Signals in DNA that influence cromatin structure in vitro and in vivo
Abstract
Chromatin is the hallmark of the genome organization in most eukaryotes, ranging from protists to cetaceans. The unit structure of chromatin is a nucleosome, composed of 147bp of DNA wrapped around an octamer of four highly conserved core histones (H2A, H2B, H3 and H4), along with 10–60bp of ‘linker’ DNA associated with the highly variable linker histone H1. Arrays of these nucleosomes, with variation in linker lengths are predicted to fold into unique higher order structures resulting in distinct chromatin domains in the nucleus. We examined the role of genomic base sequence determinants in generating different nucleosomal arrangements. Specifically, a signal in the DNA, a 10bp periodic consensus triplet VWG (non-T, A/T, G), was found to occur with highly regular oscillations reminiscent of the eukaryotic dinucleosome length (320–420bp) in some regions but not others. Using an in vitro ATP-free chromatin assembly system, it was shown that the oscillations of the signal could influence nucleosomal arrangements on DNAs obtained from the mouse adenosine deaminase and odorant receptor loci. Altering the signal's oscillations in the DNA resulted in re-arrangement of nucleosomes in a predictable way. This signal appears to function by causing nucleosomes to avoid ‘valleys’ low in period-10 VWGs and preferentially associating with ‘peaks’ enriched in period-10 VWGs. We observed that strongly ordered nucleosome arrays generated via this mechanism could propagate onto flanking DNA in the presence of the linker histone variant H5. Altering the signal resulted in a loss of such spreading. In vivo, in mouse liver nuclei, the nucleosomal arrangements correlated very well with the signal at the transcriptionally silenced odorant receptor locus, but not at the constitutively active adenosine deaminase locus. In H1-knockout embryonic stem cells (H1 depleted by 50%), chromatin structure at the adenosine deaminase locus appeared to be aberrant, suggesting that H1 may play a role in chromatin remodeling at active loci. Based on our observations, we suggest that intrinsic determinants in genomic DNA may have evolved to generate spontaneous nucleosomal arrangements, the ‘ground state’ of chromatin structure, and that this ground state may play a role in setting the stage for future activity at certain loci.
Degree
Ph.D.
Advisors
Stein, Purdue University.
Subject Area
Molecular biology|Cellular biology
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