Long-Range Nucleosome Ordering Is Associated with Gene Silencing in Drosophila melanogaster Pericentric Heterochromatin

Abstract

We have used line HS-2 of Drosophila melanogaster, carrying a silenced transgene in the pericentric heterochromatin, to investigate in detail the chromatin structure imposed by this environment. Digestion of the chromatin with micrococcal nuclease (MNase) shows a nucleosome array with extensive long-range order, indicating regular spacing, and with well-defined MNase cleavage fragments, indicating a smaller MNase target in the linker region. The repeating unit is ca. 10 bp larger than that observed for bulk Drosophila chromatin. The silenced transgene shows both a loss of DNase I-hypersensitive sites and decreased sensitivity to DNase I digestion within an array of nucleosomes lacking such sites; within such an array, sensitivity to digestion by MNase is unchanged. The ordered nucleosome array extends across the regulatory region of the transgene, a shift that could explain the loss of transgene expression in heterochromatin. Highly regular nucleosome arrays are observed over several endogenous heterochromatic sequences, indicating that this is a general feature of heterochromatin. However, genes normally active within heterochromatin (rolled and light) do not show this pattern, suggesting that the altered chromatin structure observed is associated with regions that are silent, rather than being a property of the domain as a whole. The results indicate that long-range nucleosomal ordering is linked with the heterochromatic packaging that imposes gene silencing.In eukaryotes, the very large genome size demands extensive packaging of the DNA, starting with association with histones in a nucleosome array and extending to the highly condensed chromosomes observed at mitosis. Cytological observations indicate that the genome can be subdivided into heterochromatin and euchromatin, heterochromatin being defined as that portion of the genome remaining heteropycnotic (deeply stained) as the chromosomes decondense during the passage from metaphase to interphase (31). In many species, large blocks of heterochromatin flank the centromeres and telomeres. Little gene expression is associated with this constitutive heterochromatin, which is primarily (but not exclusively) made up of repetitive sequences. Facultative heterochromatin is observed when one of the two homologues is silenced, for example, one of the X chromosomes in the somatic cells of female mammals. The inactive X chromosome is heteropycnotic, suggesting that the chromatin in such silent regions is relatively condensed. The commonly accepted model of heterochromatin is of a folded higher-order structure that might preclude access for RNA polymerase and/or other components of the transcriptional machinery. However, analyses using three-dimensional microscopy indicate that the inactive and active X chromosomes occupy the same amount of space in the mammalian nucleus, a contradiction of the simplest model (21).

Unusual DNA Structures Associated With Germline Genetic Activity in Caenorhabditis elegans

“…The most attractive hypothesis to us is that these sequences impede nucleosome rearrangement in genes for which germline expression is important. Formation of inactive heterochromatin has been shown at least in a subset of cases to be associated with the assembly of nucleosomes into arrays with a specific and uniform spacing (e.g., Sun et al 2001). Constraints on nucleosome mobility (or alternatively, strong positioning signals that failed to match the required heterochromatic spacing) would be expected to increase the energy and time needed to assemble these uniformly spaced arrays.…”

Heterochromatin protein 2 (HP2), a partner of HP1 in Drosophila heterochromatin

“…The gene is silenced in some of the cells in which it normally is expressed, resulting in a variegating phenotype (1). A silenced reporter gene shows an altered chromatin structure, including loss of accessibility in promoter regions and adoption of a repetitive nucleosome array (2,3). In addition to its role in chromatin condensation and epigenetic regulation, heterochromatin formation plays an essential role in sister chromatid cohesion at centromeres (4,5).…”

“…One of the best-characterized structural proteins, heterochromatin protein 1 (HP1), was discovered in a screen by using mAbs to analyze the distribution of proteins on polytene chromosomes (8). Loss-of-function mutations in the gene encoding HP1, Su(var) [2][3][4][5], result in loss of silencing at a reporter gene, whereas extra copies of the gene result in increased silencing (9,10). HP1 is located principally in the pericentric heterochromatin and in a banded pattern along the small fourth chromosome, with lesser immunofluorescent staining of some euchromatic sites and the telomeres (11).…”

Heterochromatin Protein 2 Interacts with Nap-1 and NURF: A Link between Heterochromatin-Induced Gene Silencing and the Chromatin Remodeling Machinery in Drosophila

“…We have used biochemical approaches to identify protein-binding partners of HP2 that may contribute to the regular array of nucleosomes that are commonly found in heterochromatin (6,7). In addition to possible interactions with enzymes that generate appropriate histone modifications [such as SU(VAR)3-9 or another critical HMT], one might anticipate identifying proteins that can bind to nucleosomes and remodel them into a regular array.…”