Limits...
A role for the IgH intronic enhancer E mu in enforcing allelic exclusion.

Li F, Eckhardt LA - J. Exp. Med. (2008)

Bottom Line: To determine E mu's subsequent functions, we created an Igh allele with assembled V(H) gene but with E mu removed.Rather, the striking breakdown in allelic exclusion took place at the pre-B to immature B cell transition.These findings reveal both an important role for E mu in influencing the fate of newly arising B cells and a second checkpoint for allelic exclusion.

View Article: PubMed Central - PubMed

Affiliation: Hunter College and Graduate Center of the City University of New York, New York, NY 10065, USA.

ABSTRACT
The intronic enhancer (E mu) of the immunoglobulin heavy chain (IgH) locus is critical for V region gene assembly. To determine E mu's subsequent functions, we created an Igh allele with assembled V(H) gene but with E mu removed. In mice homozygous for this E mu-deficient allele, B cell development was normal and indistinguishable from that of mice with the same V(H) knockin and E mu intact. In mice heterozygous for the E mu-deficient allele, however, allelic exclusion was severely compromised. Surprisingly, this was not a result of reduced suppression of V-DJ assembly on the second allele. Rather, the striking breakdown in allelic exclusion took place at the pre-B to immature B cell transition. These findings reveal both an important role for E mu in influencing the fate of newly arising B cells and a second checkpoint for allelic exclusion.

Show MeSH

Related in: MedlinePlus

Igμ transcription and cytoplasmic Igμ protein levels in pre-B cells of mutant mice. (A) B220+ lymphocytes from BM of Rag1-deficient WTb/WTb, VHEμa/WTb, and VHΔa/WTb mice were analyzed for size (forward scatter) and for CD43 expression by FACS. Data shown are representative of three individual mice of each genotype analyzed. (B) Igμ mRNA levels in cells shown in A. Data were generated by quantitative RT-PCR, normalized to hgprt1 mRNA, and included two experiments, analyzing a total of five individual animals of each genotype. Negative controls were mRNA isolated from pro-B cells of a WTb/WTb Rag1−/− littermate and mRNA from C57BL/6 heart tissue (WTb/WTb, Rag1+/+). Statistical significance (P = 0.027) was obtained by a two-tailed Student's t test. Error bars show SD. (C) Cytoplasmic Igμ levels in B220+ BM cells of Rag1−/− mice. Left, histograms of cytoplasmic Igμ. Right, mean Igμ fluorescence in pre-B cells from multiple mice (n = number of mice analyzed). Error bars show SD.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2626684&req=5

fig6: Igμ transcription and cytoplasmic Igμ protein levels in pre-B cells of mutant mice. (A) B220+ lymphocytes from BM of Rag1-deficient WTb/WTb, VHEμa/WTb, and VHΔa/WTb mice were analyzed for size (forward scatter) and for CD43 expression by FACS. Data shown are representative of three individual mice of each genotype analyzed. (B) Igμ mRNA levels in cells shown in A. Data were generated by quantitative RT-PCR, normalized to hgprt1 mRNA, and included two experiments, analyzing a total of five individual animals of each genotype. Negative controls were mRNA isolated from pro-B cells of a WTb/WTb Rag1−/− littermate and mRNA from C57BL/6 heart tissue (WTb/WTb, Rag1+/+). Statistical significance (P = 0.027) was obtained by a two-tailed Student's t test. Error bars show SD. (C) Cytoplasmic Igμ levels in B220+ BM cells of Rag1−/− mice. Left, histograms of cytoplasmic Igμ. Right, mean Igμ fluorescence in pre-B cells from multiple mice (n = number of mice analyzed). Error bars show SD.

Mentions: In an attempt to understand the mechanism through which Eμ might influence the development and selection of precursor B cells, we compared levels of μ mRNA generated from the VHEμa and VHΔa alleles in pre-B cells. To enrich for pre-B cells, VHEμa and VHΔa mice were backcrossed to Rag1−/− mice to generate VHEμa/WTb and VHΔa/WTb mice that lacked Rag-1 activity. In both of the resulting strains, B cell development was arrested at the pre-B cell stage because Ig light chain gene assembly was blocked. Age-matched mice of each genotype were killed and B220+ BM cells isolated (see Materials and methods). As expected, no IgM+ cells were present in these BM cells, and the bulk of cells were smaller and expressed lower levels of CD43 than comparable cells from WTb/WTb Rag1−/− littermates, which is consistent with their having progressed to the pre-B cell stage (Fig. 6 A). Total RNA was isolated from the B220+ BM cells of Rag1-deficient VHEμa/WTb and VHΔa/WTb mice (WTb/WTb; Rag1−/− littermates were controls) and Igμ mRNA was quantified by real-time RT-PCR (5′ primer for unique VDJ junction of VHB1-8 and 3′ primer for CH1 exon of Cμ; see Materials and methods).


