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Non-conservative homologous recombination in human B lymphocytes is promoted by activation-induced cytidine deaminase and transcription.

Mierau M, Drexler GA, Kutzera A, Braunschmidt K, Ellwart J, Eckardt-Schupp F, Fritz E, Bachl J, Jungnickel B - Nucleic Acids Res. (2008)

Bottom Line: The molecular basis for the differential use of these two pathways in different species is unclear.Employing a functional HR assay in human germinal centre like B cell lines, we detect elevated HR activity that can be enhanced by transcription and AID.Products of such recombination events mostly arise through non-conservative HR pathways, while the activity of conservative HR is low to absent.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Molecular Biology and Tumor Genetics, Helmholtz Center Munich, National Research Center for Environmental Health, D-81377 Munich.

ABSTRACT
During secondary immunoglobulin (Ig) diversification in vertebrates, the sequence of the variable region of Ig genes may be altered by templated or non-templated mechanisms. In both cases, cytidine deamination by activation-induced cytidine deaminase (AID) in the transcribed Ig loci leads to DNA lesions, which are repaired by conservative homologous recombination (HR) during Ig gene conversion, or by non-templated mutagenesis during somatic hypermutation. The molecular basis for the differential use of these two pathways in different species is unclear. While experimental ablation of HR in avian cells performing Ig gene conversion may promote a switch to somatic hypermutation, the activity of HR processes in intrinsically hypermutating mammalian cells has not been measured to date. Employing a functional HR assay in human germinal centre like B cell lines, we detect elevated HR activity that can be enhanced by transcription and AID. Products of such recombination events mostly arise through non-conservative HR pathways, while the activity of conservative HR is low to absent. Our results identify non-conservative HR as a novel DNA transaction pathway promoted by AID and suggest that somatic hypermutation in germinal centre B cells may be based on a physiological suppression of conservative HR.

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Transcription promotes recombination in Raji but not in BJAB cells. (A) Schematic structure of the vector used for the analysis of transcription effects on recombination. The bidirectional doxycycline-inducible promoter driving expression of the recombination cassette and NGFRt and elements for doxycycline dependent regulation are indicated. The vector system does not contain Ig enhancer elements. Frequencies of GFP+ cells after doxycycline addition in four representative out of 34 Raji (B) and 35 BJAB (C) subclones containing the inducible reporter. Arrowheads and different symbols indicate doxycycline addition to aliquots of each subclone. (D) Schematic illustration for calculation of the factor of transcription effect: the average number of GFP+ cells generated after transcription induction (average of all values represented by open circles) was divided by the average number of GFP+ cells detected at the beginning of transcription induction (average of all values represented by closed circles). (E) Factors of transcription effect for all Raji and BJAB subclones analysed with the inducible reporter. Each dot represents one clone. (F) Average numbers of GFP+ cells for all subclones of Raji and BJAB were calculated using the whole data set (i.e. average of all data represented by open and closed circles in D). Each dot represents one clone. The difference between the data sets is not statistically significant (P > 0.1).
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Figure 2: Transcription promotes recombination in Raji but not in BJAB cells. (A) Schematic structure of the vector used for the analysis of transcription effects on recombination. The bidirectional doxycycline-inducible promoter driving expression of the recombination cassette and NGFRt and elements for doxycycline dependent regulation are indicated. The vector system does not contain Ig enhancer elements. Frequencies of GFP+ cells after doxycycline addition in four representative out of 34 Raji (B) and 35 BJAB (C) subclones containing the inducible reporter. Arrowheads and different symbols indicate doxycycline addition to aliquots of each subclone. (D) Schematic illustration for calculation of the factor of transcription effect: the average number of GFP+ cells generated after transcription induction (average of all values represented by open circles) was divided by the average number of GFP+ cells detected at the beginning of transcription induction (average of all values represented by closed circles). (E) Factors of transcription effect for all Raji and BJAB subclones analysed with the inducible reporter. Each dot represents one clone. (F) Average numbers of GFP+ cells for all subclones of Raji and BJAB were calculated using the whole data set (i.e. average of all data represented by open and closed circles in D). Each dot represents one clone. The difference between the data sets is not statistically significant (P > 0.1).

Mentions: The human B cell lines Raji, BJAB, 721, EREB2-5 and the human T cell line Jurkat were cultured in RPMI-1640 (Invitrogen, Carlsbad, California, USA) containing 10% fetal bovine serum, penicillin, streptomycin, sodium pyruvat and glutamine at 37°C in a humidified 5% CO2 atmosphere. Three to four days after electroporation, selection was started by addition of 50–400 µg/ml hygromycin (Invitrogen) or 0.8 µg/ml puromycin (Sigma-Aldrich, St.Louis, Missouri, USA), and was typically complete 14 days after transfection. Every 2–4 days, 105 living cells were analysed for GFP+ cells on a FACScan (Becton Dickenson, Franklin lakes, NJ, USA). For analysis of transcription effects, aliquots of single cell clones containing the inducible reporter vector were induced in weekly intervals by addition of 1 µg/ml doxycycline, and the number of GFP positive cells was determined by FACS analysis over a period of 2 weeks. The factor of transcription effect was calculated for each subclone by the formula: [(1/nb)∑bi]/[(1/na)∑ai] (Figure 2D; a, closed circles; b, open circles). P-values were derived by Student's t-tests. The BJAB subclone inducible for HA-AID expression by application of doxycycline was generated by integration of the HA-AID expression vector into the genome after linearization with AscI and XmnI. This clone was analysed for transcription effect as described above by induction of AID expression and transcription in parallel.


