By
From the Department of * Cell Biology and Department of Pediatrics, Albert Einstein College of
Medicine, Bronx, New York 10461
Higher organisms have evolved unusual molecular and
genetic mechanisms to generate high affinity and
highly specific antibodies to a seemingly infinite number of
foreign antigens (1). Even though a highly diverse germline
encoded antibody repertoire is created, most of the antibodies have low affinities and the organism must find a way
to modify those antibodies so that they will bind with high
affinity and neutralize viruses and toxins. In mice and humans this is done during the course of the T dependent antibody response by introducing large numbers of point mutations (2) into the variable (V) region genes that encode
the antigen binding site. B cells expressing antibodies with
amino acid substitutions that result in a higher affinity are
selectively stimulated to proliferate and differentiate by antigen and helper T cells and these higher affinity antibodies
come to dominate the antibody response (3). Somatic V
region hypermutation occurs primarily in B cells, though
there is one report of V region mutation in T cell receptors
in germinal centers (6).
Even though somatic V region mutation was the first of
the many unusual molecular events that occur during B cell
differentiation to be documented (2), and the sequences of
hundreds of antibodies that are the products of this process
have been determined, less is known about the molecular
and biochemical mechanisms responsible for V region hypermutation than for other processes involved in the generation of antibody diversity such as V(D)J rearrangement and isotype switching. This is due in part to the lack of cultured cell systems in which the process can be studied (7, 8). Insights are now beginning to be gained from the study of
transfected genes in mice (9-13 and reviewed in 14-16)
and cultured cell systems that can carry out V region hypermutation of transfected genes have recently been reported (17).
In this issue Tumas-Brundage and Manser (21) have reported on the use of transgenic mice to examine the role of
the heavy chain promoter in the location and rate of V region hypermutation. As these authors point out, the analysis of the sequences of both endogenous heavy and light
chain genes and of transgenes that have undergone somatic
mutation have led to the belief that proteins that are recruited to the transcriptional apparatus are involved in V
region hypermutation (12, 22). The salient characteristics of the mutational process (26) are: (a) it is due primarily to point mutations that arise at rates that are estimated to be 10 Experiments first with light chain (reviewed in reference
14) and more recently with heavy chain (reviewed in reference 16) transgenic mice have provided additional information that has focused attention on the interrelationship
between transcription and V region hypermutation. The
first transgenic experiments by O'Brien et al. (9) showed
that transgenes located outside of the Ig locus could undergo what appeared to be the normal process of V region
hypermutation and suggested that the Ig gene and its immediate flanking sequences have all of the information that is necessary to target and regulate the process. Subsequent
studies revealed that non-Ig genes such as hemoglobin,
GPT, the neomycin resistance gene or CAT could replace
all or part of the V region and still undergo hypermutation
(41, 42), proving that the coding sequence of the light
chain V region did not contain any specific signals that targeted mutation to it. This suggested that the sequences
flanking the V region were responsible for targeting and
regulating the process. The essential role of flanking cisacting sequences was confirmed by showing that both the
intronic and 3 In an attempt to learn more about the detailed mechanisms responsible for V region mutation, Betz et al. (12)
showed that the hemoglobin promoter could be substituted
for the light chain promoter in light chain transgenes without having a dramatic effect on the rate of mutation. This
was an important finding since it suggested that the light
chain promoter was not required for either B cell specificity or the restriction of mutation to the Ig gene. TumasBrundage and Manser (21) have investigated the role of the
promoter further using a heavy chain V region transgene
that is not active in its ectopic location (16). However, on
rare occasions the V region transgene that they have introduced rearranges or undergoes gene conversion so that it is
now located in the heavy chain locus (16). B cells expressing this V region, now in association with the endogenous
C region, are stimulated by antigen and accessory cells to
replicate and differentiate presumably in a relatively normal
manner. Because of positive selection, there are enough of
these B cells expressing the modified transgenic V region to
be recovered as hybridomas. This system has the benefit of
allowing Tumas-Brundage and Manser, and Selsing and his
colleagues who have used a similar approach (10), to analyze the behavior of modified variable region genes that are
now in the correct chromosomal location and associated
with all of the 3 Tumas-Brundage and Manser have studied transgenes
with a minimal TATA-containing heavy chain promoter
or a non-Ig promoter that is from the gene that encodes
the Ig- Tumas-Brundage and Manser have also placed a Drosophila intron as a spacer between the promoter and the V region exon to examine whether the 3 The authors conclude that their results support an important role for transcription in V region hypermutation.
