relevant transcription factors, 6) use these clones to 
perturb expression of the transcription factors (anti- 
sense/gene-disruption methodologies) in order to 
analyze their regulatory functions, and 7) analyze 
signaling pathways that control the expression or 
activation of these transcription factors during B cell 
ontogeny. 
Dr. Singh's laboratory has been focusing on the 
regulation of transcription of genes encoding com- 
ponents of the B cell antigen-receptor complex. Spe- 
cifically, they have explored the role of the Oct-2 
protein in controlling the expression of the jii, k, and 
B29 genes. They have also undertaken an analysis of 
the control of mb l gene activity. An elucidation of 
the nature of the regulatory circuitry underlying the 
expression of these key B cell-specific genes should 
provide fundamental insight into B lymphocyte 
differentiation. 
Regulation of Immunoglobulin 
Gene Transcription 
Multiple cell-type-specific enhancer and pro- 
moter elements mediate B-lineage-restricted tran- 
scription of Ig genes. Immunoglobulin {n, k, and X) 
and B29 gene promoters contain a highly conserved 
octanucleotide element (ATTTGCAT) positioned 
~70 bp upstream of the transcriptional start site. 
The octamer element is essential for expression of Ig 
gene constructs in B cell lines, as well as in trans- 
genic mice, and also confers B cell-specific activity 
to a heterologous promoter. However, octamer ele- 
ments are also found in the promoters of ubiqui- 
tously expressed genes and function in different reg- 
ulatory capacities depending on promoter context. 
The identification of the octamer binding proteins 
Oct-1 and Oct-2 has suggested an explanation for 
how the octamer motif plays a central role in both 
lymphoid-restricted and ubiquitous gene expres- 
sion. The Oct-1 protein was detected in a variety of 
cell types, thereby suggesting its involvement in the 
regulation of ubiquitously expressed genes. Oct-2 
was detected only in B-lymphoid cell lines and was 
proposed to mediate the B cell-restricted expres- 
sion of Ig genes. 
Oct-1 and Oct-2 cDNAs have been isolated. Com- 
parison of the deduced amino acid sequences of the 
two proteins has revealed a highly conserved do- 
main (POU domain) involved in DNA recognition. 
As anticipated from biochemical analysis, the Oct-2 
gene is expressed selectively, though not exclu- 
sively, in the lymphoid system. Oct-2 expression is 
also detected in the embryonic and adult central 
nervous system. Overexpression of either Oct-1 or 
Oct-2 in HeLa cells has demonstrated that only 
Oct-2 can activate certain B cell-specific, octamer- 
260 
containing promoters. Furthermore, experiments 
with somatic cell hybrids support the conclusion 
that Oct-2 and not Oct-1 is required to activate an Ig 
promoter. In myeloma X fibroblast hybrids, the ac- 
tivity of an integrated Ig k promoter construct is ex- 
tinguished, and this correlates with suppression of 
Oct-2 expression. Transfection of an Oct-2 expres- 
sion vector into these hybrids reactivates the extin- 
guished reporter construct. 
Genetic Analysis of Oct-2 Function Reveals 
Two Cell-Type-Specific Pathways 
of Octamer-Dependent Gene Activation 
To test genetically the requirement for Oct-2 in 
the expression of Ig genes. Dr. Singh's laboratory has 
exploited a gene-targeting strategy. Both copies of 
the Oct-2 gene in the B cell line WEHI-231 have 
been successfully disrupted by the sequential use of 
promoterless neo- and gpt-hased targeting vectors. 
Disruption results in an ~ 20-fold reduction in Oct- 
2 protein levels. The low levels of residual expres- 
sion may be due to the fact that the promoterless 
targeting vectors required disruption of the first 
coding exon. Transcripts initiated upstream of the 
second exon, which also contains an in-frame AUG, 
might account for residual expression. Neverthe- 
less, given the dramatic reduction of Oct-2 protein 
levels, the phenotype of the mutant B cells is unex- 
pected. No significant alteration in the expression 
of /i, K, and B29 genes is detected in the mutant 
cells. This result represents the first genetic demon- 
stration that Oct-2 appears not to be necessary for 
the maintenance of Ig gene expression. These re- 
sults suggest the existence of an alternate pathway, 
involving the ubiquitous related protein Oct- 1 , in Ig 
gene regulation. 
To determine if the double disruptant cells are 
defective in activating different octamer-dependent 
transcription units. Dr. Singh and his colleagues 
have pursued transient transfection analysis. Simple 
octamer-containing promoters are unaffected by the 
double disruption, whereas a promoter containing a 
multiple array of octamer elements is severely com- 
promised in the mutant cells. These results provide 
strong genetic evidence for two distinct, cell-type- 
specific pathways of octamer-dependent gene acti- 
vation in B lymphocytes. 
Regulation of Oct-2 Transcription 
In pre-B cell lines, expression of the Oct-2 gene 
is transcriptionally regulated during lipopolysaccha- 
ride-induced differentiation. Dr. Singh's laboratory 
is analyzing the regulation of Oct-2 gene transcrip- 
tion in B-lineage cells, with the aim of identifying 
and analyzing other regulatory genes that constitute 
