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

Sher, Falak; Hossain, Md. Abul; Seruggia, Davide; Schoonenberg, Vivien A.C.; Yao, Qiuming; Cifani, Paolo; Dassama, Laura M. K.; Cole, Mitchel A; Ren, Chunyan; Vinjamur, Divya S.; Macias-Treviño, Claudio; Luk, Kevin; McGuckin, Connor; Schupp, Patrick G.; Canver, Matthew C.; Kurita, Ryo; Nakamura, Yukio; Fujiwara, Yuko; Wolfe, Scot A.; Pinello, Luca; Maeda, Takahiro; Kentsis, Alex; Orkin, Stuart H.; Bauer, Daniel E.

doi:10.1038/s41588-019-0453-4

Cited by 92 publications

(90 citation statements)

References 67 publications

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“…GATAD2 is also responsible for the recruitment of a CHD subunit to the complex (25), via an interaction that is mediated by the GATA-type zinc finger of GATAD2 (26). We do not observe GATAD2 binding to MTA, HDAC or RBBP (25), and thus the means by which GATAD2 prevents a second copy of MBD from binding to MTA-HDAC-RBBP remains unclear.…”

Section: Nurd Assembly Is Regulated At An Interface Between Separablecontrasting

confidence: 63%

The Nucleosome Remodeling and Deacetylase complex has an asymmetric, dynamic, and modular architecture

Low

Silva

Tabar

et al. 2020

Preprint

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The Nucleosome Remodeling and Deacetylase (NuRD) complex is essential for development in complex animals but has been refractory to biochemical analysis. We present the first integrated analysis of the architecture of the native mammalian NuRD complex, combining quantitative mass spectrometry, covalent cross-linking, protein biochemistry and electron microscopy. NuRD is built around a 2:2:4 pseudo-symmetric deacetylase module comprising MTA, HDAC and RBBP subunits. This module interacts asymmetrically with a remodeling module comprising one copy each of MBD, GATAD2 and CHD subunits. The previously enigmatic GATAD2 controls the asymmetry of the complex and directly recruits the ATP-dependent CHD remodeler. Unexpectedly, the MTA-MBD interaction acts as a point of functional switching. The transcriptional regulator PWWP2A modulates NuRD assembly by competing directly with MBD for binding to the MTA-HDAC-RBBP subcomplex, forming a 'moonlighting' PWWP2A-MTA-HDAC-RBBP complex that likely directs deacetylase activity to PWWP2A target sites. Taken together, our data describe the overall architecture of the intact NuRD complex and reveal aspects of its structural dynamics and functional plasticity. INO80(1, 2) and SWR1 complexes (3), as well as to the Snf2 (4) and Chromodomain-Helicase-DNA-binding 1 (CHD1) remodelers (5), our understanding of how such enzymes bring about remodeling is still underdeveloped. This is particularly true for the nucleosome remodeling and deacetylase (NuRD) complex.The NuRD complex is widely distributed among Metazoans and is expressed in most, if not all, tissues. It is essential for normal development (6, 7) and is a key regulator in the reprogramming of differentiated cells into pluripotent stem cells (8-10). Age-related reductions in NuRD subunit levels are strongly associated with memory loss, metastatic potential in human cancers (11), and the accumulation of chromatin defects (12, 13).The mammalian NuRD complex comprises at least six subunits (Figure S1a), and for each subunit there are at least two paralogues, giving the potential for significant compositional heterogeneity. CHD4 (and its paralogues CHD3 and -5) is the ATP-dependent DNA translocase in the complex and harbours several regulatory and targeting domains. For example, the PHD domains of CHD4 can recognize histone H3 N-terminal tails bearing methyllysine marks (14-16), and the HMG domain has been shown to bind to poly-ADP(ribose) (17). What distinguishes NuRD from many other remodelers is that it harbours a second catalytic activity, imparted by the histone deacetylases HDAC1 and -2. MBD2 and -3 can bind hydroxymethylated and/or methylated , and RBBP4 and -7 can each bind histone tails (21) and other transcriptional regulators (22,23). The metastasis-associated proteins MTA1, -2 and -3 contain several domains that are associated with nucleosome recognition, whereas GATAD2A and GATAD2B bind to both 25) and CHD proteins (25, 26) but otherwise do not have known functions. Some structural information is available for portions of t...

