GENETIC MECHANISMS INVOLVED IN THE GENERATION OF THE ANTIBODY REPERTOIRE 
Frederick W. Alt, Ph.D., Investigator 
Dr. Alt's laboratory is defining molecular factors 
involved in the development of antibody-producing 
cells. A particular focus is the elucidation of molec- 
ular mechanisms that control the genomic rear- 
rangement events involved w^ith this differentiation 
process. 
I. Regulation of Recombination Events During 
Lymphocyte Dififerentiation. 
The amino terminus of immunoglobulin (Ig) 
heavy and light chains is highly variable and is re- 
sponsible for antigen binding. The carboxyl termini 
of heavy chains have a constant amino acid se- 
quence that determines effector activities. The vari- 
able region of Ig genes is encoded by multiple 
germline elements, the y D, and J segments, which 
are assembled into complete V(D)J variable-region 
genes during the somatic differentiation of B-lin- 
eage cells. Dr. Alt's laboratory demonstrated that 
both Ig heavy- and light-chain variable-region gene 
segments, as well as related gene segments that en- 
code T cell receptor (TCR) variable regions, are as- 
sembled by a common system, VDJ recombinase. 
The constant region of the Ig heavy chain produced 
by a clonal B cell lineage can be changed as a result 
of a separate type of recombination event that jux- 
taposes one of several downstream constant-region 
genes to the expressed VDJ gene; this event is me- 
diated by a different recombination system, referred 
to as class-switch recombinase. 
Dr. Alt's past studies suggested the specific as- 
sembly of Ig heavy- and light-chain or TCR variable- 
region genes in appropriate cells and stages within 
lymphoid lineages is effected by modulating accessi- 
bility of substrate gene segments to VDJ recom- 
binase. Accessibility was correlated with transcrip- 
tion of unrearranged gene segments. To elucidate 
controlling elements, Dr. Alt and his colleagues cre- 
ated transgenic mice that carry a hybrid antigen re- 
ceptor gene minilocus composed of germline TCR 
variable -region gene segments (W, D, and J) linked 
to an Ig heavy-chain constant-region gene with or 
without a DNA segment containing the Ig heavy- 
chain transcriptional enhancer (E|jl). Transgenic 
constructs lacking E|jl did not rearrange in any tis- 
sue. In contrast, presence of the E|x segment within 
the construct dominantly targeted transgenic TCR 
D to J joining at high frequency in both B and T 
cells. However, TCR V to DJ joining within the con- 
struct occurred only in T cells and was correlated 
with transcriptional activity of the unrearranged 
TCR V gene segment. Therefore Dr. Alt's group has 
demonstrated elements that can control two sepa- 
rate aspects of VDJ rearrangement. The E|x element 
(and possibly associated sequences) acts as a domi- 
nant recombinational enhancer to initiate lym- 
phoid-specific D to J rearrangement within the 
transgenic construct; this suggests that enhancer el- 
ements associated with various endogenous Ig and 
TCR loci may act to target those loci for rearrange- 
ment. In addition, an element associated with the V 
segment in the construct provides T cell-specific 
control of V to DJ rearrangement. During the past 
year, new transgenic lines have been generated that 
contain constructs in which various lymphoid en- 
hancer or promoter elements were mutated or ex- 
changed to define their activity precisely with re- 
spect to VDJ recombination. 
Heavy-chain class-switch recombination allows a 
clonal B cell lineage to express the same variable 
(VDJ) region in association with a different con- 
stant region. Dr. Alt's group defined transcription 
units that initiate upstream of class-switch recombi- 
nation target sequences of four different germline 
heavy-chain genes, including 7I, 72b, 73, and e. In 
addition, they have demonstrated that treatment of 
B-lineage cells with the polyclonal activator bacte- 
rial lipopolysaccharide (LPS) induces transcription 
from the germline 72b and 73 promoters, followed 
by induction of switch recombination to these 
genes. Conversely, they demonstrated that addition 
of the T cell factor interleukin-4 (IL-4) simulta- 
neously with LPS inhibits germline 72b and 73 tran- 
scription and switching but induces germline 7I 
and e transcription, followed by switching to those 
constant genes. They also demonstrated that pre- 
cursor B cells inherit a precommitment to switch to 
72b and 73 but that treatment with IL-4 can alter 
that commitment, leading to switching to e even at 
this early stage. This evidence suggests that class 
switching may also be controlled by modulating ac- 
cess of different heavy-chain constant genes to a 
common class-switch recombinase. Accessibility can 
be modulated by treatment of cells with external 
agents and correlates with effects on transcription 
of the target gene sequences. Recently the group 
found several proteins that bind to germline con- 
stant-gene promoter regions and, as a result, may 
be involved in regulating switching. 
Continued 
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