Developmental Control of Gene Expression 
Rudolf Grosschedl, Ph.D. — Assistant Investigator 
Dr. Grosschedl is also Associate Professor of Microbiology and Immunology and of Biochemistry and 
Biophysics at the University of California, San Francisco. He completed his undergraduate studies on the 
replication of lambdoid bacteriophages in the laboratory of Gerd Hobom in Freiburg, West Germany. His 
graduate studies, on the regulation of histone gene expression, were carried out in the laboratory of Max 
Birnstiel in Zurich, Switzerland. Dr. Grosschedl spent his postdoctoral years with David Baltimore at the 
Massachusetts Institute of Technology and the Whitehead Institute. 
THE process of terminal differentiation turns a 
multipotential cell into a cell that carries out 
a particular function or synthesizes a specific 
product. The lymphoid B cell lineage ultimately 
generates a cell that secretes antibody. During 
cell differentiation, genes that encode the anti- 
body or associated proteins are expressed in a de- 
fined cell type-specific and temporally ordered 
pattern. 
Transcription of the m immunoglobulin (Ig) 
gene encoding the heavy chain of the antibody 
can be detected in virtually all lymphocytes. By 
contrast, the k immunoglobulin light-chain gene 
is transcribed only in late-stage B cells, and the 
mb l gene encoding an antibody-associated pro- 
tein is expressed only in early-stage B cells. The 
goal of our research is to gain some insight into 
the molecular mechanisms that mediate the de- 
velopmental control of lymphoid-specific gene 
expression. 
Tissue-Specific Regulation of Ig Gene 
Expression in a Transgenic Model 
We are attempting to understand how multiple 
cis-acting regulatory information of a gene is in- 
tegrated to govern the correct developmental 
pattern of expression. Toward this goal, we trans- 
ferred rearranged wild-type and mutated Ig 
genes into the mouse germline and analyzed ex- 
pression in various tissues and developmental 
stages. Our first set of experiments suggested that 
lymphoid-specific expression is dependent upon 
multiple tiers of regulation. 
We found that the intragenic enhancer directs 
high-level expression in lymphoid tissues and ba- 
sal expression in most but not all nonlymphoid 
tissues. Basal expression appears to be governed 
by negative regulation, since mutations of the 
juE5- and ME2-binding sites in the enhancer in- 
creased At gene expression in many nonlymphoid 
tissues to levels similar to those found in lym- 
phoid tissues. However, the "off-state" of ex- 
pression observed in a few other nonlymphoid 
tissues was not affected by these mutations, sug- 
gesting that another tier of negative regulation 
may be involved in further decreasing basal 
expression. 
An additional set of experiments suggested that 
the Oct-binding site in the promoter contributes 
to the positive regulation of ^l gene expression 
during B cell differentiation. A mutation in the 
Oct-binding site decreased ti gene expression in 
fetal pre-B cells (which represent early B cells) 
by a factor of 5, but decreased ^L gene expression 
in stimulated splenocytes (representing late- 
stage B cells) by a factor of 100 to 200. Together 
these experiments indicate that the multiple 
modes of regulation are necessary to maximize 
the difference between the on-state and off-state 
of gene expression in vivo. 
In a second set of experiments, we addressed 
the regulation of the accessibility of binding sites 
for nuclear factors in native chromatin. With the 
aim of uncoupling changes in chromatin struc- 
ture from the process of transcription, we linked 
DNA fragments comprising the m enhancer region 
to the promoter for bacteriophage T7 RNA poly- 
merase and incorporated the gene construct into 
the mouse germline. Subsequently, we examined 
the accessibility of the T7 promoter in isolated 
pre-B cell nuclei by adding exogenous T7 RNA 
polymerase and measuring the synthesis of 
TT-specific RNA. 
The T7 promoter was accessible in 7/7 lines 
when linked to the ti enhancer. By contrast, the 
T7 promoter alone was only weakly accessible in 
two lines and not accessible in six. Because the 
enhancer fragment used in this experiment 
lacked any known promoter activity, we are 
currently using this assay to examine which se- 
quences confer accessibility in native chromatin. 
Novel Lymphoid-Specific Regulators 
of Gene Expression 
To identify and clone novel lineage-specific 
transcriptional regulators, we adopted two ap- 
proaches. First, we included in our analysis of 
lymphoid-specific gene expression the mb-1 
promoter, which has a cell type-specific pattern 
of activity distinct from that of the Ig promoters. 
We found that the mb- 1 promoter is functional in 
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