genes. (B) Representative FACS plots of erythroid cell surface markers …

https://doi.org/10.7554/eLife.44080.005
Figure 2 with 2 supplements
Summary Characterization of shRNA Screen Outcomes.

(A) Kernel density plot showing library representation as log2 shRNA CPM across all hairpins. (B) shRNA abundance log2 fold changes from day 4 to day 16. Represented values are the mean of hairpin …

https://doi.org/10.7554/eLife.44080.008
Figure 2—figure supplement 1
shRNA abundance log2 fold changes from day four to each of the other time points.

Represented values are the mean of hairpin abundance log2 fold changes across hairpins for each gene and two standard deviations.

https://doi.org/10.7554/eLife.44080.009
Figure 2—figure supplement 2
Scatter plots showing agreement of replicate observations across independent CD34+ donor populations.
https://doi.org/10.7554/eLife.44080.010
Figure 3 with 3 supplements
Statistical Modeling of Gene Effect Accounting for Off-target shRNA Confounders.

(A) Bar graph showing the 38 of 75 loci in the screen with at least one corresponding statistically significant (FDR < 0.1, β >0.1) gene effect causing either a positive or negative log2 fold change …

https://doi.org/10.7554/eLife.44080.011
Figure 3—source data 1

Table containing the R model output for each gene.

https://doi.org/10.7554/eLife.44080.015
Figure 3—figure supplement 1
Additional Characterization of Modeling Outcomes.

(A) Histogram showing the number of gene hits identified at each of the 40 loci with at least one significant gene effect detected. Statistical model output for each gene in screen available in Figur…

https://doi.org/10.7554/eLife.44080.012
Figure 3—figure supplement 2
K562 Essentiality Scores Comparing Hit Genes vs.Genes Implicated by Other Traits.

(A) Permuted enrichment of essentiality among the set of hit genes vs. randomly chosen sets of genes from the human genome. (B) Permuted enrichment of essentiality among the set of hit genes vs. …

https://doi.org/10.7554/eLife.44080.013
Figure 3—figure supplement 3
Heat map depicting strength of expression (as z scores within each gene) for each of the 77 identified hit genes throughout the specific stages of fetal erythropoiesis.

Purple boxes highlight the cell types that were enriched for expression of hit genes.

https://doi.org/10.7554/eLife.44080.014
Analysis of Interactions Among Members of the Hit Set Identifies Signaling/Transcription, Membrane, and mRNA Translation-Related Subnetworks Important to Erythropoiesis.

STRING interaction network analysis identifies signaling/transcription, membrane, and mRNA translation-related subnetworks important to erythropoiesis embedded in the genes identified in the screen …

https://doi.org/10.7554/eLife.44080.016
Figure 5 with 1 supplement
Transferrin receptor two is a Negative Regulator of Human Erythropoiesis.

(A) Quantitative RT-PCR and (B) Western blot showing the expression of TFR2 in human CD34+ cells five days post-infection with the respective lentiviral shRNAs targeting TFR2 (TFR2 sh1 and sh2) and …

https://doi.org/10.7554/eLife.44080.017
Figure 5—figure supplement 1
Additional Analysis Showing Transferrin Receptor two is a Negative Regulator of Human Erythropoiesis.

(A) Representative FACS plots of alternate erythroid cell surface markers CD49d (α4 integrin) and CD235a (Glycophorin A) expression at various time points during erythroid differentiation. (B) …

https://doi.org/10.7554/eLife.44080.018
Figure 6 with 2 supplements
SF3A2 is a Key regulator of Human Erythropoiesis and Modulates Erythropoiesis Defects in a Murine Model of MDS.

(A) Quantitative RT-PCR and (B) Western blot showing the expression of SF3A2 in human CD34+ cells five days post-infection with the respective lentiviral shRNAs targeting SF3A2 (sh1-4) and a control …

https://doi.org/10.7554/eLife.44080.019
Figure 6—source data 1

Table containing the DESeq2 output for differentially expressed genes in cells undergoing SF3A2 knockdown or control shRNA treatment.

https://doi.org/10.7554/eLife.44080.022
Figure 6—source data 2

Table containing the DESeq2 output for differentially expressed genes in MDS patients with and without mutations in SF3B1.

https://doi.org/10.7554/eLife.44080.023
Figure 6—source data 3

Tables containing the GO component (Table 1) and function (Table 2) enrichments calculated using GOrilla for cells undergoing SF3A2 knockdown or control shRNA treatment.

https://doi.org/10.7554/eLife.44080.024
Figure 6—source data 4

Tables containing the GO component (Table 1) and function (Table 2) enrichments calculated using GOrilla for MDS patient samples with and without mutations in SF3B1.

https://doi.org/10.7554/eLife.44080.025
Figure 6—source data 5

Tables containing the differential splicing analysis for cells undergoing SF3A2 knockdown or control shRNA treatment.

