Heterochromatin Abstracts

Leung, W, CD Shaffer, T Cordonnier, J Wong, MS Itano, EE Slawson-Tempel, E Kellmann, DM Desruisseau, C Cain, R Carrasquillo, TM Chusak, K Falkowska, KD Grim, R Guan, J Honeybourne, S Khan, L Lo, R McGaha, J Plunkett, JM Richner, R Richt, L Sabin, A Shah, A Sharma, S Singhal, F Song, C Swope, CB Wilen, J Buhler, ER Mardis, SCR Elgin

Department of Biology, Washington University, St. Louis, MO 63130, USA.

Brower-Toland, B., Riddle, N.C., Jiang, H., Huisinga, K.L., and Elgin, S.C.R.

Department of Biology, Washington University, St. Louis, MO 63130, USA.

Biochem Cell Biol. 2009 Feb;87(1):229-41.

A lot about a little dot - lessons learned from Drosophila melanogaster chromosome 4.

Riddle NC, Shaffer CD, and Elgin SC.
Department of Biology, Washington University, St. Louis, MO 63130, USA.

The fourth chromosome of Drosophila melanogaster has a number of unique properties that make it a convenient model for the study of chromatin structure. Only 4.2 Mb overall, the 1.2 Mb distal arm of chromosome 4 seen in polytene chromosomes combines characteristics of heterochromatin and euchromatin. This domain has a repeat density of ~35%, comparable to some pericentric chromosome regions, while maintaining a gene density similar to that of the other euchromatic chromosome arms. Studies of position-effect variegation have revealed that heterochromatic and euchromatic domains are interspersed on chromosome 4, and both cytological and biochemical studies have demonstrated that chromosome 4 is associated with heterochromatic marks, such as heterochromatin protein 1 and histone 3 lysine 9 methylation. Chromosome 4 is also marked by POF (painting-of-fourth), a chromosome 4-specific chromosomal protein, and utilizes a dedicated histone methyltransferase, EGG. Studies of chromosome 4 have helped to shape our understanding of heterochromatin domains and their establishment and maintenance. In this review, we provide a synthesis of the work to date and an outlook to the future.

PubMed

Genetics Vol 178, 1177-1191, 2008.

Nicole C. Riddle, Wilson Leung, Karmella A. Haynes, Howard Granok, Jo Wuller, and Sarah C. R. Elgin

The banded portion of Drosophila melanogaster chromosome four exhibits euchromatic and heterochromatic characteristics. Reminiscent of heterochromatin, it contains a high percentage of repetitive elements, does not undergo recombination, and exhibits high levels of HP1 nd histone 2 lysine 9 dimethylation. However, in the distal 1.2 Mb, the gene density is typical of euchromatin, and the region is polytene in salivary gland nuclei. Using P element reporters carrying a copy of hsp70-white, alternative chromatin packaging domains can be distinguished by the eye color phenotype. Mapping studies identified the repetitive element 1360 as a candidate for heterochromatin targeting tin the fourth chromosome Hcf region. We report her two new screens using this reporter to look for additional heterochromatin target sites. We confirm that reporter elements within 10kb of 1360 are usually packaged as heterochromatin however, heterochromatin packaging occurs in the sv region in the absence of 1360. Analyses of the sequences adjacent to P element reporters show no simple association between specific repeated elements and transgene expression phenotype on a whole chromosome level. The data require that heterochromatin formation as a a whole depend on a more complex pattern of sequence organization rather than the presence of a single sequence element.

Genetics. 2007 Mar;175(3):1539-42. Epub 2006 Dec 28.

Haynes KA, Gracheva E, Elgin SC.
Department of Biology, Washington University, St. Louis, Missouri 63130, USA

Studies of transcriptional gene silencing in Drosophila melanogaster suggest that most of chromosome 4 resembles pericentric heterochromatin. However, some modifiers of position-effect variegation, including chromosome 4 dosage and loss of SU(VAR)3-9, have different effects on silencing in pericentric vs. distal arm chromosome 4 heterochromatin, distinguishing these two heterochromatin types.

Chromosome Research Vol 14, 405-416, 2006.

