The primary aim of this proposal is to examine the processes whereby the sequential binding of developmentally regulated transcription factors organizes the cis-acting sequences which make up the enhancer of the serum albumin gene into a series of three translationally phased nucleosome-like particles encompassing the essential enhancer factor binding sites in liver chromatin. This will be accomplished using three different approaches. First, the assembly of the albumin enhancer into an active higher-order nucleoprotein complex during liver development will be followed by comparison of nucleosome phasing at the albumin enhancer in liver chromatin at successive developmental stages. These observations will be combined with recently completed in vivo DMS protection analyses which have identified the transcription factors responsible for activating the albumin enhancer. Second, the ability of purified transcription factors alone and in combination to bind to and perturb nucleosomes reconstituted in vitro from albumin enhancer fragments and core histones will be determined, in order to determine the mechanisms whereby transcription factors specify the nucleosome structure of the albumin enhancer. Finally, the role played by specific transcription factors in the chromatin remodeling of the albumin enhancer will be assessed by studying the activity and chromatin structure of albumin enhancer transgenes containing mutations in selected enhancer binding sites during liver development in transgenic mice. These studies should further our understanding of the role played by chromatin architecture in transcriptional regulation as well as lend insight into the mechanisms whereby regulatory factors dictate normal liver growth and development.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32DK009529-03
Application #
2733937
Study Section
Special Emphasis Panel (ZRG2-MBY (01))
Program Officer
Podskalny, Judith M,
Project Start
1998-07-01
Project End
Budget Start
1998-07-01
Budget End
1999-06-30
Support Year
3
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Brown University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
001785542
City
Providence
State
RI
Country
United States
Zip Code
02912
Cirillo, Lisa Ann; Zaret, Kenneth S (2004) Preparation of defined mononucleosomes, dinucleosomes, and nucleosome arrays in vitro and analysis of transcription factor binding. Methods Enzymol 375:131-58
Cirillo, Lisa Ann; Lin, Frank Robert; Cuesta, Isabel et al. (2002) Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4. Mol Cell 9:279-89