Categories of splice mutations presented in each table are alternative 3’ splice sites, alternative 5’ splice sites, mutually exclusive exons, retrained introns, and skipped exons, respectively.

https://doi.org/10.7554/eLife.44080.026
Figure 6—source data 6

Tables containing the differential splicing analysis for MDS patient patient samples with and without mutations in SF3B1.

Categories of splice mutations presented in each table are alternative 3’ splice sites, alternative 5’ splice sites, mutually exclusive exons, retrained introns, and skipped exons, respectively.

https://doi.org/10.7554/eLife.44080.027
Figure 6—figure supplement 1
Additional Analysis Showing SF3A2 is Required for Human Erythropoiesis.

(A) shRNAs targeting SF3A2 co-expressing a reporter GFP gene was infected into human CD34+ cells and cultured in erythroid conditions. GFP expression at various time points from three independent …

https://doi.org/10.7554/eLife.44080.020
Figure 6—figure supplement 2
Additional Analysis of Erythropoiesis Defects Observed in Sf3b1K700E Murine Erythroid Cells upon SF3A2 knockdown.

(A) Knockdown efficiency of shRNAs targeting SF3A2 in murine erythroleukemia (MEL) cells by western blot. (B) Total cell numbers of GFP +shRNA expressing bone marrow cells from wildtype (WT) and Sf3b…