The dot chromosome of Drosophila: Insights into chromatin states and their change over evolutionary time

Nicole C. Riddle and Sarah C. R. Elgin

Historically, chromatin has been subdivided into heterochromatin, transcriptionally inactive regions that remain densely packaged throughout the cell cycle, and euchromatin, transcriptionally active regions that take on a diffuse appearance as the cell enters interphase. The banded portion of the small fourth chromosome (dot chromosome) of Drosophila melanogaster is unusual in exhibiting many characteristics of heterochromatic domains, and at the same time maintaining a gene density typical of euchromatin. Similar to genes embedded in pericentric heterochromatin, many of the dot chromosome genes have adapted to a heterochromatic environment. Little is known about the regulation of these genes and less about their evolution in a chromatin context. Interestingly, most of the genes from the D. melanogaster fourth chromosome remain clustered on a small chromosome throughout the genus Drosophila; yet the dot chromosome appears euchromatic in some species, such as D. virilis. Existing genomic sequence data allow an exploration of the underlying differences in DNA sequence organization between species. Here we review the available data describing the dot chromosome, which derives primarily from D. melanogaster. With its unusual and changing nature, the dot chromosome in the genus Drosophila provides a unique opportunity for the examination of transitions between chromatin states during evolution.

Current Biology Vol 16, 2222-27, 2006

Element 1360 and RNAi Components Contribute to HP1-Dependent Silencing of a Pericentric Reporter

Karmella A. Haynes, Amy A. Caudy, Lynne Collins, and Sarah C.R. Elgin

In eukaryotes, distinct regions of the genome are packaged as euchromatin (less condensed, more active) or heterochromatin (condensed, silenced). Studies in yeast, plants, and flies suggest that RNA interference (RNAi) is linked to heterochromatin formation and transcriptional silencing of transposable element (TE) sequences. We previously reported that insertion of a mobile hsp70-white reporter within 10 kb of a 1360 element on chromosome four of Drosophila melanogaster correlates with variegation (silencing). Here, we report small RNAs (23 nt) corresponding to 1360, indicating processing by the RNAi machinery. To directly test the ability of 1360 to silence a nearby gene in vivo, we introduced a P element construct carrying a single copy of 1360 upstream of the hsp70-white reporter into flies. This 1360 element contributes to HP1-dependent variegation at a pericentric insertion site, as demonstrated by a decrease in silencing after FLP-mediated removal of 1360. In euchromatin, 1360 is not sufficient to induce silencing, suggesting that proximity to pericentric heterochromatin and/or a high local TE density contributes to heterochromatin formation. Silencing of the 1360, hsp70-white reporter is sensitive to mutations in RNAi components. Our results implicate 1360 as a target for sequence-specific heterochromatic silencing through an RNAi-dependent mechanism.

Mol Cell Biol. 2004 Sep;24(18):8210-20.

Sun FL, Haynes K, Simpson CL, Lee SD, Collins L, Wuller J, Eissenberg JC, Elgin SC.
Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

The heterochromatic domains of Drosophila melanogaster (pericentric heterochromatin, telomeres, and the fourth chromosome) are characterized by histone hypoacetylation, high levels of histone H3 methylated on lysine 9 (H3-mK9), and association with heterochromatin protein 1 (HP1). While the specific interaction of HP1 with both H3-mK9 and histone methyltransferases suggests a mechanism for the maintenance of heterochromatin, it leaves open the question of how heterochromatin formation is targeted to specific domains. Expression characteristics of reporter transgenes inserted at different sites in the fourth chromosome define a minimum of three euchromatic and three heterochromatic domains, interspersed. Here we searched for cis-acting DNA sequence determinants that specify heterochromatic domains. Genetic screens for a switch in phenotype demonstrate that local deletions or duplications of 5 to 80 kb of DNA flanking a transposon reporter can lead to the loss or acquisition of variegation, pointing to short-range cis-acting determinants for silencing. This silencing is dependent on HP1. A switch in transgene expression correlates with a switch in chromatin structure, judged by nuclease accessibility. Mapping data implicate the 1360 transposon as a target for heterochromatin formation. We propose that heterochromatin formation is initiated at dispersed repetitive elements along the fourth chromosome and spreads for approximately 10 kb or until encountering competition from a euchromatic determinant.

Curr Biol, 2003 Dec 2;13(23):R895-8.

Heterochromatin: silence is golden

Elgin, S.C.R, and Grewal, S.I.S.

