Conserved Sequence Motifs in Immunoglobulin Loci
Potential Role for Hypermutation Recruitment
Background:
Betz, 1994. Elements regulating somatic hypermutation of an immunoglobulin
kappa gene: critical role for the intron enhancer/matrix attachment region.
(Milstein lab)
Conclusion: The VDJ promoter was replaced with an Hb promoter, and
it was found that hypermutation was unaffected in kappa TG. Therefore,
the promoter does not specifically target hypermutation.
Yelamos, 1995. Targeting of non-Ig sequences in place of the V segment
by somatic hypermutation. (Milstein/Neuberger lab)
Conclusion: The V gene sequences do not recruit somatic hypermutation
to the VDJ region, as mutations were found in the beta globin gene when
it replaced the V gene in the Ig kappa transgene.
Lin, 1998. (Scharff lab) The effects of E mu, 3 alpha and 3 kappa
enhancers on mutation of an Ig-VDJ-Cgamma2a Ig heavy gene in cultured B
cells
Conclusion: The intronic enhancer was replaced with an SV40 or CMV
enhancer region, in both orientations. The hypermutation frequency was
unaffected, but the transcription was 4 20 times lower than with the
endogenous Ei. The addition of the 3 kappa or 3 alpha enhancer regions
did not increase the frequency of hypermutation.
Kenny, 2001. Mutational analysis of immunoglobulin germline-derived
vlambda4b light chains in rheumatoid arthritis. .
Conclusion: Lambda chains also undergo hypermutation.
Bachl, 2001. Increased transcription levels induce higher mutation rates
in a hypermutating cell line.
Conclusion: The rate of transcription correlate directly with the rate
of hypermutation. AID is transcribed in the hypermutating cell line.
Summary:
The rearranged Ig genes in both the IgH and the IgL loci undergo somatic
hypermutation in activated B cells following stimulation with antigen.
The hypermutating region starts at the promoter, and continues downstream
for about 2 kB. Transcription of the gene is essential for hypermutation.
Rate of transcription is correlated with the rate of hypermutation. There
are several promoters found in the Ig loci: kappa Ei, 3E kappa, E lambda,
heavy Ei, 3 E alpha. The enhancers are essential for hypermutation, however,
if they are replaced with viral enhancers, hypermutation still occurs.
Likewise, the promoter is also essential, but can be replaced. The identity
of the gene that is being hypermutated is also irrelevant. Activation-induced
cytosine deaminase (AID) is essential for both somatic hypermutation as
well as switch recombination. This implies that similar mechanisms are
at play in the two phenomena. However, switch recombination occurs independently
of hypermutation, and vice versa.
The plan:
It seems that a consensus sequence for recruiting hypermutation
should exist, but it has not been found yet. However, the above experiments
have ruled out certain regions:
a. The promoter and the actual gene.
b. The Ei and the MARs on the heavy chain. The experiments in the IgH
have not been repeated in the IgL loci, so it is not certain that the kappa
and lambda promoters do not participate in hypermutation recruitment.
c. The 3E alpha promoter is an interesting conundrum. The transgene
that was tested for hypermutation did not contain the 3 alpha enhancer.
In the endogenous IgH locus, the 3Ea is around 200 kB from the V(D)J.
Could this element act over 200 kB, or perhaps even across chromosomes?
It is unlikely. Indeed, if it did act across chromosomes, its unlikely
that the 3Ea confers specificity to recruit hypermutation. Thus, this
also cant be the hypermutation consensus sequence (HCS).
With the above taken into account, there are several possible locations
for the HCS in the IgH and IgL kappa loci:
1. 3 of J and 5 of the Ei region. However, Klix (1998) made an IgL
kappa transgene where 1.07 kB of this region was deleted. A normal rate
of mutation was found in this transgene. Another thing to consider is that
this region still undergoes hypermutation. Thus, it would not be the ideal
place for a conserved sequence.
2. 3 of Ei and 5 of the switch region in IgH r 5 of Ck in Ig kappa.
In the same kappa transgene, Klix had also deleted 0.43 kB of this region,
leaving a very small amount of sequence. Thus, its once again unlikely
that this region contains the HCS, but it should still be investigated.
3. 3 of switch region and 3 of C. This is a very large region (up
to 60kB in IgG3). However, this region is also present in every constant
region. There are 11 constant regions in the human IgH. These can all be
compared for conserved pattern motifs.
The experiment:
1. Obtain the following sequences of IgH (and alter for IgL):
a. 3 of J to 5 of C.
b. 3 of C to 5 of the next C.
2. Divide the a) sequence into the following segments:
1. 3 of J to 5 of Ei/MAR
2. 3 of Ei/MAR to 5 of the switch region.
3. 3 of switch to 5 of C.
3. Compare the sequence chunks. i.e. all the 3 J to 5 Ei/MAR should
be compared to each other, etc. Isolate conserved sequence motifs.
4. If one of the samples doesnt contain the conserved sequence, look
for the sequence in the other parts of that sample.
5. To compare, use: Teiresias, MACAW, Gibbs. Also ompare IgH and IgL.
6. Later, try TUNA.
Positive control
There is no known HCS, but the HCS must have the following properties:
1. It must be present in all the genes that undergo hypermutation,
IH, Igkappa and Iglambda.
2. It should be fairly conserved.
3. A protein should bind to the HCS.
4. When it is deleted, no hypermutation should occur.
1 and 2 can be tested by bioinformatics, but 3 is for the wet lab,
and a cell line-transgene experiments, with the 18.8 or NSO cell line.
Negative control
It is difficult to have an absolute negative control, as the hypermutation
machinery might use a specific combination of proteins. Some of the individual
proteins might be found in other tissues, but the specific combination
is not. Thus, the key for the negative control lies in selecting a gene
that has many of the similar regulatory elements as the Ig, is expressed
at the same time as the Ig, but does not hypermutate.
I believe that an excellent choice for the negative control gene is the Ig beta gene. It is expressed in B cells, and it is transcribed during the period hypermutation occurs. It is also under the control of many of the same regulatory elements as the IgH and IgL genes. However, it does not hypermutate. Thus, it should not ontain the HCS.
Another possibility is the TcR gene. These undergo recombination, but not somatic hypermutation. However, they are expressed in T cells, and not B cells. In B cells, they are in the unrearranged form, so hypermutation would not ccur even if the HCS was present. Nonetheless, from the looks of it, the somatic hypermutation and switch recombination are quite unique to the B cells, so might it be that the HCS is also unique? This gene will serve as an interesting quasi-negative control.