Recombinase-Mediated Cassette Exchange as a Novel Method To Study Somatic Hypermutation in Ramos Cells

Baughn, Linda B.; Kalis, Susan L.; MacCarthy, Thomas; Wei, Lan; Fan, Mingming; Bergman, Aviv; Scharff, Matthew D.

doi:10.1128/mbio.00186-11

Cited by 8 publications

(19 citation statements)

References 67 publications

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“…In addition, the AGCT at residues 9 and 10 have more mutations than OHS1 and as many as the AGCT at residues 104/105. As has been reported previously for the endogenous Ramos V region (58,59), 90% of the mutations were in G:C sites, and 10% were in A:T residues, most of which were in Polη hotspots. This relatively lower frequency of mutation in A:T residues in the Ramos cell line has been reported by others (4,58,60).…”

Section: The Lack Of the Ohss Decreases The Mutation Frequency In Ramosmentioning

confidence: 54%

“…As has been reported previously for the endogenous Ramos V region (58,59), 90% of the mutations were in G:C sites, and 10% were in A:T residues, most of which were in Polη hotspots. This relatively lower frequency of mutation in A:T residues in the Ramos cell line has been reported by others (4,58,60). Consistent with this lower frequency of mutations at A:T residues in Ramos, there are many fewer mutations in the A:T residues just 3′ to OHS2 and at the 3′ end of the V region (Fig.…”

Section: The Lack Of the Ohss Decreases The Mutation Frequency In Ramosmentioning

confidence: 54%

“…The Ramos cells provide a good cellular model for the initial mutational events that occur in vivo because AID is constantly expressed and the relative susceptibility of the different AID hotspots to mutation is similar to that seen in mice (69). However, the frequency of AID-induced mutation is 20-50 times lower in the Ramos cells than is estimated to occur in vivo (4,(58)(59)(60). Because the frequency of mutation was so much lower than in vivo and almost all of the mutated V regions had only one mutation, it was easier to see that the OHS2 had an ∼10 times higher frequency of mutation than the other AGCTs and an ∼100 times higher frequency of mutation than at many other sites in the V region (Fig.…”

Section: Discussionmentioning

confidence: 98%

“…The Ramos Burkitt's lymphoma cell line with RMCE was generated (58) and grown in Iscove's modified Dulbecco's medium (BioWhittaker) supplemented with 10% (vol/vol) FBS (Atlanta Biologicals), 100 U/mL penicillin-streptomycin (Mediatech), and 0.6 mg/mL hygromycin (Calbiochem). Soft agar cloning was performed and Ramos 6.25 subclone was used as IgM+ control.…”

Section: Methodsmentioning

confidence: 99%

“…In an attempt to identify the location of the initial deaminations and the effect of those events on the rest of the V region, we examined mutations of the same germ line IGHV3-23*01 in the Ramos cultured human B-cell line that is like the unselected memory B-cell database and does not undergo antigen selection. We used recombinase-mediated cassette exchange (RMCE) (38,58) to introduce the WT IGHV3-23*01 (3-23WT) and two modified versions of IGHV3-23*01 (3-23Mod and 3-23AATT) as single copies in the right orientation into the endogenous Ig heavy chain V-region locus of the Ramos human Burkitt's lymphoma cell line (58). The Ramos cells are a germinal center-like B-cell line that constitutively expresses AID at a low level (59) and undergoes SHM at a rate that is 20-50 times lower than the estimates for the rate of mutation in vivo (4,(58)(59)(60).…”

Section: The Lack Of the Ohss Decreases The Mutation Frequency In Ramosmentioning

confidence: 99%

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Overlapping hotspots in CDRs are critical sites for V region diversification

