TY - JOUR
T1 - Elucidation of a downstream boundary of the 3′ IgH regulatory region
AU - Manis, John P.
AU - Michaelson, Jennifer S.
AU - Birshtein, Barbara K.
AU - Alt, Frederick W.
N1 - Funding Information:
This work was supported by NIH grants AI31541 (to F.W.A.), and AI13509 (to B.K.B.), and by a grant from the Hood Foundation Grant, and the Cure for Lymphoma Foundation (to J.P.M.). F.W.A. is an Investigator of the Howard Hughes Medical Institute. The authors wish to thank Laurie Davidson and Daniel Foy for their assistance in generation and maintenance of the chimeric mice.
PY - 2003/1
Y1 - 2003/1
N2 - Class switch recombination (CSR) changes the immunoglobulin heavy chain (IgH) constant region gene (CH) in B cells from IgM to IgG, IgA, or IgE, without modifying the variable region gene segment. This process requires transcription through switch (S) regions located upstream of the CH genes targeted for CSR, a process that relies on the activity of an uncharacterized regulatory region at the 3′ end of the CH locus (3′ IgH RR) that has been implicated via the effects of pgk-neo cassettes inserted into the locus. The 30kb region just downstream of the most 3′ CH gene (Ca) contains four known enhancer elements including HS3a, HS1,2, HS3b, and HS4. Replacement of either of the proximal two enhancer elements (HS3a or HS1,2) with a pgk-neo gene cassette disrupted germline transcription of and CSR to most CH genes. However, replacement of either of the enhancers with a loxP sequence had no effect on CSR indicating that these elements are not critical for CSR. Insertion of a pgk-neo cassette at various sites within the CH locus inhibited CSR to upstream, but not downstream CH genes, supporting the notion that the pgk-neo cassette insertion into the locus short-circuits the ability of the 3′ RR to facilitate CSR of dependent CH genes upstream of the insertion. These analyses also indicated that the key elements of the 3′ IgH RR were downstream from HS1,2. In this study, we have sought to localize the 3′ IgH RR by defining its 3′ boundary. For this purpose, a pgk-neo gene cassette was targeted 2kb downstream of the HS4 element in ES cells that had normal ability to undergo CSR. We then employed Rag-2 deficient blastocyst complementation to generate chimeric mice that harbored B cells homozygous for this mutation. Such chimeras exhibited normal reconstitution of the splenic compartment and had normal serum immunoglobulin levels. Upon in vitro activation, transcription from the pgk-neo cassette was induced in B cells, however, CSR to all measured IgH isotypes occurred at normal levels. These findings, coupled with previous pgk-neo insertion studies, suggest that key elements of the 3′ IgH RR lie within a 17kb region between HS1,2 and 2kb downstream of HS4.
AB - Class switch recombination (CSR) changes the immunoglobulin heavy chain (IgH) constant region gene (CH) in B cells from IgM to IgG, IgA, or IgE, without modifying the variable region gene segment. This process requires transcription through switch (S) regions located upstream of the CH genes targeted for CSR, a process that relies on the activity of an uncharacterized regulatory region at the 3′ end of the CH locus (3′ IgH RR) that has been implicated via the effects of pgk-neo cassettes inserted into the locus. The 30kb region just downstream of the most 3′ CH gene (Ca) contains four known enhancer elements including HS3a, HS1,2, HS3b, and HS4. Replacement of either of the proximal two enhancer elements (HS3a or HS1,2) with a pgk-neo gene cassette disrupted germline transcription of and CSR to most CH genes. However, replacement of either of the enhancers with a loxP sequence had no effect on CSR indicating that these elements are not critical for CSR. Insertion of a pgk-neo cassette at various sites within the CH locus inhibited CSR to upstream, but not downstream CH genes, supporting the notion that the pgk-neo cassette insertion into the locus short-circuits the ability of the 3′ RR to facilitate CSR of dependent CH genes upstream of the insertion. These analyses also indicated that the key elements of the 3′ IgH RR were downstream from HS1,2. In this study, we have sought to localize the 3′ IgH RR by defining its 3′ boundary. For this purpose, a pgk-neo gene cassette was targeted 2kb downstream of the HS4 element in ES cells that had normal ability to undergo CSR. We then employed Rag-2 deficient blastocyst complementation to generate chimeric mice that harbored B cells homozygous for this mutation. Such chimeras exhibited normal reconstitution of the splenic compartment and had normal serum immunoglobulin levels. Upon in vitro activation, transcription from the pgk-neo cassette was induced in B cells, however, CSR to all measured IgH isotypes occurred at normal levels. These findings, coupled with previous pgk-neo insertion studies, suggest that key elements of the 3′ IgH RR lie within a 17kb region between HS1,2 and 2kb downstream of HS4.
KW - 3′ Immunoglobulin heavy chain regulatory region
KW - Class switch recombination
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U2 - 10.1016/S0161-5890(02)00256-0
DO - 10.1016/S0161-5890(02)00256-0
M3 - Article
C2 - 12531286
AN - SCOPUS:0037230164
SN - 0161-5890
VL - 39
SP - 753
EP - 760
JO - Molecular Immunology
JF - Molecular Immunology
IS - 12
ER -