The goal of this proposal is to determine how """"""""pioneer"""""""" transcription factors, the first to bind genes in development, engage target sites in silent, compacted chromatin and promote entry of subsequent proteins. These initial steps are among the most decisive in eukaryotic gene control, yet little is known about the underlying mechanisms. Previously we showed that chromatin sites for the transcription factors HNF3 and GATA-4 are engaged in undifferentiated endoderm, prior to the activation of the albumin gene during liver specification. In the past grant period, we discovered that HNF3 and GATA-4, but not various other DNA binding factors, can bind their target sites within highly compacted chromatin in vitro and make the local nucleosomal domain accessible to other proteins. These activities do not require ATP-dependent nucleosome remodeling complexes. We further discovered a protein domain on HNF3alpha that interacts with a subset of core histones and is critical for loosening inter-nucleosomal interactions, thereby """"""""decompacting"""""""" chromatin. In the next grant period, we will combine biochemical and transgenic approaches to understand how HNF3 and GATA factors initiate regulatory events at target sites in chromatin, with the following specific aims:1. To determine how chromatin binding by HNF3alpha and GATA factors disrupts inter-nucleosomal interactions and creates a locally open domain in defined chromatin templates in vitro.2. To determine how wild type and mutant HNF3alpha and GATA-4, when inducibly expressed in transgenic mice, open chromatin in a diverse array of silent chromatin contexts.3. To determine how HNF3 and GATA-4 binding leads to linker histone displacement, changes in nucleosome linker DNA, arid the recruitment of accessory proteins that help activate transcription.Understanding how chromatin structure is modified during the earliest stages of gene regulation will provide fundamental insight into genetic processes that are essential for growth, development, and pathogenesis in humans.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM047903-13
Application #
6936520
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Carter, Anthony D
Project Start
1994-09-01
Project End
2007-08-31
Budget Start
2005-09-01
Budget End
2007-08-31
Support Year
13
Fiscal Year
2005
Total Cost
$242,250
Indirect Cost
Name
Institute for Cancer Research
Department
Type
DUNS #
064367329
City
Philadelphia
State
PA
Country
United States
Zip Code
19111
Cuesta, Isabel; Zaret, Kenneth S; Santisteban, Pilar (2007) The forkhead factor FoxE1 binds to the thyroperoxidase promoter during thyroid cell differentiation and modifies compacted chromatin structure. Mol Cell Biol 27:7302-14
Sekiya, Takashi; Zaret, Kenneth S (2007) Repression by Groucho/TLE/Grg proteins: genomic site recruitment generates compacted chromatin in vitro and impairs activator binding in vivo. Mol Cell 28:291-303
Cirillo, Lisa A; Zaret, Kenneth S (2007) Specific interactions of the wing domains of FOXA1 transcription factor with DNA. J Mol Biol 366:720-4
Xu, Jian; Pope, Scott D; Jazirehi, Ali R et al. (2007) Pioneer factor interactions and unmethylated CpG dinucleotides mark silent tissue-specific enhancers in embryonic stem cells. Proc Natl Acad Sci U S A 104:12377-82
Holmqvist, Per-Henrik; Belikov, Sergey; Zaret, Kenneth S et al. (2005) FoxA1 binding to the MMTV LTR modulates chromatin structure and transcription. Exp Cell Res 304:593-603
Greger, James G; Katz, Richard A; Taganov, Konstantin et al. (2004) Transduction of terminally differentiated neurons by avian sarcoma virus. J Virol 78:4902-6
Chaya, Dina; Zaret, Kenneth S (2004) Sequential chromatin immunoprecipitation from animal tissues. Methods Enzymol 376:361-72
Taganov, Konstantin D; Cuesta, Isabel; Daniel, Rene et al. (2004) Integrase-specific enhancement and suppression of retroviral DNA integration by compacted chromatin structure in vitro. J Virol 78:5848-55
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

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