A role for the IgH intronic enhancer E mu in enforcing allelic exclusion.

Li F, Eckhardt LA - J. Exp. Med. (2008)

Igμ transcription and cytoplasmic Igμ protein levels in pre-B cells of mutant mice. (A) B220+ lymphocytes from BM of Rag1-deficient WTb/WTb, VHEμa/WTb, and VHΔa/WTb mice were analyzed for size (forward scatter) and for CD43 expression by FACS. Data shown are representative of three individual mice of each genotype analyzed. (B) Igμ mRNA levels in cells shown in A. Data were generated by quantitative RT-PCR, normalized to hgprt1 mRNA, and included two experiments, analyzing a total of five individual animals of each genotype. Negative controls were mRNA isolated from pro-B cells of a WTb/WTb Rag1−/− littermate and mRNA from C57BL/6 heart tissue (WTb/WTb, Rag1+/+). Statistical significance (P = 0.027) was obtained by a two-tailed Student's t test. Error bars show SD. (C) Cytoplasmic Igμ levels in B220+ BM cells of Rag1−/− mice. Left, histograms of cytoplasmic Igμ. Right, mean Igμ fluorescence in pre-B cells from multiple mice (n = number of mice analyzed). Error bars show SD.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2626684&req=5

fig6: Igμ transcription and cytoplasmic Igμ protein levels in pre-B cells of mutant mice. (A) B220+ lymphocytes from BM of Rag1-deficient WTb/WTb, VHEμa/WTb, and VHΔa/WTb mice were analyzed for size (forward scatter) and for CD43 expression by FACS. Data shown are representative of three individual mice of each genotype analyzed. (B) Igμ mRNA levels in cells shown in A. Data were generated by quantitative RT-PCR, normalized to hgprt1 mRNA, and included two experiments, analyzing a total of five individual animals of each genotype. Negative controls were mRNA isolated from pro-B cells of a WTb/WTb Rag1−/− littermate and mRNA from C57BL/6 heart tissue (WTb/WTb, Rag1+/+). Statistical significance (P = 0.027) was obtained by a two-tailed Student's t test. Error bars show SD. (C) Cytoplasmic Igμ levels in B220+ BM cells of Rag1−/− mice. Left, histograms of cytoplasmic Igμ. Right, mean Igμ fluorescence in pre-B cells from multiple mice (n = number of mice analyzed). Error bars show SD.
Mentions: In an attempt to understand the mechanism through which Eμ might influence the development and selection of precursor B cells, we compared levels of μ mRNA generated from the VHEμa and VHΔa alleles in pre-B cells. To enrich for pre-B cells, VHEμa and VHΔa mice were backcrossed to Rag1−/− mice to generate VHEμa/WTb and VHΔa/WTb mice that lacked Rag-1 activity. In both of the resulting strains, B cell development was arrested at the pre-B cell stage because Ig light chain gene assembly was blocked. Age-matched mice of each genotype were killed and B220+ BM cells isolated (see Materials and methods). As expected, no IgM+ cells were present in these BM cells, and the bulk of cells were smaller and expressed lower levels of CD43 than comparable cells from WTb/WTb Rag1−/− littermates, which is consistent with their having progressed to the pre-B cell stage (Fig. 6 A). Total RNA was isolated from the B220+ BM cells of Rag1-deficient VHEμa/WTb and VHΔa/WTb mice (WTb/WTb; Rag1−/− littermates were controls) and Igμ mRNA was quantified by real-time RT-PCR (5′ primer for unique VDJ junction of VHB1-8 and 3′ primer for CH1 exon of Cμ; see Materials and methods).

Bottom Line: To determine E mu's subsequent functions, we created an Igh allele with assembled V(H) gene but with E mu removed.Rather, the striking breakdown in allelic exclusion took place at the pre-B to immature B cell transition.These findings reveal both an important role for E mu in influencing the fate of newly arising B cells and a second checkpoint for allelic exclusion.

View Article: PubMed Central - PubMed

Affiliation: Hunter College and Graduate Center of the City University of New York, New York, NY 10065, USA.

ABSTRACT
The intronic enhancer (E mu) of the immunoglobulin heavy chain (IgH) locus is critical for V region gene assembly. To determine E mu's subsequent functions, we created an Igh allele with assembled V(H) gene but with E mu removed. In mice homozygous for this E mu-deficient allele, B cell development was normal and indistinguishable from that of mice with the same V(H) knockin and E mu intact. In mice heterozygous for the E mu-deficient allele, however, allelic exclusion was severely compromised. Surprisingly, this was not a result of reduced suppression of V-DJ assembly on the second allele. Rather, the striking breakdown in allelic exclusion took place at the pre-B to immature B cell transition. These findings reveal both an important role for E mu in influencing the fate of newly arising B cells and a second checkpoint for allelic exclusion.

Show MeSH
Related in: MedlinePlus