Rational targeting of a NuRD subcomplex guided by comprehensive in situ mutagenesis

Abstract

Developmental silencing of fetal globins serves as both a paradigm of spatiotemporal gene regulation and an opportunity for therapeutic intervention of β-hemoglobinopathy. The nucleosome remodeling and deacetylase (NuRD) chromatin complex participates in γ-globin repression. We used pooled CRISPR screening to disrupt NuRD protein coding sequences comprehensively in human adult erythroid precursors. Essential for fetal hemoglobin (HbF) control is a non-redundant subcomplex of NuRD protein family paralogs, whose composition we corroborated by affinity chromatography and proximity labeling mass spectrometry proteomics. Mapping top functional guide RNAs identified key protein interfaces where in-frame alleles resulted in loss-of-function due to destabilization or altered function of subunits. We ascertained mutations of CHD4 that dissociate its requirement for cell fitness from HbF repression in both primary human erythroid precursors and transgenic mice. Finally we demonstrated that sequestering CHD4 from NuRD phenocopied these mutations. These results indicate a generalizable approach to discover protein complex features amenable to rational biochemical targeting.

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Fig. 1: Dense mutagenesis of NuRD genes by CRISPR-Cas9 pooled screening.
Fig. 2: NuRD subcomplex expression.
Fig. 3: Maps of functional NuRD subcomplex.
Fig. 4: In-frame deletions disrupt MTA2 function.
Fig. 5: Targeting CHD4 CHDCT2 uncouples HbF induction from cytotoxicity.
Fig. 6: GATAD2A ZF sequesters CHD4 from NuRD.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. Data and analysis are included in the article and Supplementary Note. Mass spectrometery raw data is accessable from proteomecentral under accession no. PXD009793. Next-generation sequencing (NGS) data (RNA-seq and CRISPR screen) are available from NCBI SRA portal under accession No. PRJNA496556.

Code availability

For analysis and visualization of functional readout from tiled pooled CRISPR screen, we used a custom computational pipeline available at https://gitlab.com/bauerlab/crispro.

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Acknowledgements

We thank X. Wang, C. Brendel, E. C. Smith, A. Gutierrez, G. D. Ginder and D. C. Williams for useful discussions and J. Desimini for graphical assistance. V.A.C.S. was supported by an Individual Travel Grant (ITG) from Radboud University. D.S.V. was supported by the Cooley’s Anemia Foundation. L.M.K.D. was supported by NHLBI (no. 5T32HL007574-36) and a Burroughs Wellcome Fund Postdoctoral Enrichment Grant (PDEP 1015098). S.A.W. and K.L. were supported by NIAID (no. R01AI117839) and NIGMS (no. R01GM115911). L.P. was supported by a National Human Genome Research Institute (NHGRI) Career Development Award (no. R00HG008399). T.M. was supported by NIH (no. 5R01DK111455) and JSPS Grant-in-Aid for Scientific Research A (no. 17H01567). A.K. is the Damon Runyon-Richard Lumsden Foundation Clinical Investigator and acknowledges support of the St. Baldrick’s Arceci Innovation Award, and NCI grant nos. R01 CA204396 and P30 CA008748. Generation of the mouse model was supported by a NIDDK Cooperative Centers of Excellence in Hematology (CCEH) award (no. U54DK110805) to S.H.O. S.H.O. was supported by the Doris Duke Charitable Foundation and is an Investigator of the Howard Hughes Medical Institute. D.E.B. was supported by NIDDK (grant nos. K08DK093705 and R03DK109232), NHLBI (nos. DP2OD022716 and P01HL032262), the Doris Duke Charitable Foundation, Burroughs Wellcome Fund, the American Society of Hematology and an Epigenetics Seed Grant from Harvard Medical School.

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F.S., T.M. and D.E.B. conceptualized the study. F.S., M.H., D.S., L.P., T.M., A.K., S.H.S. and D.E.B. provided methodology. V.A.C.S., M.A.C., Q.Y., C.M.T., P.G.S., M.C.C., L.P. and D.E.B. provided software. F.S., M.H., D.S., D.S.V., C.R., P.C., L.M.K.D., K.L., C.M.G. and Y.F. validated the project. F.S., V.A.C.S., Q.Y., M.A.C., L.P., T.M., A.K., S.H.O. and D.E.B. performed formal analysis. F.S., M.H., D.S., D.S.V., L.M.K.D., C.R., C.M.G. and Y.F. were investigators. C.R., K.L., R.K., Y.N., S.A.W., L.P., T.M., A.K., S.H.O. and D.E.B. provided resources. F.S., M.H., D.S., Q.Y. and D.E.B. curated data. F.S. and D.E.B. wrote the original draft. All authors reviewed and edited the article. F.S., M.H., D.S., V.A.C.S., M.A.C., Q.Y. and D.E.B. provided visualization. L.P., T.M., A.K., S.H.O. and D.E.B. supervised the research. D.E.B. was the project administrator. T.M., A.K., S.H.O. and D.E.B acquired funding.

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Correspondence to Daniel E. Bauer.

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Supplementary information

Supplementary Information

Supplementary Note and Supplementary Figs. 1–9

Reporting Summary

Supplementary Table 1

Quantitative analysis of mass spectrometery data.

Supplementary Table 2

Functional scores of CRISPR library (all sgRNAs) against various protein-level sequence annotations.

Supplementary Table 3

List of oligonucleotides and PCR primer sequences used in the study.

Supplementary Table 4

Key resources.

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Sher, F., Hossain, M., Seruggia, D. et al. Rational targeting of a NuRD subcomplex guided by comprehensive in situ mutagenesis. Nat Genet 51, 1149–1159 (2019). https://doi.org/10.1038/s41588-019-0453-4

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