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ChIP-chip Experiments Improve Understanding of Transcriptional Control

NEW YORK (GenomeWeb News) – Scientists today reported using both genomic and gene-specific experiments to uncover a nucleosome-binding protein’s role in genome-wide transcriptional control.
 
Using chromatin immunoprecipitation and microarrays, along with more targeted functional experiments, researchers at Cornell University discovered that the nucleosome-binding protein PARP-1 not only localizes to an unexpected number of promoters, but seems to interact with a histone protein called H1 to regulate the expression of some of these genes. The findings were published online today in the journal Science.
 
“Our research won’t necessarily find cures for human diseases, but it provides molecular insight into the regulation of gene expression that will give us clues where to look next,” senior author Lee Kraus, a molecular biologist at Cornell University, said in a statement.
 
PARP-1 was originally studied for its role in the DNA damage response. It is also known to bind nucleosomes and influence chromatin structure in a manner similar to the histone H1. But no one was quite sure where these proteins were on chromatin — or what they did there.
 
To begin to answer these questions, Kraus and his team used chromatin immunoprecipitation with DNA from MCF-7 breast cancer cells using PARP-1 and H1 specific antibodies coupled with NimbleGen custom microarrays of just over 1,500 promoter, transcribed, or intergenic regions.
 
Surprisingly, they discovered that PARP-1 and histone H1 often localize to promoters, but not simultaneously. Their results suggest PARP-1 and H1 jockey for position on the promoters with H1 often turning off genes and PARP-1 keeping them on. In regions where the two factors function together, PARP-1 seems to block H1’s access to the chromatin under normal circumstances.
 
“We were surprised PARP-1 was localized to promoters,” Kraus told GenomeWeb Daily News. “Not only is there reciprocal binding [between PARP-1 and H1], there appears to be a functional interaction.”
 
Interestingly, neither PARP-1 nor H1 seems to associate with chromatin in a sequence-specific manner, leaving the researchers perplexed as to how they are targeted. “That’s the million-dollar question,” Kraus said. “We looked and we haven’t really found anything.”
 
Even so, Kraus says, only a subset of the PARP-1 that’s localized to promoters actually seems to have a functional role, suggesting widespread genomic associations are not always indicative of widespread genomic function. For the genes that were controlled by PARP-1, though, knocking down PARP-1 with short hairpin RNA allowed H1 back onto the promoters and turned the genes off.
 
PARP-1 also seems to vamoose when cells are exposed to certain stress signals such as nicotinamide adenine dinucleotide following damage to DNA. This, in turn, weakens PARP-1’s association with chromatin.
 
 “Under conditions of severe DNA damage, it’s likely that PARP-1 is going to leave the promoters,” Kraus said. That understanding may eventually provide new insights into the way gene expression changes in response to cellular stress events.
 
The study also may one day let scientists turn off a large subset of genes simultaneously by targeting PARP-1. “Think of PARP-1 as a key regulator of gene expression in response to normal signals and harmful stresses,” Kraus said in a statement. “If you could control most of the traffic lights in a city’s street grid with one hand, this is analogous to controlling gene expression across the genome with PARP-1. Under really adverse conditions, you can set all the lights to stop.”

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