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Section: Nurd Assembly Is Regulated At An Interface Between Separablecontrasting

confidence: 63%

The Nucleosome Remodeling and Deacetylase complex has an asymmetric, dynamic, and modular architecture

Low

Silva

Tabar

et al. 2020

Preprint

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“…For ZBTB7A we observed a positive fitness score, suggesting cells mutated at this gene accumulated in the population, consistent with its known requirement for terminal erythroid maturation 11 . In addition, we validated prior findings that a NuRD subcomplex including CHD4, MTA2, GATAD2A, MBD2, and HDAC2 was required for HbF control 6 . Editing CHD4 led to potent HbF induction but was associated with negative cell fitness.…”

Section: Crispr Screen For Novel Transcriptional Regulators Of Hbf Levelsupporting

confidence: 83%

“…Induction of fetal -globin 4 gene expression could bypass the underlying -globin molecular defects and ameliorate the 5 pathophysiological cascades that result in elevated morbidity and mortality. Critical regulators of 6 the switch from fetal to adult globin gene expression include the DNA-binding transcription 7 factors (TFs) BCL11A and ZBTB7A and the nucleosome remodeling and deacetylase (NuRD) 8 chromatin complex [4][5][6][7] . BCL11A and ZBTB7A each bind to unique sites at the proximal 9 promoters of the duplicated fetal -globin genes HBG1 and HBG2 and each physically interact with NuRD 5,[8][9][10] .…”

Section: Introduction 1mentioning

confidence: 99%

ZNF410 represses fetal globin by devoted control of CHD4/NuRD

Vinjamur¹,

Yao²,

Cole³

et al. 2020

Preprint

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Major effectors of adult-stage fetal globin silencing include the transcription factors (TFs) BCL11A and ZBTB7A/LRF and the NuRD chromatin complex, although each has potential on-target liabilities for rational β-hemoglobinopathy therapeutic inhibition. Here through CRISPR screening we discover ZNF410 to be a novel fetal hemoglobin (HbF) repressing TF. ZNF410 does not bind directly to the γ-globin genes but rather its chromatin occupancy is solely concentrated at CHD4, encoding the NuRD nucleosome remodeler, itself required for HbF repression. CHD4 has two ZNF410-bound regulatory elements with 27 combined ZNF410 binding motifs constituting unparalleled genomic clusters. These elements completely account for ZNF410’s effects on γ-globin repression. Knockout of ZNF410 reduces CHD4 by 60%, enough to substantially de-repress HbF while avoiding the cellular toxicity of complete CHD4 loss. Mice with constitutive deficiency of the homolog Zfp410 are born at expected Mendelian ratios with unremarkable hematology. ZNF410 is dispensable for human hematopoietic engraftment potential and erythroid maturation unlike known HbF repressors. These studies identify a new rational target for HbF induction for the β-hemoglobin disorders with a wide therapeutic index. More broadly, ZNF410 represents a special class of gene regulator, a conserved transcription factor with singular devotion to regulation of a chromatin subcomplex.

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“…To allow denser tiling of regions with mutations, CRISPR-nucleases with alternative or dispensable PAM requirements could be used 17,53,54 . Although indels are applicable to regulatory regions and even coding sequences [55][56][57] , point mutagenesis would enable fi ne mapping of regulatory nucleotides and amino acids. This exciting possibility could be opened up by implementing hyperactive base [58][59][60][61] -or prime editors [62][63][64] .…”

Section: Discussionmentioning

confidence: 99%

Parallel genetics of regulatory sequencesin vivo

Froehlich

Uyar

Herzog

et al. 2020

Preprint

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Understanding how regulatory sequences control gene expression is fundamental to explain how phenotypes arise in health and disease. Traditional reporter assays inform about function of individual regulatory elements, typically in isolation. However, regulatory elements must ultimately be understood by perturbing them within their genomic environment and developmental- or tissue-specific contexts. This is technically challenging; therefore, few regulatory elements have been characterized in vivo. Here, we used inducible Cas9 and multiplexed guide RNAs to create hundreds of mutations in enhancers/promoters and 3′ UTRs of 16 genes in C. elegans. To quantify the consequences of mutations on expression, we developed a targeted RNA sequencing strategy across hundreds of mutant animals. We were also able to systematically and quantitatively assign fitness cost to mutations. Finally, we identified and characterized sequence elements that strongly regulate phenotypic traits. Our approach enables highly parallelized, functional analysis of regulatory sequences in vivo.

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

Cited by 92 publications

References 67 publications

The Nucleosome Remodeling and Deacetylase complex has an asymmetric, dynamic, and modular architecture

The Nucleosome Remodeling and Deacetylase complex has an asymmetric, dynamic, and modular architecture

ZNF410 represses fetal globin by devoted control of CHD4/NuRD

Parallel genetics of regulatory sequencesin vivo

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