Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter

Abstract

Differential chromatin structure and accessibility, particularly at the promoter, have long been recognized as characteristics that distinguish active from inactive genes. Active genes are in general more accessible to regulatory factors than inactive genes, as indicated by nuclease sensitivity. In addition, the promoters of active genes often exhibit marked DNase I hypersensitivity, especially in the vicinity of transcription factor binding sites (10,20). This hypersensitivity is postulated to be due to changes in the chromatin architecture of the promoter and may represent nucleosomal remodeling or displacement, stretches of single-stranded DNA, torsionally stressed DNA, or other distortions in chromatin structure arising from factor binding (18,20,62).The functional effect of nucleosomes on transcription initiation is thought to be repressive since in vitro assembly of nucleosomal arrays on DNA templates drastically reduces the capacity of these templates to support basal transcription (25,35,36,57). Furthermore, the differential accessibility and transcriptional potential of chromatin structure in active versus inactive promoters are often associated with differential nucleosomal organization (3,5,23,30,46,62). Thus, remodeling the nucleosomal architecture of a promoter is likely to be an integral feature of mechanisms of gene activation and/or silencing, which may involve histone acetylation and chromatinremodeling complexes such as SWI/SNF (15,20,60).The organization of genomic DNA into nucleosomal arrays is defined by both the translational position of the nucleosome relative to the linear nucleotide sequence and the rotational orientation of the DNA helix relative to the surface of the histone octamer. The translational position of nucleosomes on a DNA template (i.e., the linear position of the nucleosome relative to the DNA sequence [46]) has been shown to affect the accessibility of cis-acting elements in the promoter to various transcription factors as well as the basal transcription complex. Whether a transcription factor binding site is incorporated into a nucleosomal core or is in the linker region between nucleosomes can dramatically affect its accessibility to its cognate binding protein(s) in vitro (25,58). However, the extent to which incorporating a transcription factor binding site into a nucleosomal core reduces the accessibility of that site can vary widely among transcription factors (4, 25). The rotational orientation of the DNA helix wound around a nucleosomal core also affects the accessibility of that DNA to transcription factors (25). For instance, the rotational orientation of the TATA box, the glucocorticoid response element, and the thyroid response element within a nucleosome strongly affects the accessibility of these elements to their cognate binding factors (14,24,59). These findings suggest that both the translational position and rotational orientation of cis-acting

Combinatorial, site-specific requirement for heterochromatic silencing factors in the elimination of nucleosome-free regions

“…Such a model offers a simple physical explanation for transcriptional repression that does not require invoking a closed higherorder structure. Additionally, our results are also consistent with anecdotal reports of promoter-localized changes in silenced regions in S. cerevisiae (Vega-Palas et al 1998), Drosophila (Sun et al 2001), and humans (Chen and Yang 2001). Given that nucleosomes flanking NFRs are more periodic than those elsewhere in the genome (Yuan et al 2005), NFRs may act to promote periodicity of nucleosomes in euchromatin, and thus the lower periodicity observed in S. pombe heterochromatin could be a consequence of NFR elimination.…”

Role of the Promoter in Maintaining Transcriptionally Active Chromatin Structure and DNA Methylation Patterns In Vivo

“…Two independent cell lines were constructed in which 323 bp of the active endogenous HPRT promoter region in cultured male fibrosarcoma cells was replaced with plasmid sequences. The promoter mutation deleted sequences that colocalize with the major DNase I-hypersensitive site in the 5Ј region of the HPRT gene (7,30). In mutated cells, the major hypersensitive site was abolished; however, a new weak hypersensitive site was detected just downstream of the targeted promoter mutation.…”

“…For Southern blots, genomic DNA (10 g) was digested with the appropriate restriction enzyme and was size fractionated on a 0.8% agarose gel in 1ϫ Tris-acetate-EDTA (40 mM Tris-acetate and 2 mM EDTA). Capillary transfer to nylon membranes (Hybond Nϩ; Amersham), hybridization, and washing were performed as described previously (7).…”

Constitutive DNase I Hypersensitivity of p53-Regulated Promoters

“…For example, the hypoxanthine phosphoribosyltransferase gene is X-linked. On the active X chromosome, where the gene is transcribed, the promoter is nucleosome-free (6) and is hypersensitive to DNase I (6,53). In contrast, the hypoxanthine phosphoribosyltransferase gene on the inactive X chromosome is not transcribed, and its promoter is occupied by nucleosomes, and it is not DNase I hypersensitive.…”

“…Thus, inactive genes usually have promoters that are nucleosomal, are insensitive to DNase I, and are regarded as being in a closed confirmation whereas active genes usually have core promoters that are nucleosome-free, hypersensitive to DNase I, and in an open configuration. Consequently, one of the hallmarks of gene induction mechanisms is the remodeling of the nucleosomal architecture of a promoter as it is activated (5,6,8).…”