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Nucleosomes are assembled at the MP2 and M5 sequences in vivo. Lanes 1 and 2, bp amplification band with MP2-specific primers using the mononucleosomes as the template; lanes 3 and 4, bp amplification band with spacer-specific primers using the mononucleosomes as the template; lane 5, bp DNA ladder to 1, bp ; lane 6, bp and bp amplification bands with MP2-specific primers using the genomic DNA gDNA from the MP2-MP2 knock-in clones; lane 7, bp amplification band with spacer-specific primers using the genomic DNA from the MP2-MP2 knock-in clones as the template.
The histograms represent the relative abundance of either the MP2 or the M5 positioning sequence compared to the respective spacer regions, as analyzed by Q-PCR. The values are normalized for the copy number and primer efficiencies. The data represent means and SD of two independent experiments. In the strong nucleosome positioning sequences MP2 , we observed a reduction in mutation frequencies compared with the flanks Fig. The number of mutations was 2- to 4-fold reduced in both copies of the MP2 nucleosome positioning sequence compared with the spacer region and the neighboring IgL V region.
Moreover, mutation frequencies per AID hot spot in the two nucleosome positioning sequences were also considerably lower, despite a similar proportion of AID hot spots Fig. We observed a predominance of single-nucleotide substitutions with few insertions and deletions see Fig. S1A in the supplemental material.
S1B and S2 in the supplemental material , as was found by others in DT40 cells 2. These results indicate that the presence of stably positioned nucleosomes in the immunoglobulin gene significantly affects the accessibility of AID to and the mutation patterns within Ig genes. Ig light chain sequence analysis of the nucleosome positioning sequence knock-in clones. A Map of lg gene with 2 MP2s not to scale ; the triangle represents the two recombined loxP sites. B, D, F, and H Mutations in 1. C, E, G, and I Summary of mutations. CDR, complementarity determining region.
Mutation events in the MP2 and the M5 sequences. The ratios of transitions ts to transversions tv are also shown. C and D Histograms showing distribution of mutations across the bp region of either MP2 or M5 sequence. The bp positioning sequence was divided into three bp regions I, II, and III , and the total number of mutation events is shown in each region.
Top All mutation events. We also observed significant increases in the percentage of mutations in the neighboring regions of M5 Fig. Finally, when we replaced both copies of the MP2 sequence with M5, we observed a significant increase in the percentage of mutations in both copies of M5 Fig.
Replacing the MP2 sequence with the M5 sequence also changed the mutation pattern within the nucleosome. In contrast, the distribution of mutations within the M5 sequence remained relatively constant Fig. S1A and S2 in the supplemental material. S1B in the supplemental material ; two independent cell clones for each of the four types of nucleosome combinations were very similar data not shown.
In the total 1. However, the M5 sequence has more mutation events in the central bp region, although both the MP2 and the M5 sequences have the same number seven of AID hotspots in this region Fig. Thus, we conclude that when we replaced a strong nucleosome positioning sequence MP2 with a weak positioning sequence M5 , mutations in M5 were much higher than in MP2.
The rules for nucleosome assembly deduced from total-genome analyses 22 , 32 enabled us to change the MP2 sequence with high affinity for histone cores to the low-affinity M5 sequence. While regulatory mechanisms also play a major role in chromatin structure 16 , 43 , the striking difference in the biophysical properties of MP2 and M5 nucleosomes validates previous conclusions that the primary DNA sequence can considerably affect the propensity for its assembly into nucleosomes 22 , The findings show that both the presence and stability of the nucleosome strongly influence mutation patterns during SHM: We conclude that the stability of nucleosomes in the IgL gene significantly affects the outcome of the somatic hypermutation process.
In this model, the mutation rate should remain constant through the nucleosome positioning sequence, since mutations occur only when the DNA is nucleosome free. These nucleosome alterations expose DNA that is originally wrapped into a nucleosome. Transcription through a nucleosome 13 and nucleosome remodeling could induce nucleosome repositioning 5 and enhance nucleosomal-DNA unwrapping fluctuations, which occur rapidly, many times a second A The nucleosome can be disassembled and reassembled, which requires all of the DNA-histone contacts to be broken.
Both of these models maintain DNA-histone contacts. M5 has both a reduced affinity for the histone octamer and reduced nucleosome positioning strength relative to MP2. Therefore, M5 has the ability to enhance mutations by both nucleosome repositioning and disassembly. Furthermore, the mutation patterns within MP2 and M5 indicate that both mechanisms occur. This mutation pattern suggests that the dominant mechanism by which AID gains access to highly stable nucleosomes is by either the unwrapping Fig. Repositioning would likely be affected by the neighboring nucleosomes.
However, the mutation frequency within the M5 sequence is relatively constant, in relative concordance with the AID hot spot distributions across M5 Fig.
