Independent and complementary methods for large-scale structural analysis of mammalian chromatin.
The fundamental building block of chromatin, the nucleosome, occupies 150 bp of DNA in a spaced arrangement that is a primary determinant in regulation of the genome. The nucleosomal organization of some regions of the human genome has been described, but mapping of these regions has been limited to a few kilobases. We have explored two independent and complementary methods for the high-throughput analysis of mammalian chromatin structure. Through adaptations to a protocol used to map yeast chromatin structure, we determined sites of nucleosomal protection over large regions of the mammalian genome using a tiling microarray. By modifying classical primer extension methods, we localized specific internucleosomally cleaved mammalian genomic sequences using a capillary electrophoresis sequencer in a manner that allows high-throughput nucleotide-resolution characterization of nucleosome protection patterns. We developed algorithms for the automated and unbiased analysis of the resulting data, a necessary step toward large-scale analysis. We validated these assays using the known positions of nucleosomes on the mouse mammary tumor virus LTR, and additionally, we characterized the previously unreported chromatin structure of the LCMT2 gene. These results demonstrate the effectiveness of the combined methods for reliable analysis of mammalian chromatin structure in a high-throughput manner.
Dennis
Jonathan H
JH
Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.
Fan
Hua-Ying
HY
Reynolds
Sheila M
SM
Yuan
Guocheng
G
Meldrim
James C
JC
Richter
Daniel J
DJ
Peterson
Daniel G
DG
Rando
Oliver J
OJ
Noble
William S
WS
Kingston
Robert E
RE
D000465:Algorithms; D002874:Chromosome Mapping; D006801:Humans; D009707:Nucleosomes / Q000737:chemistry / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D017422:Sequence Analysis, DNA
D009707:Nucleosomes