https://doi.org/10.7554/eLife.44080.021

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Biological sample (Homo sapiens)CD34 + mobilized peripheral bloodFred Hutchinson Cancer Research Center
Cell line (Homo sapiens)UT-7/EPONARRID:CVCL_5202maintained in Sankaran laboratory
Cell line (Mus musculus)MELNAmaintained in Sankaran laboratory
Genetic reagent (Mus musculus)Sf3b1K700EObeng et al., 2016Dr. Benjamin L. Ebert (Brigham Women's Hospital, Boston MA)
Recombinant DNA reagent (lentiviral shRNA)PLKO.1-Puro (plasmid)Sigma-AldrichRRID
:Addgene_10878
Pol III based shRNA backbone
Recombinant DNA reagent (lentiviral shRNA)PLKO-GFP (plasmid)this paperGFP version of pLKO.1-Puro
Recombinant DNA reagent (lentiviral shRNA)SFFV-Venus-mir30 shRNA (plasmid)this paperPol II based shRNA backbone
Antibodymouse monoclonal anti-human CD235a-APCThermo Fisher ScientificCat#: 17-9987-42; RRID:AB_2043823FACS (5 ul per test)
Antibodymouse monoclonal anti-human CD71-FITCThermo Fisher ScientificCat#: 11-0719-42; RRID:AB_1724093FACS (5 ul per test)
Antibodymouse monoclonal anti-human CD71-PEcy7Thermo Fisher ScientificCat#: 25-0719-42; RRID:AB_2573366FACS (5 ul per test)
Antibodymouse monoclonal ant-human CD49d-PEMiltenyi BiotecCat#: 130-093-282; RRID:AB_1036224FACS (10 ul per test)
Antibodymouse monoclonal anti-human CD41a-PEThermo Fisher ScientificCat#: 12-0419-42; RRID:AB_10870785FACS (5 ul per test)
Antibodymouse monoclonal anti-human CD11b-PEThermo Fisher ScientificCat#: 12-0118-42; RRID:AB_2043799FACS (5 ul per test)
AntibodyRat monoclonal anti-mouse Ter119-APCThermo Fisher ScientificCat#: 17-5921-82; RRID:AB_469473FACS (0.25 ug/test)
AntibodyRat monoclonal anti-mouse CD71-PEThermo Fisher ScientificCat#: 12-0711-82; RRID:AB_465740FACS (0.5 ug/test)
Antibodymouse monoclonal anti-phospho STAT5 Alexa Fluor-647BD BioscienceCat#: 612599; RRID:AB_399882FACS (1:20)
Antibodymouse monoclonal anti-GAPDHSanta Cruz Biotechnologysc-32233;
RRID:AB_627679
Western (1:20,000)
Antibodymouse monoclonal anti-TFR2Santa Cruz Biotechnologysc-32271; RRID:AB_628395Western (1:200)
Antibodymouse monoclonal anti-SF3A2Santa Cruz Biotechnologysc-390444Western (1:1000)
Sequence-based reagentshLUCSigma-AldrichTRCN00000722595’- CGCTGAGTACTTCGAAATGTC-3’
Sequence-based
reagent
TFR2 sh1 (human)Sigma-AldrichTRCN00000636285’-GCCAGATCACTACGTTGTCAT-3’
Sequence-based reagentTFR2 sh2 (human)Sigma-AldrichTRCN00000636325-CAACAACATCTTCGGCTGCAT-3’
Sequence-based reagentSF3A2 sh1 (human)Sigma-AldrichTRCN00000000605’-CTACGAGACCATTGCCTTCAA-3’
Sequence-based reagentSF3A2 sh2 (human)Sigma-AldrichTRCN00000000615’-CCTGGGCTCCTATGAATGCAA-3’
Sequence-based reagentSF3A2 sh3 (human)Sigma-AldrichTRCN00000000625’-CAAAGTGACCAAGCAGAGAGA-3’
Sequence-based reagentSF3A2 sh4 (human)Sigma-AldrichTRCN00000000635’-ACATCAACAAGGACCCGTACT-3’
Commercial assay or kitRNeasy Mini KitQIAGENCat#: 74104
Commercial assay or kitiScript cDNA synthesis KitBio-RadCat#: 1708891
Commercial assay or kitiQ SYBR Green SupermixBio-RadCat#: 170–8882
Commercial assay or kitNucleoSpin Blood XL-Maxi kitClonetchCat#: 740950.1
Commercial assay or kitLineage Cell Depletion Kit (mouse)MiltenyiCat#: 130-090-858
Commercial assay or kitNextera XT DNA Library Preparation KitIlluminaCat#: FC-131–1096
Commercial assay or kitNextSeq 500/550 High Output Kit v2.5 (75 Cycles)IlluminaCat#: 20024906
Commercial assay or kitBioanalyzer High Sensitivity DNA AnalysisAgilentCat#: 5067–4626
Commercial assay or kitAgencourt AMPure XPBeckman-CoulterCat#: A63881
Commercial assay or kitTaKaRa Ex TaqDNA PolymeraseTakaraCat#: RR001B
Commercial assay or kitQubit dsDNA HS Assay KitThermo FisherCat#: Q32854
Chemical compound, drugHuman Holo-TransferrinSigma AldrichCat#: T0665-1G
Peptide, recombinant proteinHumulin R (insulin)LillyNDC 0002-8215-01
Peptide, recombinant proteinHeparinHospiraNDC 00409-2720-01
Peptide, recombinant proteinEpogen (recombinant erythropoietin)AmgenNDC 55513-267-10
Peptide, recombinant proteinRecombinant human stem cell factor (SCF)PeprotechCat#: 300–07
Peptide, recombinant proteinRecombinant human interleukin-3 (IL-3)PeprotechCat#: 200–03
Peptide, recombinant proteinRecombinant mousestem cell factor (SCF)R&D systemsCat# 455-MC-010
Peptide, recombinant proteinrecombinant mouse Insulin like Growth Factor 1 (IGF1)R&D systemsCat# 791 MG-050
Chemical compound, drugHoechst 33342Life TechnologiesCat#: H1399FACS (1:1000)
Chemical compound, drugFixation BufferBD BioscienceCat#: 554655
Chemical compound, drugPerm Buffer IIIBD BioscienceCat#: 558050
Chemical compound, drugMay-Grünwald StainSigma-AldrichCat#: MG500
Chemical compound, drugGiemsa StainSigma-AldrichCat#: GS500
Software, algorithmSTARDobin et al., 2013RRID:SCR_015899
Software, algorithmMISOKatz et al., 2010RRID:SCR_003124
Software, algorithmRThe R FoundationRRID:SCR_001905
Software, algorithmSalmonPatro et al., 2017RRID:SCR_017036
Software, algorithmGOrillaEden et al., 2009RRID:SCR_006848
Software, algorithmVEPMcLaren et al., 2016RRID:SCR_007931
Software, algorithmFlowJo version 10FlowJoRRID:SCR_008520
Software, algorithmGraphPad Prism 7GraphPad Software IncRRID:SCR_002798
Software, algorithmPython 2, 3Python Software FoundationRRID:SCR_008394
Software, algorithmPLINKChang et al., 2015RRID:SCR_001757
Software, algorithmPoolQBroad Institutehttps://portals.broadinstitute.org/gpp/public/software/poolq

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