DEVELOPMENTAL CONTROL OF GENE EXPRESSION 
Rudolf Grosschedl, Ph.D., Assistant Investigator 
The research in Dr. Grosschedl's laboratory is fo- 
cused on the molecular mechanisms that underlie 
the developmental regulation of immunoglobulin 
(Ig) gene expression. Ig |jl heavy-chain and k light- 
chain genes are expressed only in B lymphocytes. 
Transcripts from these genes are generated in a 
temporally ordered manner during B cell differenti- 
ation, M^ith the appearance of |jl transcripts preced- 
ing the synthesis of k mRNA. 
I. Tissue-specific and Temporal Regulation of Immu- 
noglobulin Gene Expression in Transgenic Mice. 
To examine the function of known regulatory se- 
quence elements and individual factor-binding sites 
for the developmental expression pattern of Ig 
genes in vivo and to identify new sequence ele- 
ments, Dr. Grosschedl and his colleagues introduced 
wild-type and mutated |jl genes into the mouse germ- 
line. Deletion of the enhancer abrogated expression 
of the exogenous |x Ig gene at any stage of the B 
cell lineage. The Ig heavy-chain (IgH) enhancer in- 
teracts with many nuclear factors. Although most of 
the Ig enhancer-binding factors are present in all 
tested cell types, one of these factors, Oct-2, is 
found only in B cells. To examine the role of Oct-2 
in the establishment of the correct developmental 
expression pattern in vivo, the laboratory intro- 
duced |x genes carrying point mutations in the Oct- 
2-binding sites into the mouse germline. Mutations 
in the Oct-2 -binding sites of the promoter and en- 
hancer decreased the level of |x gene expression in 
B cells by two orders of magnitude. The mutations 
in the Oct-2 -binding site, however, did not abro- 
gate the cell-type-specificity of |jl gene expression. 
These data suggest that Oct-2 is an important, but 
not the only, determinant for the lymphoid-specific 
expression of the |jl gene. The occupancy of the wild- 
type and mutated enhancer was analyzed by in vivo 
genomic footprinting to examine whether the point 
mutations in the Oct-2-binding site of the enhancer 
abrogate binding of any of the other factors. In the 
mutated enhancer of the transgene, another factor- 
binding site that, by itself, is not affected by the mu- 
tation was also found to be unoccupied, indicating 
that the binding of a factor to this specific site re- 
quires an interaction with Oct-2. 
To determine the molecular basis for the sequen- 
tial expression of Ig heavy- and light-chain genes 
during B cell differentiation. Dr. Grosschedl and his 
colleagues constructed hybrid Ig genes in which 
individual regulatory sequences of either |jl heavy- 
or K light-chain genes were interchanged. After 
gene transfer into the mouse germline, the tempo- 
ral expression pattern of the transgenes was exam- 
ined. Expression of the hybrid transgene in which 
the |JL heavy-chain enhancer was replaced with the k 
light-chain enhancer resulted in a change in the 
temporal expression pattern. During mouse devel- 
opment, expression of the hybrid transgene was de- 
layed by at least one day gestation. Moreover, in 
pre-B cells representing the early stage of the B cell 
lineage, expression of the hybrid transgene was low 
when compared with the intact |x gene. In contrast, 
in more mature B cells the level of expression of 
the hybrid gene was similar to that of the |x gene. 
Thus the differential activity of the k enhancer in 
pre-B and mature B cells appears to determine, at 
least in part, the sequential expression of Ig genes 
in vivo. 
11. Role of Enhancer Sequences in Maintenance of 
the Active Transcriptional State During Cell 
Proliferation. 
The pattern of gene expression of mammalian 
cells can be stably propagated from mother to 
daughter cells. The maintenance of the transcrip- 
tional state has been attributed to the stable propa- 
gation of transcription complexes and to stable 
modifications of DNA or chromatin. If transcrip- 
tional complexes persist during DNA replication, 
and if modifications of DNA or chromatin are inher- 
itable, the transcriptional state of a gene could be 
propagated in the absence of the genetic elements 
required for establishment of its expression pat- 
tern. In particular, enhancer elements have been 
considered only transiently required to establish 
the assembly of a transcription complex. 
An experiment was devised to test whether the 
enhancer can confer upon a gene "memory" of its 
active transcriptional state. Pre-B cell lines were 
generated carrying a transfected |x gene inserted 
stably at various chromosomal locations. The trans- 
fected |JL gene construct contained an Ig enhancer 
positioned between D and J recombination signals. 
These signals are recognized by a site-specific 
recombinase in pre-B cells and are joined together, 
resulting in a deletion of internal sequences. Be- 
cause the deletion of the enhancer in the trans- 
Continued 
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