Wei

Chahwan

Wang

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Activation-induced deaminase (AID) mediates the somatic hypermutation (SHM) of Ig variable (V) regions that is required for the affinity maturation of the antibody response. An intensive analysis of a published database of somatic hypermutations that arose in the IGHV3-23*01 human V region expressed in vivo by human memory B cells revealed that the focus of mutations in complementary determining region (CDR)1 and CDR2 coincided with a combination of overlapping AGCT hotspots, the absence of AID cold spots, and an abundance of polymerase eta hotspots. If the overlapping hotspots in the CDR1 or CDR2 did not undergo mutation, the frequency of mutations throughout the V region was reduced. To model this result, we examined the mutation of the human IGHV3-23*01 biochemically and in the endogenous heavy chain locus of Ramos B cells. Deep sequencing revealed that IGHV3-23*01 in Ramos cells accumulates AID-induced mutations primarily in the AGCT in CDR2, which was also the most frequent site of mutation in vivo. Replacing the overlapping hotspots in CDR1 and CDR2 with neutral or cold motifs resulted in a reduction in mutations within the modified motifs and, to some degree, throughout the V region. In addition, some of the overlapping hotspots in the CDRs were at sites in which replacement mutations could change the structure of the CDR loops. Our analysis suggests that the local sequence environment of the V region, and especially of the CDR1 and CDR2, is highly evolved to recruit mutations to key residues in the CDRs of the IgV region.A fter an encounter with antigen and subsequent migration into the germinal centers of the secondary lymphoid organs, B cells undergo a regulated cascade of mutational events that occur at a very high frequency and are largely restricted to the variable (V) and switch (S) regions of the Ig heavy chain locus and the V region of the light chain locus. These mutagenic events are responsible for the somatic hypermutation (SHM) of the V regions and the class switch recombination of the constant (C) regions that are required for protective antibodies (1, 2). Both SHM and class switch recombination are initiated by activationinduced deaminase (AID) that preferentially deaminates the dC residues in WRC (W = A/T, R = A/G) hotspot motifs at frequencies 2-10-fold higher than SYC (S = G/C; Y = C/T) cold spots (3-7). During V region SHM, the resulting dU:G mismatch can then be replicated during S-phase to produce transition mutations, be processed by uracil-DNA glycosylase 2 and apurinic/ apyrimidinic endonucleases through the base excision repair pathway to produce both transitions and transversions (8-10), or be recognized by MutS homolog (MSH)2/MSH6 of the mismatch repair (MMR) complex that recruits the low-fidelity polymerase eta (Polη) to generate additional mutations at neighboring A:T residues (11).The specificity of AID targeting to the Ig gene has been under intense investigation. Studies have shown that AID deamination and mutagenesis targets single-stranded DNA substrates generated during ...

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Section: The Lack Of the Ohss Decreases The Mutation Frequency In Ramosmentioning

confidence: 54%

Section: The Lack Of the Ohss Decreases The Mutation Frequency In Ramosmentioning

confidence: 54%

Section: Discussionmentioning

confidence: 98%

Section: Methodsmentioning

confidence: 99%

Section: The Lack Of the Ohss Decreases The Mutation Frequency In Ramosmentioning

confidence: 99%

See 3 more Smart Citations

Overlapping hotspots in CDRs are critical sites for V region diversification

Wei

Chahwan

Wang

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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J H 6 downstream intronic sequence is dispensable for RNA polymerase II accumulation and somatic hypermutation of the variable gene in Ramos cells

Castiblanco

Norton

Maul

et al. 2018

Molecular Immunology

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Activation-induced deaminase (AID) introduces nucleotide substitutions within the variable region of immunoglobulin genes to promote antibody diversity. This activity, which is limited to 1.5 kb downstream of the variable gene promoter, mutates both the coding exon and downstream intronic sequences. We recently reported that RNA polymerase II accumulates in these regions during transcription in mice. This build-up directly correlates with the area that is accessible to AID, and manipulation of RNA polymerase II levels alters the mutation frequency. To address whether the intronic DNA sequence by itself can regulate RNA polymerase II accumulation and promote mutagenesis, we deleted 613 bp of DNA downstream of the J6 intron in the human Ramos B cell line. The loss of this sequence did not alter polymerase abundance or mutagenesis in the variable gene, suggesting that most of the intronic sequence is dispensable for somatic hypermutation.

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A source of the single-stranded DNA substrate for activation-induced deaminase during somatic hypermutation

et al. 2014

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During somatic hypermutation (SHM), activation-induced deaminase (AID) mutates deoxycytidine on single-stranded DNA (ssDNA) generated by the transcription machinery, but the detailed mechanism remains unclear. Here we report a higher abundance of RNA polymerase II (Pol II) at the immunoglobulin heavy chain variable (Igh-V) region compared to the constant region and partially transcribed Igh RNAs, suggesting a slower Pol II progression at Igh-V that could result in some early/premature transcription termination after prolonged pausing/stalling of Pol II. Knocking down RNA exosome complexes, which could decrease premature transcription termination, leads to decreased SHM. Knocking down Spt5, which can augment premature transcription termination, leads to increases in both SHM and the abundance of ssDNA substrates. Collectively, our data support the model that, following the reduction of Pol II progression (pausing or stalling) at the Igh-V, additional steps such as premature transcription termination are involved in providing ssDNA substrates for AID during SHM.

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Recombinase-Mediated Cassette Exchange as a Novel Method To Study Somatic Hypermutation in Ramos Cells

Cited by 8 publications

References 67 publications

Overlapping hotspots in CDRs are critical sites for V region diversification

Overlapping hotspots in CDRs are critical sites for V region diversification

J H 6 downstream intronic sequence is dispensable for RNA polymerase II accumulation and somatic hypermutation of the variable gene in Ramos cells

A source of the single-stranded DNA substrate for activation-induced deaminase during somatic hypermutation

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