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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 ?-hemoglobinopathy. The nucleosome remodeling and deacetylase (NuRD) chromatin complex participates in ?-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.

SUBMITTER: Sher F

PROVIDER: S-EPMC6650275 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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

Sher Falak F Hossain Mir M Seruggia Davide D Schoonenberg Vivien A C VAC Yao Qiuming Q Cifani Paolo P Dassama Laura M K LMK Cole Mitchel A MA Ren Chunyan C Vinjamur Divya S DS Macias-Trevino Claudio C Luk Kevin K McGuckin Connor C Schupp Patrick G PG Canver Matthew C MC Kurita Ryo R Nakamura Yukio Y Fujiwara Yuko Y Wolfe Scot A SA Pinello Luca L Maeda Takahiro T Kentsis Alex A Orkin Stuart H SH Bauer Daniel E DE

Nature genetics 20190628 7

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 ...[more]

PMID: 31253978

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Project description:Myeloperoxidase (MPO) is a member of the mammalian heme peroxidase (MHP) multigene family. Whereas all MHPs oxidize specific halides to generate the corresponding hypohalous acid, MPO is unique in its capacity to oxidize chloride at physiologic pH to produce hypochlorous acid (HOCl), a potent microbicide that contributes to neutrophil-mediated host defense against infection. We have previously resolved the evolutionary relationships in this functionally diverse multigene family and predicted in silico that positive Darwinian selection played a major role in the observed functional diversities (Loughran NB, O'Connor B, O'Fagain C, O'Connell MJ. 2008. The phylogeny of the mammalian heme peroxidases and the evolution of their diverse functions. BMC Evol Biol. 8:101). In this work, we have replaced positively selected residues asparagine 496 (N496), tyrosine 500 (Y500), and leucine 504 (L504) with the amino acids present in the ancestral MHP and have examined the effects on the structure, biosynthesis, and activity of MPO. Analysis in silico predicted that N496F, Y500F, or L504T would perturb hydrogen bonding in the heme pocket of MPO and thus disrupt the structural integrity of the enzyme. Biosynthesis of the mutants stably expressed in human embryonic kidney 293 cells yielded apoproMPO, the heme-free, enzymatically inactive precursor of MPO, that failed to undergo normal maturation or proteolytic processing. As a consequence of the maturational arrest at the apoproMPO stage of development, cells expressing MPO with mutations N496F, Y500F, L504T, individually or in combination, lacked normal peroxidase or chlorinating activity. Taken together, our data provide further support for the in silico predictions of positive selection and highlight the correlation between positive selection and functional divergence. Our data demonstrate that directly probing the functional importance of positive selection can provide important insights into understanding protein evolution.

Dataset Information

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

Publications

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

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