However, it is still not obvious how transcription actually
contributes to mutation. For example, although it is true
that TFIIH is part of both the transcriptional initiation apparatus and of the apparatus that carries out nucleotide excision repair (reviewed in 43 and 44), the available evidence indicates that TFIIH is lost from the transcriptional
process within 30-70 bases after initiation (44). It is possible that there is pausing of transcription at certain places in
the V region, perhaps marked by the hot spot motifs, and
that TFIIH, or a similar group of proteins, are then recruited to that site and carry out a process that is error
prone because of some B cell- and Ig-specific factor (23).
However, it still seems equally possible that the high rate of
transcription is merely making the V region accessible to
other factors that are not directly connected to transcription since a role for error prone replication has not been
ruled out. This sort of argument is reminiscent of the discussions of the role of germline transcripts of the C region
in isotype switching (46) which is still unresolved, though
recent transgenic experiments suggest splicing of the germline transcript is a critical event in this process (47). It is
clear that high rates of transcription per se are not sufficient
for V region hypermutation since there is little mutation in
T independent responses even though large amounts of Ig
are produced (48).
The experiments described by Tumas-Brundage and
Manser (21) demonstrate the benefits of using an in vivo
system but also illustrate why it will be difficult to resolve
the questions that are raised if only transgenic systems are
used. These are very demanding experiments and it is virtually impossible in transgenic systems to do all of the controls and to examine the many different constructs that are
required to identify the essential elements in these cis-acting
sequences. Nevertheless, it is just those sorts of experiments
that will be required to dissect out the role of particular cisacting elements in transcription and mutation. This will require in vitro systems in which V region mutation occurs at
the same high rates and through the same mechanisms as in
vivo (19, 20). In the meantime, studies such as those reported by Tumas-Brundage and Manser (21) are contributing important information on normal mechanisms of V region hypermutation against which all in vitro systems will
have to validated.
5 to 10
3/base pair/generation and are 4-6 orders of
magnitude higher than the rate of mutation of housekeeping genes in higher organisms. This results in the accumulation of 5-15 base changes in the V regions of most antibodies that have been selected for during the late primary
and secondary response. However, large numbers of mutations are also seen in passenger transgenes (27) and in the 3
untranslated regions immediately flanking the V region (28), so the high frequency of point mutations is not an artifact of selection. There is even a report of 40-70 point
mutations in V regions associated with
constant regions,
but these antibodies do not appear to play a role in the normal response (29); (b) high rates of mutation occur in already rearranged heavy and light chain variable region
genes and their immediate flanking sequences. Few mutations are found 5
to the promoter and the mutational process extends 3
from the promoter for 1-1.5 kb with the
maximum accumulation of mutations in the coding exon
and its immediate 3
flanking region (24, 25, 28, 30).
The fact that the mutational process begins at the site of
initiation of transcription and extends in the direction of
transcription for a limited distance has suggested to many
that transcription is involved in V region hypermutation
(12, 22); (c) the highest frequency of somatic V region
mutations is found in centroblasts in the dark zone of the
germinal center (reviewed in reference 33). V region hypermutation appears to occur at about the same time or just
before isotype switching but is distinct from and does not
depend on that process (34). It is unclear whether somatic mutation is limited to one stage in B cell differentiation or if it can occur at lower rates in pre-B cells or in
more differentiated plasma cells since most of the relevant
studies have used sequencing techniques that have sufficiently high error rates so that mutation below a rate of 10
5 could not be detected: (d) although point mutations
are found throughout the V region and its immediate
flanking sequences, there are triplets such as the AGC and
TAC and their inverted repeats, GTA and GCT that are
preferred targets for the mutational process (27, 28, 38),
and more extended versions of some of those triplets such
as a purine, a G, a pyrimidine and an A or T (RGYW)
have been recognized (27, 28, 38). These hot spots for mutation are not an artifact of selection since they can be deduced from silent base changes and from mutations in untranslated regions (27, 28). In addition, there appears to
have been evolutionary selection for hot spot motifs in the complementary determining regions of the V region that
encode the contact residues with antigen (39 and reviewed
in reference 15). The mutational process results in transitions more often than transversions and appears to have a
bias for the transcribed strand (27, 28, 40).
