Influence of deficiency of the histone gene-containing 38B-40 region on X-chromosome template activity and the White gene position effect variegation in Drosophila melanogaster

Khesin, R. B.; Leibovitch, Boris A.

doi:10.1007/bf00268858

Cited by 27 publications

(15 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The homogenate was placed at 4°C overnight and then warmed at 50°C for 5 min. After centrifugation in a microcentrifuge for 10 min, the supernatant was recovered, and the optical density at 480 nm was recorded (37). Female y w 67c23 flies were used to establish background values.…”

Section: Methodsmentioning

confidence: 99%

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

Sun

Cuaycong

Elgin

2001

Molecular and Cellular Biology

123

View full text Add to dashboard Cite

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).

show abstract

Section: Methodsmentioning

confidence: 99%

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

Sun

Cuaycong

Elgin

2001

Molecular and Cellular Biology

123

View full text Add to dashboard Cite

show abstract

“…The level of eye pigmentation of the hemizygous female V line flies with the transgene in a y w background was used as the standard control; siblings containing the balancer chromosome were not used for comparison, because the balancers frequently have accumulated mutations, including some that modify PEV. To measure eye pigmentation, five hemizygous adult female flies (4-5 days after emergence) were homogenized in 0.5 ml of 0.01 M HCl in ethanol (30). The homogenate was placed at 4°C overnight and then warmed at 50°C for 5 min.…”

Section: Methodsmentioning

confidence: 99%

The fourth chromosome of Drosophila melanogaster : Interspersed euchromatic and heterochromatic domains

Sun

Cuaycong

Craig

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

124

106

View full text Add to dashboard Cite

The small fourth chromosome of Drosophila melanogaster (3.5% of the genome) presents a puzzle. Cytological analysis suggests that the bulk of the fourth, including the portion that appears banded in the polytene chromosomes, is heterochromatic; the banded region includes blocks of middle repetitious DNA associated with heterochromatin protein 1 (HP1). However, genetic screens indicate 50 -75 genes in this region, a density similar to that in other euchromatic portions of the genome. Using a P element containing an hsp70-white gene and a copy of hsp26 (marked with a fragment of plant DNA designated pt), we have identified domains that allow for full expression of the white marker (R domains), and others that induce a variegating phenotype (V domains). In the former case, the hsp26-pt gene shows an accessibility and heatshock-inducible activity similar to that seen in euchromatin, whereas in the latter case, accessibility and inducible expression are reduced to levels typical of heterochromatin. Mapping by in situ hybridization and by hybridization of flanking DNA sequences to a collection of cosmid and bacterial artificial chromosome clones shows that the R domains (euchromatin-like) and V domains (heterochromatin-like) are interspersed. Examination of the effect of genetic modifiers on the variegating transgenes shows some differences among these domains. The results suggest that heterochromatic and euchromatic domains are interspersed and closely associated within this 1.2-megabase region of the genome.

show abstract

“…Males from these crosses are then collected, aged to three to five days post-eclosion, and photographed. Pigment assays were performed by extracting pigment and measuring OD at 480 nm (30). Fifteen male fly heads were used in one preparation for pigment quantification.…”

Section: Drosophila Melanogaster Stocks and Crosses For Nap-1 Pev Assaysmentioning

confidence: 99%

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

et al. 2006

View full text Add to dashboard Cite

Heterochromatin Protein 2 is a nonhistone chromosomal protein from Drosophila melanogaster that binds to HP1 and has been implicated in heterochromatin-induced gene silencing. Heretofore, HP1 has been the only known binding partner of HP2, a large protein devoid of sequence motifs other than a pair of AT-hooks. In an effort to identify proteins that interact with HP2 and assign functions to its various domains, nuclear proteins were fractionated under non-denaturing conditions. On separation of nuclear proteins, Nap-1, or Nucleosome assembly protein 1, has an overlapping elution profile with HP2 (assayed by Western blot) and has been identified by mass spectrometry in fractions with HP2. Upon probing fractions in which HP2 and Nap-1 are both present, we find that NURF, an ISWI-dependent chromatin remodeling complex, is also present. Results from coimmunoprecipitation experiments suggest that HP2 interacts with Nap-1 as well as with NURF; NURF appears to interact directly with both HP2 and Nap-1. Three distinct domains within HP2 mediate the interaction with NURF, allowing us to assign NURF binding domains in addition to the AT-hooks and HP1 binding domains already mapped in HP2. Mutations in Nap-1 are shown to suppress position effect variegation, suggesting that Nap-1 functions to help assemble chromatin into a closed form, as does HP2. Based on these interactions, we speculate that HP2 may cooperate with these factors in the remodeling of chromatin for silencing.Heterochromatin Protein 2 (HP2) was originally identified based on its ability to bind to Heterochromatin Protein 1 (HP1 1 ), one of the best-characterized nonhistone chromosomal proteins, in a yeast two-hybrid assay (1). HP2 colocalizes with HP1 at the pericentric heterochromatin of Drosophila polytene chromosomes, coimmunoprecipitates with HP1 from a Drosophila embryo extract, and is recruited to ectopic sites upon mislocalization of HP1. Analysis of the structure of the gene coding for HP2, Su(var)2-HP2, reveals two isoforms, a consequence of alternative splicing. Both proteins are large; the larger isoform of HP2 (HP2-L) is 356 kDa and the smaller isoform of HP2 (HP2-S) is 175 kDa. Both proteins are devoid of recognizable sequence motifs, except for two AT-hooks that are present only in the larger isoform. [AT-hooks are small, 9-aa DNA binding motifs which preferentially interact with ATCorrespondence to be sent to:

show abstract

Influence of deficiency of the histone gene-containing 38B-40 region on X-chromosome template activity and the White gene position effect variegation in Drosophila melanogaster

Cited by 27 publications

References 8 publications

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

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

The fourth chromosome of Drosophila melanogaster : Interspersed euchromatic and heterochromatic domains

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

Contact Info

Product

Resources

About