Biased dA/dT somatic hypermutation as regulated by the heavy chain intronic iEμ enhancer and 3′Eα enhancers in human lymphoblastoid B cells

Atsumasa Komori, Zhenming Xu, Xiaoping Wu, Hong Zan, Paolo Casali

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Somatic hypermutation (SHM) in immunoglobulin gene (Ig) variable (V) regions is critical for the maturation of the antibody response. It is dependent on the expression of activation-induced cytidine deaminase (AID) and translesion DNA polymerases in germinal center B cells as well as Ig V transcription, as regulated by the Ig heavy chain (H) intronic enhancer (iEμ) and the 3′ enhancer (3′Eα) region. We analyzed the role of these cis elements in SHM by stably transfecting Ramos human lymphoblastoid B cells with a rearranged human IgH chain VD (diversity) J (joining) DNA construct containing a VH promoter at the 5′ end and CH1 and CH2 exons of Cγ1 at the 3′ end. In this construct, mutations preferentially targeted dA/dT basepairs in the RGYW/WRCY hotspot. Most of the dA/dT mutations and accompanying dC/dG mutations were transitions. Deletion of iEμ resulted in decreased SHM which could be partially restored by insertion of the IgH hs1,2 enhancer. Other two 3′Eα enhancers, hs3-hs4, did not significantly increase the mutation frequency, but further strengthened the dA/dT bias. The frequency and spectrum of the mutations were independent of the genomic integration of the transgene or V gene transcription level. Thus, we have established a novel in vitro system to analyze SHM and identify the role of multiple cis-regulatory elements in regulating dA/dT biased SHM. This model system will be useful to further address the role of other cis-regulating elements and recruited trans-acting factors in expressing the modalities of SHM.

Original languageEnglish (US)
Pages (from-to)1817-1826
Number of pages10
JournalMolecular Immunology
Volume43
Issue number11
DOIs
StatePublished - Apr 1 2006
Externally publishedYes

Fingerprint

Immunoglobulin Genes
B-Lymphocytes
Mutation Rate
Mutation
Immunoglobulin Variable Region
Immunoglobulin Heavy Chain Genes
Germinal Center
Trans-Activators
DNA-Directed DNA Polymerase
Transgenes
Antibody Formation
Exons
DNA
Genes
In Vitro Techniques
AICDA (activation-induced cytidine deaminase)

Keywords

  • 3′ Heavy chain enhancer
  • cis-Regulatory elements
  • iEμ
  • Intronic Ig μ enhancer
  • SHM
  • Somatic hypermutation

ASJC Scopus subject areas

  • Immunology
  • Molecular Biology

Cite this

Biased dA/dT somatic hypermutation as regulated by the heavy chain intronic iEμ enhancer and 3′Eα enhancers in human lymphoblastoid B cells. / Komori, Atsumasa; Xu, Zhenming; Wu, Xiaoping; Zan, Hong; Casali, Paolo.

In: Molecular Immunology, Vol. 43, No. 11, 01.04.2006, p. 1817-1826.

Research output: Contribution to journalArticle

@article{905dd6245acc4a99bd13c8249a36901f,
title = "Biased dA/dT somatic hypermutation as regulated by the heavy chain intronic iEμ enhancer and 3′Eα enhancers in human lymphoblastoid B cells",
abstract = "Somatic hypermutation (SHM) in immunoglobulin gene (Ig) variable (V) regions is critical for the maturation of the antibody response. It is dependent on the expression of activation-induced cytidine deaminase (AID) and translesion DNA polymerases in germinal center B cells as well as Ig V transcription, as regulated by the Ig heavy chain (H) intronic enhancer (iEμ) and the 3′ enhancer (3′Eα) region. We analyzed the role of these cis elements in SHM by stably transfecting Ramos human lymphoblastoid B cells with a rearranged human IgH chain VD (diversity) J (joining) DNA construct containing a VH promoter at the 5′ end and CH1 and CH2 exons of Cγ1 at the 3′ end. In this construct, mutations preferentially targeted dA/dT basepairs in the RGYW/WRCY hotspot. Most of the dA/dT mutations and accompanying dC/dG mutations were transitions. Deletion of iEμ resulted in decreased SHM which could be partially restored by insertion of the IgH hs1,2 enhancer. Other two 3′Eα enhancers, hs3-hs4, did not significantly increase the mutation frequency, but further strengthened the dA/dT bias. The frequency and spectrum of the mutations were independent of the genomic integration of the transgene or V gene transcription level. Thus, we have established a novel in vitro system to analyze SHM and identify the role of multiple cis-regulatory elements in regulating dA/dT biased SHM. This model system will be useful to further address the role of other cis-regulating elements and recruited trans-acting factors in expressing the modalities of SHM.",
keywords = "3′ Heavy chain enhancer, cis-Regulatory elements, iEμ, Intronic Ig μ enhancer, SHM, Somatic hypermutation",
author = "Atsumasa Komori and Zhenming Xu and Xiaoping Wu and Hong Zan and Paolo Casali",
year = "2006",
month = "4",
day = "1",
doi = "10.1016/j.molimm.2005.10.018",
language = "English (US)",
volume = "43",
pages = "1817--1826",
journal = "Molecular Immunology",
issn = "0161-5890",
publisher = "Elsevier Limited",
number = "11",

