Base editing of key residues in the BCL11A-XL-specific zinc finger domains derepresses fetal globin expression
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved..
BCL11A-XL directly binds and represses the fetal globin (HBG1/2) gene promoters, using 3 zinc-finger domains (ZnF4, ZnF5, and ZnF6), and is a potential target for β-hemoglobinopathy treatments. Disrupting BCL11A-XL results in derepression of fetal globin and high HbF, but also affects hematopoietic stem and progenitor cell (HSPC) engraftment and erythroid maturation. Intriguingly, neurodevelopmental patients with ZnF domain mutations have elevated HbF with normal hematological parameters. Inspired by this natural phenomenon, we used both CRISPR-Cas9 and base editing at specific ZnF domains and assessed the impacts on HbF production and hematopoietic differentiation. Generating indels in the various ZnF domains by CRISPR-Cas9 prevented the binding of BCL11A-XL to its site in the HBG1/2 promoters and elevated the HbF levels but affected normal hematopoiesis. Far fewer side effects were observed with base editing- for instance, erythroid maturation in vitro was near normal. However, we observed a modest reduction in HSPC engraftment and a complete loss of B cell development in vivo, presumably because current base editing is not capable of precisely recapitulating the mutations found in patients with BCL11A-XL-associated neurodevelopment disorders. Overall, our results reveal that disrupting different ZnF domains has different effects. Disrupting ZnF4 elevated HbF levels significantly while leaving many other erythroid target genes unaffected, and interestingly, disrupting ZnF6 also elevated HbF levels, which was unexpected because this region does not directly interact with the HBG1/2 promoters. This first structure/function analysis of ZnF4-6 provides important insights into the domains of BCL11A-XL that are required to repress fetal globin expression and provide framework for exploring the introduction of natural mutations that may enable the derepression of single gene while leaving other functions unaffected.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:32 |
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| Enthalten in: |
Molecular therapy : the journal of the American Society of Gene Therapy - 32(2024), 3 vom: 06. März, Seite 663-677 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Rajendiran, Vignesh [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 11.03.2024 Date Revised 25.04.2024 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/j.ymthe.2024.01.023 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM367640651 |
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| 245 | 1 | 0 | |a Base editing of key residues in the BCL11A-XL-specific zinc finger domains derepresses fetal globin expression |
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| 500 | |a Date Revised 25.04.2024 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved. | ||
| 520 | |a BCL11A-XL directly binds and represses the fetal globin (HBG1/2) gene promoters, using 3 zinc-finger domains (ZnF4, ZnF5, and ZnF6), and is a potential target for β-hemoglobinopathy treatments. Disrupting BCL11A-XL results in derepression of fetal globin and high HbF, but also affects hematopoietic stem and progenitor cell (HSPC) engraftment and erythroid maturation. Intriguingly, neurodevelopmental patients with ZnF domain mutations have elevated HbF with normal hematological parameters. Inspired by this natural phenomenon, we used both CRISPR-Cas9 and base editing at specific ZnF domains and assessed the impacts on HbF production and hematopoietic differentiation. Generating indels in the various ZnF domains by CRISPR-Cas9 prevented the binding of BCL11A-XL to its site in the HBG1/2 promoters and elevated the HbF levels but affected normal hematopoiesis. Far fewer side effects were observed with base editing- for instance, erythroid maturation in vitro was near normal. However, we observed a modest reduction in HSPC engraftment and a complete loss of B cell development in vivo, presumably because current base editing is not capable of precisely recapitulating the mutations found in patients with BCL11A-XL-associated neurodevelopment disorders. Overall, our results reveal that disrupting different ZnF domains has different effects. Disrupting ZnF4 elevated HbF levels significantly while leaving many other erythroid target genes unaffected, and interestingly, disrupting ZnF6 also elevated HbF levels, which was unexpected because this region does not directly interact with the HBG1/2 promoters. This first structure/function analysis of ZnF4-6 provides important insights into the domains of BCL11A-XL that are required to repress fetal globin expression and provide framework for exploring the introduction of natural mutations that may enable the derepression of single gene while leaving other functions unaffected | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a BCL11A-XL | |
| 650 | 4 | |a base editing | |
| 650 | 4 | |a engraftment | |
| 650 | 4 | |a erythroid cells | |
| 650 | 4 | |a erythroid maturation | |
| 650 | 4 | |a fetal hemoglobin | |
| 650 | 4 | |a genome editing | |
| 650 | 4 | |a globin regulation | |
| 650 | 4 | |a hematopoietic stem cells | |
| 650 | 4 | |a zinc finger domains | |
| 650 | 7 | |a gamma-Globins |2 NLM | |
| 650 | 7 | |a Repressor Proteins |2 NLM | |
| 650 | 7 | |a Fetal Hemoglobin |2 NLM | |
| 650 | 7 | |a 9034-63-3 |2 NLM | |
| 650 | 7 | |a BCL11A protein, human |2 NLM | |
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| 700 | 1 | |a Ravi, Nithin Sam |e verfasserin |4 aut | |
| 700 | 1 | |a Panigrahi, Lokesh |e verfasserin |4 aut | |
| 700 | 1 | |a Paul, Joshua |e verfasserin |4 aut | |
| 700 | 1 | |a Gopalakrishnan, Chandrasekar |e verfasserin |4 aut | |
| 700 | 1 | |a Wyman, Stacia |e verfasserin |4 aut | |
| 700 | 1 | |a Ariudainambi, Keerthiga |e verfasserin |4 aut | |
| 700 | 1 | |a Mahalingam, Gokulnath |e verfasserin |4 aut | |
| 700 | 1 | |a Periyasami, Yogapriya |e verfasserin |4 aut | |
| 700 | 1 | |a Prasad, Kirti |e verfasserin |4 aut | |
| 700 | 1 | |a George, Anila |e verfasserin |4 aut | |
| 700 | 1 | |a Sukumaran, Dhiyaneshwaran |e verfasserin |4 aut | |
| 700 | 1 | |a Gopinathan, Sandhiya |e verfasserin |4 aut | |
| 700 | 1 | |a Pai, Aswin Anand |e verfasserin |4 aut | |
| 700 | 1 | |a Nakamura, Yukio |e verfasserin |4 aut | |
| 700 | 1 | |a Balasubramanian, Poonkuzhali |e verfasserin |4 aut | |
| 700 | 1 | |a Ramalingam, Rajasekaran |e verfasserin |4 aut | |
| 700 | 1 | |a Thangavel, Saravanabhavan |e verfasserin |4 aut | |
| 700 | 1 | |a Velayudhan, Shaji R |e verfasserin |4 aut | |
| 700 | 1 | |a Corn, Jacon E |e verfasserin |4 aut | |
| 700 | 1 | |a Mackay, Joel P |e verfasserin |4 aut | |
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| 700 | 1 | |a Crossley, Merlin |e verfasserin |4 aut | |
| 700 | 1 | |a Mohankumar, Kumarasamypet M |e verfasserin |4 aut | |
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