transcriptional enhancers of the light chain
were required for mutation (12). This requirement for these
transcriptional regulatory elements drew additional attention to the potential role of transcription in V region hypermutation. This was reinforced by the growing appreciation that factors such as TFIIH, that were part of the basal
transcriptional apparatus, also play a role in excision repair
in eukaryotic cells in general (43, 44). Peters and Storb (13)
provided a dramatic illustration of the importance of the
initiation of transcription in V region mutation by introducing a promoter in front of the C region, which is
thought not to undergo mutation normally, and showing
that the initiation of transcription proximal to the C region
resulted in C region mutations in transgenic mice.
elements that are normally present. In addition, the B cells expressing this transgene are in a mouse
that also has B cells expressing unmodified endogenous V
regions so that V regions under the control of variant and
wild type promoters can be compared in the same mouse.
The studies described in this paper (21) and in their previous work (16) indicate that the sequences 3
to the V region are important in targeting and regulating V region hypermutation. This is consistent with the findings of others
that there are important regulatory sequences in the intron
between J and C (29) and associated with the C regions (19).
polypeptide (45), a part of the complex through
which membrane Ig receptors signal. This B29 promoter
lacks a TATA box but shares at least six transcriptional regulatory elements, including one for Oct 2, with the Ig promoter and confers B cell specific expression, so it could recruit many of the same transcriptional factors as the Ig
promoter (45). There is no evidence, however, that Ig-
chains undergo somatic mutation. With both the minimal
Ig and the B29 promoters, V region somatic mutation occurs, though perhaps at a lower frequency (21). TumasBrundage and Manser also suggest that in the studies by
Betz at al. (12) there may have been a lower frequency of
mutation in the transgenes under control of the hemoglobin promoter. It may be difficult to make reliable quantitative comparisons between transgenes, or even between the
transgenes and a comparable endogenous gene, because differences in expression could result in differences in selection and mutation. In fact, differences in expression could
explain why B cells expressing the modified transgenic V region do not participate fully in the memory response
(21). The important point is that mutation occurs in Ig
genes under the control of a variety of promoters. We cannot be certain that a transcriptional factor, or factors, that
normally interact with the Ig promoter is not also interacting with these variant promoters and recruiting some B cell
and Ig gene specific factor that is required for mutation.
Nevertheless, these studies, along with those of Betz et al.
(12), suggest that the role of the promoter is non-specific in
that transcription is required but there is not a particular
regulatory sequence in the promoter that is necessary for
mutation.
border of mutation is
determined by its distance from the promoter. Previous
studies with endogenous V regions with different distances
between the promoter and the V region suggested that the
distance from the promoter determined the 3
border of
mutation (25). Tumas-Brundage and Manser found the highest frequency of mutations in the spacer and a somewhat lower frequency in its immediate 3
region, which is
now the displaced coding region of V (21). These findings
confirm that mutation can occur in non-Ig sequences (41,
42) and suggest that the 3
border of mutation is determined by its distance from the promoter.
Address correspondence to Matthew D. Scharff, Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461.
Received for publication 3 December 1996
The work of the authors that is discussed here was supported by grants to M.D. Scharff from the NCI (R35 CA39838) and to N.S. Green from the NCI (5K11CA01635), James S. McDonnell Foundation and American Society for Hematology.1. | Weill, J.-C., and C.A. Reynaud. 1996. Rearrangement/hypermutation/gene conversion: when, where and why? Immunol. Today. 17: 92-97 [Medline] . |
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