Since the M5 sequence has a lower affinity for the histone octamer, this DNA sequence could reduce nucleosome occupancy by both enhancing the rate of nucleosome disassembly and reducing reassembly. The combination of these results strongly suggests that AID accesses less stable nucleosomes largely by the disassembly model. These in vivo findings are interesting, given our previous results with in vitro assays of the mutability by AID of MP2 embedded in a supercoiled circular plasmid, pKMP2 However, there were ample mutations in the MP2 nucleosome sequences when the plasmids were transcribed.
The arrangement and sequences of the two MP2 and spacer elements were the same as used in this paper. The conclusion was that AID cannot access nucleosomes unless they are transcribed Clearly the Ig lambda gene in the DT40 cells used here is continuously transcribed and MP2 is mutated, although at a reduced frequency, compared with the flanking DNAs.
It is not simple to make a direct comparison between MP2 and flanking DNAs in the in vitro experiments; some regions without a defined nucleosome were slightly more mutable than MP2, while others had very few mutations see Fig. Interestingly, in the in vitro experiments, the pKMP2 plasmid was transcribed by T7 RNA polymerase that is considerably smaller than the pol II operating in vertebrate cells. It has been shown that nucleosomes containing a T7 promoter are completely displaced by T7 pol The results reported here show that RNA polymerase Pol II can deal with nucleosomes more efficiently than AID and suggest that subtle epigenetic events may be best investigated in vivo.
Our previous data support the idea that negative supercoils behind the RNA polymerases pol extrude single-stranded C's as AID targets The propagation of negative supercoils is probably inhibited at the next nucleosome. This is supported by comparing the processivity of AID in cell-free assays with that in vivo Thus, the average length of the spacers between nucleosomes apparently allows sufficiently large stretches of negative supercoils to develop and become accessible to AID. We find that while nucleosome occupancy influences SHM within the Ig locus, the level of transcription is not significantly influenced by a strong NPS.
Only a 2-fold change in transcription was also observed in budding yeast when the high-affinity NPS, , was inserted at the beginning of the CUP1 gene Interestingly, Gaykalova et al.
In addition, a high-throughput sequencing study 11 found that a NPS inserted in the EF1a promoter of the human factor IX gene contained well-positioned nucleosomes that then became depositioned as the gene was silenced. An important difference between these previous studies and ours is the location of the NPS with respect to the transcription start site. We inserted the two NPSs into the transcribed region of the gene and bp, respectively, from the transcription start site. The sequence in the studies by Gaykalova et al.
The combinations of these results are consistent with the idea that chromatin remodeling may selectively influence the nucleosome position near the promoter regions of genes. Interestingly, in vitro measurements by Gaykolova et al. SHM experiments in mice showed a certain periodicity of the mutation patterns in a highly mutable Ig transgene, RS The results were consistent with the conclusion that the Ig gene was organized into nucleosomes but that different cells had different nucleosome phasings and that the nucleosome pattern was relatively stable for a given cell for several generations throughout the hypermutation process.
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Clearly, in the current study, the inserted MP2 must have caused rather stable nucleosome phasing. Even the M5 sequence is slightly more nucleosomal than the spacer Fig. We found that the free energy of the MP2 sequence, which is a variant of the sequence, is 1. This indicates that the MP2 sequence is at the extreme of high-affinity nucleosome positioning sequences in vivo and that it is not representative of the typical nucleosomal DNA in vivo. These sequences are about 0. This indicates that the M5 sequence is representative of typical nucleosomal DNA in vivo and that our observations of the influence of nucleosomes on SHM at the M5 sequence may apply to nucleosomes in the native Ig locus.
Thus, this study suggests that the limits of nucleosome positioning for Ig genes may be below MP2 stability and around or below that of M5. It will be interesting to investigate the propensity for nucleosome positioning of endogenous Ig genes in mice and humans. It seems possible that the variable regions of Ig genes have evolved for low nucleosome stability to enhance the chance for increased access to AID, DNA repair factors, and error-prone DNA polymerases and hence the creation of maximal variability by somatic hypermutation.
We are greatly indebted to J. Hall for DNA sequencing, and R. Duggan for flow cytometric cell sorting. We are grateful to B. Kee for technical advice and use of her instrument for real-time PCR. Martin for constructive discussions of these experiments and critical reading of the paper.
Published ahead of print 5 March Supplemental material for this article may be found at http: National Center for Biotechnology Information , U.
The biochemistry of somatic hypermutation. - Semantic Scholar
Journal List Mol Cell Biol v. North , c Michael G. Poirier , c, d, e and Ursula Storb a, b. Author information Article notes Copyright and License information Disclaimer. Address correspondence to Ursula Storb, ude.
