Regulation of Gene Activity During 
B Cell Development 
Harinder Singh, Ph.D. — Assistant Investigator 
Dr. Singh is also Assistant Professor in the Department of Molecular Genetics and Cell Biology at the 
University of Chicago. He received his Ph.D. degree in biochemistry, molecular biology, and cell biology 
with Lawrence Dumas at Northwestern University. His postdoctoral research was done with Phillip Sharp 
at the Massachusetts Institute of Technology, as a Jane Coffin Childs fellow. He remained at MIT as a 
research associate until his move to Chicago. 
MY research interests are focused on the 
analysis of transcriptional regulatory cir- 
cuits that turn genes on or off during the growth 
and differentiation of B lymphocytes, cells that 
produce antibodies. The B cell lineage is a very 
useful model for exploring the molecular basis of 
differential gene activity in mammalian develop- 
ment. We are seeking answers to the following 
questions. What is the nature of the genetic ele- 
ment (s) linked to a target gene that controls its 
transcriptional activity? What is the nature of the 
regulatory protein (s) that recognizes this genetic 
element? How upon binding near its target gene 
does the regulatory protein modulate the activity 
of the enzyme complex that transcribes the gene? 
What is the mechanism by which a growth or de- 
velopmental signal is transduced by the regula- 
tory protein (s) to effect gene activity? How does 
the structure of the chromatin within which the 
target gene is packaged influence the function of 
the regulatory protein (s)? 
The heavy- and light-chain genes encoding the 
immunoglobulin (Ig) molecule are selectively 
transcribed in B cells. These genes are assembled 
from gene segments through an ordered series of 
somatic recombination events that occur in a de- 
veloping B cell. The heavy-chain gene locus is the 
first to undergo recombination and transcrip- 
tional activation, thereby defining the pre-B cell 
developmental state. Subsequently one of two 
light-chain gene loci is recombined and ex- 
pressed, resulting in the development of a mature 
B cell. Ig genes contain multiple cis-acting tran- 
scriptional regulatory elements that restrict their 
expression to appropriate stages in the B lineage. 
Previous work by various research groups has 
resulted in the identification, characterization, 
and cloning of a B cell-specific regulatory pro- 
tein, Oct-2. This protein recognizes the octanu- 
cleotide sequence ATTTGCAT, which confers B 
cell specificity to Ig gene promoters. The same 
sequence motif is also a functional component of 
the heavy-chain and K-light-chain gene en- 
hancers. Oct-2 can activate transcription of a re- 
porter gene linked to an Ig promoter in a non-B 
cell. Thus Oct-2 appears to be both necessary and 
sufficient for regulating the activity of Ig pro- 
moters. The deduced amino acid sequence of 
Oct-2 reveals a region of similarity that is shared 
with three other regulatory proteins: Pit- 1 , Oct- 1 , 
and unc-86. This region is termed the POU box 
and includes a subdomain related to the 
homeobox. 
The levels of the Oct-2 protein are regulated 
during B cell differentiation. The Oct-2 protein is 
expressed at low levels in pre-B cells. Oct-2 lev- 
els are elevated 5- to 10-fold upon pre-B cell 
differentiation, and higher Oct-2 protein levels 
correlate with activation of the Ig /c-light-chain 
gene locus. Increased expression of Oct-2 in 
pre-B cells is induced by signaling with the B cell 
mitogen, bacterial lipopolysaccharide, as well as 
the lymphokine interleukin-1 (IL-1). Transform- 
ing growth factor-/8, an inhibitor of k gene induc- 
tion in pre-B cells, blocks the up-regulation of 
Oct-2 but not the activation of NF-kB. The latter is 
another regulatory protein that has been previ- 
ously implicated in controlling the activity of the 
K locus. We have proposed a model in which the 
concerted action of increased levels of Oct-2 and 
activated NF-kB controls the proper stage-specific 
expression of the k locus. To test genetically the 
function of Oct-2 in regulating immunoglobulin 
gene transcription, we have used gene targeting 
to disrupt sequentially the two copies of the 
Oct-2 gene in a B cell. The analysis of the mutant 
and wild-type cells is currently under way. 
Expression of the Oct-2 gene has been shown 
to be regulated at the level of transcription dur- 
ing pre-B cell differentiation. By studying the 
control of Oct-2 expression, regulatory proteins 
interacting with Oct-2 in the genetic hierarchy 
underlying B cell difi'erentiation can be identi- 
fied and isolated. A promoter controlling tran- 
scription of the Oct-2 gene has been identified. 
This region is being analyzed and will enable the 
characterization of transcription factors that 
regulate Oct-2 expression as well as B cell 
development. 
B cells need to activate the expression of two 
other genes, mb- 1 and B29, to function as anti- 
gen-recognizing cells. These genes encode mem- 
brane proteins that associate with the antibody 
379 