Non-conservative homologous recombination in human B lymphocytes is promoted by activation-induced cytidine deaminase and transcription.

Mierau M, Drexler GA, Kutzera A, Braunschmidt K, Ellwart J, Eckardt-Schupp F, Fritz E, Bachl J, Jungnickel B - Nucleic Acids Res. (2008)

Transcription promotes recombination in Raji but not in BJAB cells. (A) Schematic structure of the vector used for the analysis of transcription effects on recombination. The bidirectional doxycycline-inducible promoter driving expression of the recombination cassette and NGFRt and elements for doxycycline dependent regulation are indicated. The vector system does not contain Ig enhancer elements. Frequencies of GFP+ cells after doxycycline addition in four representative out of 34 Raji (B) and 35 BJAB (C) subclones containing the inducible reporter. Arrowheads and different symbols indicate doxycycline addition to aliquots of each subclone. (D) Schematic illustration for calculation of the factor of transcription effect: the average number of GFP+ cells generated after transcription induction (average of all values represented by open circles) was divided by the average number of GFP+ cells detected at the beginning of transcription induction (average of all values represented by closed circles). (E) Factors of transcription effect for all Raji and BJAB subclones analysed with the inducible reporter. Each dot represents one clone. (F) Average numbers of GFP+ cells for all subclones of Raji and BJAB were calculated using the whole data set (i.e. average of all data represented by open and closed circles in D). Each dot represents one clone. The difference between the data sets is not statistically significant (P > 0.1).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2553578&req=5

Figure 2: Transcription promotes recombination in Raji but not in BJAB cells. (A) Schematic structure of the vector used for the analysis of transcription effects on recombination. The bidirectional doxycycline-inducible promoter driving expression of the recombination cassette and NGFRt and elements for doxycycline dependent regulation are indicated. The vector system does not contain Ig enhancer elements. Frequencies of GFP+ cells after doxycycline addition in four representative out of 34 Raji (B) and 35 BJAB (C) subclones containing the inducible reporter. Arrowheads and different symbols indicate doxycycline addition to aliquots of each subclone. (D) Schematic illustration for calculation of the factor of transcription effect: the average number of GFP+ cells generated after transcription induction (average of all values represented by open circles) was divided by the average number of GFP+ cells detected at the beginning of transcription induction (average of all values represented by closed circles). (E) Factors of transcription effect for all Raji and BJAB subclones analysed with the inducible reporter. Each dot represents one clone. (F) Average numbers of GFP+ cells for all subclones of Raji and BJAB were calculated using the whole data set (i.e. average of all data represented by open and closed circles in D). Each dot represents one clone. The difference between the data sets is not statistically significant (P > 0.1).
Mentions: The human B cell lines Raji, BJAB, 721, EREB2-5 and the human T cell line Jurkat were cultured in RPMI-1640 (Invitrogen, Carlsbad, California, USA) containing 10% fetal bovine serum, penicillin, streptomycin, sodium pyruvat and glutamine at 37°C in a humidified 5% CO2 atmosphere. Three to four days after electroporation, selection was started by addition of 50–400 µg/ml hygromycin (Invitrogen) or 0.8 µg/ml puromycin (Sigma-Aldrich, St.Louis, Missouri, USA), and was typically complete 14 days after transfection. Every 2–4 days, 105 living cells were analysed for GFP+ cells on a FACScan (Becton Dickenson, Franklin lakes, NJ, USA). For analysis of transcription effects, aliquots of single cell clones containing the inducible reporter vector were induced in weekly intervals by addition of 1 µg/ml doxycycline, and the number of GFP positive cells was determined by FACS analysis over a period of 2 weeks. The factor of transcription effect was calculated for each subclone by the formula: [(1/nb)∑bi]/[(1/na)∑ai] (Figure 2D; a, closed circles; b, open circles). P-values were derived by Student's t-tests. The BJAB subclone inducible for HA-AID expression by application of doxycycline was generated by integration of the HA-AID expression vector into the genome after linearization with AscI and XmnI. This clone was analysed for transcription effect as described above by induction of AID expression and transcription in parallel.

Bottom Line: The molecular basis for the differential use of these two pathways in different species is unclear.Employing a functional HR assay in human germinal centre like B cell lines, we detect elevated HR activity that can be enhanced by transcription and AID.Products of such recombination events mostly arise through non-conservative HR pathways, while the activity of conservative HR is low to absent.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Molecular Biology and Tumor Genetics, Helmholtz Center Munich, National Research Center for Environmental Health, D-81377 Munich.

ABSTRACT
During secondary immunoglobulin (Ig) diversification in vertebrates, the sequence of the variable region of Ig genes may be altered by templated or non-templated mechanisms. In both cases, cytidine deamination by activation-induced cytidine deaminase (AID) in the transcribed Ig loci leads to DNA lesions, which are repaired by conservative homologous recombination (HR) during Ig gene conversion, or by non-templated mutagenesis during somatic hypermutation. The molecular basis for the differential use of these two pathways in different species is unclear. While experimental ablation of HR in avian cells performing Ig gene conversion may promote a switch to somatic hypermutation, the activity of HR processes in intrinsically hypermutating mammalian cells has not been measured to date. Employing a functional HR assay in human germinal centre like B cell lines, we detect elevated HR activity that can be enhanced by transcription and AID. Products of such recombination events mostly arise through non-conservative HR pathways, while the activity of conservative HR is low to absent. Our results identify non-conservative HR as a novel DNA transaction pathway promoted by AID and suggest that somatic hypermutation in germinal centre B cells may be based on a physiological suppression of conservative HR.

Show MeSH
Related in: MedlinePlus