This article has been cited by other articles in PMC. Associated Data Supplementary Materials Supplemental material. Open in a separate window. Flow cytometric analysis and cell sorting. Identification of somatic mutations. Electrophoresis mobility shift assay EMSA. Nucleosomes significantly influence the mutation pattern of the IgL locus. Reduction in nucleosome stability alters the mutation pattern within the IgL locus. Supplementary Material Supplemental material: Click here to view. We have no conflicting financial interests.
Footnotes Published ahead of print 5 March Supplemental material for this article may be found at http: Anderson JD, Widom J. Sequence and position-dependence of the equilibrium accessibility of nucleosomal DNA target sites. Y — [ PubMed ]. Activation-induced cytidine deaminase initiates immunoglobulin gene conversion and hypermutation by a common intermediate. Betz AG, et al. Elements regulating somatic hypermutation of an immunoglobulin kappa gene: Chaudhuri J, et al. The biology of chromatin remodeling complexes. Conley ME, et al. Primary B cell immunodeficiencies: Gaykalova DA, et al.
A polar barrier to transcription can be circumvented by remodeler-induced nucleosome translocation. Gracey LE, et al. An in vitro-identified high-affinity nucleosome-positioning signal is capable of transiently positioning a nucleosome in vivo. As a consequence, high-efficiency B cells are selected during the immune response in a process known as affinity maturation [ 5 ].
Unlike B cells, T cells lack the capacity to mutate their TCR genes after T-cell activation, and thus classical affinity maturation does not take place in T cells. They found that only primed T cells produced IL-2 and proliferated in vitro in response to TCR triggering induced by anti-CD3 antibodies and monocytes [ 14 ]. Similar observations were later reported by others [ 7 , 9 — 13 , 15 ]. This finding was supported in a subsequent study, where Croft et al.
Mechanisms behind Functional Avidity Maturation in T Cells
In an equivalent study also examining T-cell responses to infection, Pihlgren et al. An overview of studies indicating the existence of functional avidity maturation is given in Table 1. Today, it is widely accepted that T-cell activation should not be considered as a single signal process, but as a sum of interdependent signals. TCR signaling takes place at the interface between the T-cell and the antigen presenting cell. At this contact zone, often referred to as the immunological synapse IS , TCR-signaling components including the TCR itself as well as intracellular-signaling molecules are continuously accumulated during antigen contact [ 26 ].
Although somewhat controversial [ 26 , 27 ], formation of an IS correlates with generation of a robust immune response, and is considered a prerequisite for T-cell activation [ 28 , 29 ]. Even so, new insight into the biology of immunological synapses has revealed that TCR signaling is already initiated in TCR microclusters prior to IS formation. At the IS, CD28 signaling both induces structural stabilization and enlargement of the area itself [ 32 , 33 ].
Eventhough the exact implication of CD28 signaling in T-cell activation is still elusive, it is generally agreed that CD28 amplifies intracellular signaling induced by antigen-triggering of the TCR through modulation of morphological features and TCR signals [ 32 , 33 ]. CD45 is a transmembrane tyrosine phosphatase that maintains Lck activity by promoting dephosphorylation of an inhibitory carboxy-terminal tyrosine residue of Lck.
The biochemistry of somatic hypermutation
Lck activity is a necessity for initiation of TCR signal transduction [ 39 ]. This finding parallels the study of Kersh et al. The vast majority of studies contributing to the current model for TCR signaling were performed using immortal T-cell lines or primed T cells propagated in vitro. The discrepancy between the two human studies might be due to two different primed T-cell populations studied effector and memory cells, resp. Adachi and Davis used high concentrations of soluble anti-CD3 and anti-CD28 antibodies cross-linked by secondary antibodies to stimulate the T cells.
By using cross-linked antibodies for stimulation, a very strong receptor signaling is achieved. As illustrated in a series of mouse virus studies, the strength of TCR signaling determines the requirement for additional activation signals like CD28 signaling and also results in somewhat different responses [ 19 ].
Both scenarios could be relevant for human immunity where a wide range of pathogens with different origins is encountered. Unfortunately, mouse and man seem to differ when it comes to some of the signaling molecules involved in TCR signaling. Early studies describing a need for a third-signal cytokine came from a series of in vitro and in vivo experiments performed by Mesher and co-workers.
Eventhough IL has a role in skewing the CD4 T-cell response, it has no effect on CD4 T-cell proliferation and differentiation in response to antigen. This includes both the requirement for the three antigenic-induced signals as well as intrinsic differences in the signaling machinery. In this way, the signaling machinery is already optimized for signal transduction in primed T cells prior to antigen reencounter. As a result, primed T cells respond much faster and stronger when an antigen is eventually engaged.
It therefore seems as the T cells retain a permanent imprint of a prior response to antigen. But how is such an imprint formed?