}

TY - JOUR

T1 - Biased dA/dT somatic hypermutation as regulated by the heavy chain intronic iEμ enhancer and 3′Eα enhancers in human lymphoblastoid B cells

AU - Komori, Atsumasa

AU - Xu, Zhenming

AU - Wu, Xiaoping

AU - Zan, Hong

AU - Casali, Paolo

PY - 2006/4/1

Y1 - 2006/4/1

N2 - Somatic hypermutation (SHM) in immunoglobulin gene (Ig) variable (V) regions is critical for the maturation of the antibody response. It is dependent on the expression of activation-induced cytidine deaminase (AID) and translesion DNA polymerases in germinal center B cells as well as Ig V transcription, as regulated by the Ig heavy chain (H) intronic enhancer (iEμ) and the 3′ enhancer (3′Eα) region. We analyzed the role of these cis elements in SHM by stably transfecting Ramos human lymphoblastoid B cells with a rearranged human IgH chain VD (diversity) J (joining) DNA construct containing a VH promoter at the 5′ end and CH1 and CH2 exons of Cγ1 at the 3′ end. In this construct, mutations preferentially targeted dA/dT basepairs in the RGYW/WRCY hotspot. Most of the dA/dT mutations and accompanying dC/dG mutations were transitions. Deletion of iEμ resulted in decreased SHM which could be partially restored by insertion of the IgH hs1,2 enhancer. Other two 3′Eα enhancers, hs3-hs4, did not significantly increase the mutation frequency, but further strengthened the dA/dT bias. The frequency and spectrum of the mutations were independent of the genomic integration of the transgene or V gene transcription level. Thus, we have established a novel in vitro system to analyze SHM and identify the role of multiple cis-regulatory elements in regulating dA/dT biased SHM. This model system will be useful to further address the role of other cis-regulating elements and recruited trans-acting factors in expressing the modalities of SHM.

AB - Somatic hypermutation (SHM) in immunoglobulin gene (Ig) variable (V) regions is critical for the maturation of the antibody response. It is dependent on the expression of activation-induced cytidine deaminase (AID) and translesion DNA polymerases in germinal center B cells as well as Ig V transcription, as regulated by the Ig heavy chain (H) intronic enhancer (iEμ) and the 3′ enhancer (3′Eα) region. We analyzed the role of these cis elements in SHM by stably transfecting Ramos human lymphoblastoid B cells with a rearranged human IgH chain VD (diversity) J (joining) DNA construct containing a VH promoter at the 5′ end and CH1 and CH2 exons of Cγ1 at the 3′ end. In this construct, mutations preferentially targeted dA/dT basepairs in the RGYW/WRCY hotspot. Most of the dA/dT mutations and accompanying dC/dG mutations were transitions. Deletion of iEμ resulted in decreased SHM which could be partially restored by insertion of the IgH hs1,2 enhancer. Other two 3′Eα enhancers, hs3-hs4, did not significantly increase the mutation frequency, but further strengthened the dA/dT bias. The frequency and spectrum of the mutations were independent of the genomic integration of the transgene or V gene transcription level. Thus, we have established a novel in vitro system to analyze SHM and identify the role of multiple cis-regulatory elements in regulating dA/dT biased SHM. This model system will be useful to further address the role of other cis-regulating elements and recruited trans-acting factors in expressing the modalities of SHM.

KW - 3′ Heavy chain enhancer

KW - cis-Regulatory elements

KW - iEμ

KW - Intronic Ig μ enhancer

KW - SHM

KW - Somatic hypermutation

UR - http://www.scopus.com/inward/record.url?scp=33644938791&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33644938791&partnerID=8YFLogxK

U2 - 10.1016/j.molimm.2005.10.018

DO - 10.1016/j.molimm.2005.10.018

M3 - Article

C2 - 16412510

AN - SCOPUS:33644938791

VL - 43

SP - 1817

EP - 1826

JO - Molecular Immunology

JF - Molecular Immunology

SN - 0161-5890

IS - 11

ER -