REGULATION OF GENE ACTIVITY IN B CELLS 
Harinder Singh, Ph.D., Assistant Investigator 
Differential gene activity is a fundamental feature 
of cellular differentiation. Research in the labora- 
tory of Dr. Singh focuses on transcriptional regula- 
tory circuits that underlie the establishment and 
maintenance of cell-specific patterns of gene ex- 
pression during mammalian development. The lab- 
oratory uses transformed B lymphocytes represent- 
ing various stages of B cell differentiation as models 
to explore the molecular basis of differential gene 
activity in a defined lineage. 
The heavy- and light-chain genes encoding the 
immunoglobulin (Ig) molecule are selectively tran- 
scribed in B cells. These genes are assembled from 
gene segments through an ordered series of so- 
matic recombination events that occur in a devel- 
oping B cell. Three B cell-specific, cis-acting tran- 
scriptional regulatory elements have been 
identified. Two of these elements are located in the 
introns between the variable and constant regions 
of the heavy- and K-light-chain genes and act as en- 
hancers. The third element is found upstream of 
both heavy- and K-light-chain gene promoters. 
The elaboration of a sensitive DNA-binding assay 
has led to the identification and characterization of 
two B cell-specific regulatory proteins. One of 
these proteins, Oct-2 (NF-A2), recognizes the oc- 
tanucleotide sequence ATTTGCAT, which confers 
B cell specificity to Ig gene promoters. The same se- 
quence motif is also a functional component of the 
heavy-chain gene enhancer. The second B cell-spe- 
cific regulatory protein, NF-kB, recognizes the se- 
quence GGGGACTTTCC, which appears to dictate 
the B cell specificity of the K-gene enhancer. In con- 
trast with the lineage-restricted Oct-2 protein, 
NF-kB exists in an inactive but inducible form in a 
variety of non-B cell types. 
A novel expression screening strategy that uses 
recognition site probes was previously developed 
by Dr. Singh and his colleagues to enable the rapid 
isolation of genes encoding transcriptional regula- 
PUBLICATIONS 
tors. This strategy was used to isolate cDNA clones 
for an NF-KB-like protein and the Oct-2 protein. 
The deduced amino acid sequences of these pro- 
teins reveal that the former belongs to the family of 
zinc finger proteins, whereas the latter contains a 
domain related to the homeobox. 
Two different human Oct-2 cDNA clones have 
been isolated in the laboratories of Drs. David Balti- 
more and Phillip Sharp. The clones encode nearly 
identical proteins that differ at their carboxyl ter- 
mini by 12 amino acids. In addition to the 
homeodomain, the predicted proteins contain a re- 
gion, termed the POU-specific box, that defines a 
new class of regulatory proteins, as well as a puta- 
tive leucine zipper domain. At least five different 
Oct-2 transcripts, ranging in size from 1.5 to 7 kb, 
are expressed specifically in various B cell lines. 
These transcripts appear to represent alternatively 
spliced mRNAs. 
Because the regulatory functions of the various 
Oct-2 gene products, as well as the regulation of 
Oct-2 expression, can be explored more thoroughly 
in the murine system, this laboratory has initiated 
the isolation of murine cDNA and genomic clones. 
Two types of murine cDNA clones have been iso- 
lated that encode proteins similar to their human 
counterparts. Surprisingly, however, the two types 
of clones appear to represent transcripts of diffier- 
ent genes, since they differ in nucleotide sequence 
within regions encoding identical protein seg- 
ments. This interpretation is consistent with the 
isolation of two types of genomic clones encoding 
the Oct-2 POU domain, using the polymerase chain 
reaction. Genomic analysis and cloning is being 
pursued rigorously to resolve this structural differ- 
ence between the murine and human Oct-2 loci. 
Dr. Singh is also Assistant Professor of Molecular 
Genetics and Cell Biology at The University of 
Chicago. 
Article 
Singh, H., Clerc, R.G., and LeBowitz, J. 1989. Molecular cloning of sequence-specific DNA binding proteins 
using recognition site probes. BioTechniques 7:252-261. 
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