Control of Gene Expression During the Cell Cycle 
and in the Developing Mammalian Cerebellum 
Nathaniel Heintz, Ph.D. — Associate Investigator 
Dr. Heintz is also Associate Professor at the Rockefeller University. He received his Ph.D. degree at the State 
University of New York at Albany, where he studied the genetics and biochemistry of bacteriophage SPOl 
gene expression. During postdoctoral studies with Robert Roeder at Washington University, St. Louis, he 
initiated his work on histone gene expression during the cell cycle. Continuation of these studies and ex- 
amination of the developing mammalian cerebellum are his current research interests. 
MOST interesting biological transitions, 
whether during the life of a single cell or 
the development of a complex tissue, are usually 
accompanied by underlying changes in the ex- 
pression of genes. Knowledge of molecular 
events that result in activation of these genes can 
lead to a detailed understanding of such transi- 
tions. We are using a molecular and biochemical 
approach to examine specific transitions that oc- 
cur in very different contexts: the mammalian 
cell division cycle and the developing mouse 
cerebellum. 
Control of Gene Expression During 
the Cell Cycle 
We have established over the past several years 
that coordinate induction of histone H4, H2b, 
and H 1 gene expression during the S phase of the 
cell cycle is achieved through the agency of dis- 
tinct transcription factors that interact with 
highly conserved subtype-specific consensus ele- 
ments within each of the promoters. That the 
transcription of these genes is accomplished by 
different proteins suggests that their coordinate 
induction is due to a pleiotropic regulatory mech- 
anism that directly participates in this activation 
step during the transition from Gl to S phase. Our 
efforts are focused on discovering the nature 
of this regulatory step and determining its 
specificity. 
During the past year progress in elucidating 
this regulatory step has been achieved through 
in-depth analysis of the regulatory protein OTFl 
(Octl, NFIII) for the H2b cell cycle. Thus high- 
titer polyclonal and monoclonal antibodies spe- 
cific for Oct 1 have been raised and employed to 
examine the chemical nature of this protein as 
the division cycle progresses. Our results demon- 
strate that multiple forms of Octl exist in mam- 
malian cells and that their distribution is dramati- 
cally regulated during the cell cycle. 
Further analysis has established that differen- 
tial phosphorylation of Octl during the cycle is 
at least partially responsible for the observed 
changes in this protein as cells proceed toward 
division. In particular, a peptide-mapping exper- 
iment using Octl pulse-labeled with P ortho- 
phosphate in vivo has resulted in identification 
of at least six phosphopeptides that are specifi- 
cally labeled in metaphase cells. Preliminary re- 
sults indicate that purified Octl is a substrate for 
CDC2 kinase in vitro, but that these phosphory- 
lations may be due to several protein kinases. 
Our present efforts are focused on discovery of 
the enzymes that post-translationally modify 
Octl during the cell cycle, and demonstration 
that these modifications are functionally relevant 
to histone gene transcription. Similar analyses of 
a second histone-specific cell cycle regulatory 
factor, H1TF2, have begun to determine whether 
its properties are similarly modulated during the 
cycle. Demonstration that the timing and nature 
of the post-translational modifications on Octl 
and H1TF2 are similar in vivo would establish 
the existence of the proposed pleiotropic regula- 
tory mechanism for regulation of transcription 
during S phase. 
One fundamental question that has arisen from 
this work is whether the initiation of S phase- 
specific transcription and chromosomal DNA syn- 
thesis are mechanistically coupled. To address 
this issue, we have focused on two specific ques- 
tions: Are the regulatory proteins for S phase his- 
tone gene transcription directly involved in chro- 
mosomal DNA synthesis? Might proteins that 
regulate initiation of DNA synthesis at specific 
chromosomal origins of replication be activated 
by the same mechanisms that modulate those 
transcription factors? 
In collaboration with Nicholas Heintz (Univer- 
sity of Vermont Medical School), we have re- 
cently identified a cellular protein complex 
(RIP60/RIP100) with several properties ex- 
pected of replication-initiating proteins. Thus 
the purified complex binds specifically to two 
well-characterized origins of DNA replication at 
sites thought to be important for origin function, 
and shows ATP-dependent DNA helicase activity. 
Our present efforts are directed toward a defini- 
tive demonstration that RIP60/RIP 1 00 can partic- 
ipate in DNA replication, and toward generation 
of appropriate tools to determine whether the ac- 
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