The large genomes of higher eukaryotes suggest a need for stable packaging, particularly as most of the DNA does not code for proteins, and much consists of repetitious sequences, including remnants of invading retrotransposons, transposable elements and the like. Cytological studies first demonstrated that much of the repetitous DNA is packaged in a condensed form referred to as heterochromatin, and indicated that such packaging limits transcription. During the last few years, remarkable progress has been made in identifying the biochemical characteristics of heterochromatin, suggesting mechanisms by which heterochromatin formation is targeted and maintained.

Curr Opin Genet Dev. 2002 Apr;12(2):178-87.

Grewal, S.I.S., and Elgin, S.C.R.

Significant portions of the eukaryotic genome are heterochromatic, made up largely of repetitious sequences and possessing a distinctive chromatin structure associated with gene silencing. New insights into the form of packaging, the associated histone modifications, and the associated nonhistone chromosomal proteins of heterochromatin have suggested a mechanism for providing an epigenetic mark that allows this distinctive chromatin structure to be maintained following replication and to spread within a given domain.

Proc Natl Acad Sci USA. 2000 May 9;97(10):5340-5.

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

Sun FL, Cuaycong MH, Craig CA, Wallrath LL, Locke J, Elgin SC.
Department of Biology, Washington University, St. Louis, MO 63130, USA.

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 heat-shock-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.

Mol Cell Biol 2001 Apr;21(8):2867-79.

Long-range nucleosome ordering is associated with gene silencing in Drosophila melanogaster pericentric heterochromatin

Sun FL, Cuaycong MH, Elgin SC.

Department of Biology, Washington University, St. Louis, Missouri 63130, USA.

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.

The EMBO Journal Vol 18, 3724-3735, 1999.

Silencing at Drosophila telomeres: nuclear organization and chromatin structure play critical roles

Diane E. Cryderman, Eric J. Morris, Harald Biessmann, Sarah C.R. Elgin and Lori L. Wallrath

Transgenes inserted into the telomeric regions of Drosophila melanogaster chromosomes exhibit position effect variegation (PEV), a mosaic silencing characteristic of euchromatic genes brought into juxtaposition with heterochromatin. Telomeric transgenes on the second and third chromosomes are flanked by telomeric associated sequences (TAS), while fourth chromosomes telomeric transgenes are most often associated with repetitious transposable elements. Telomeric PEV on the second and third chromosomes is suppressed by mutations in Su(z)2, but not mutations in Su(var)2-5 (encoding HP1), while the converse is true for telomeric PEV on the fourth chromosome. This genetic distinction allowed for a spatial and molecular analysis of telomeric PEV. Reciprocal translocations between the fourth chromosome telomeric region containing a transgene and a second chromosome telomeric region result in a change in nuclear location of the transgene. While the variegating phenotype of the white transgene is suppressed, sensitivity to a mutation in HP1 is retained. Corresponding changes in the chromatin structure and inducible activity of an associated hsp26 transgene are observed. The data indicate that both nuclear organization and local chromatin structure play a role in this telomeric PEV.

Methods Enzymol. 1999;304:462-96.

Analysis of Drosophila chromatin structure in vivo.

Cartwright IL, Cryderman DE, Gilmour DS, Pile LA, Wallrath LL, Weber JA, Elgin SC.
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Ohio 45267, USA.

Abstract not available.

Genes Dev 1995 May 15;9(10):1263-1277.

Position effect variegation in Drosophila is associated with an altered chromatin structure

Wallrath LL, Elgin SC.

Department of Biology, Washington University, St. Louis, Missouri 63130, USA.

A euchromatic gene placed in the vicinity of heterochromatin by a chromosomal rearrangement generally exhibits position effect variegation (PEV), a clonally inherited pattern showing gene expression in some somatic cells but not in others. The mechanism responsible for this loss of gene expression is investigated here using fly lines carrying a P element containing the Drosophila melanogaster white and hsp26 genes. Following mobilization of the P element, a screen for variegation of white expression recovered inserts at pericentric, telomeric, and fourth chromosome regions. Previously identified suppressors of PEV suppressed white variegation of pericentric and fourth chromosome inserts but not telomeric inserts on the second and third chromosomes. This implies a difference in the mechanism for gene repression at telomeres. Heat shock-induced hsp26 expression was reduced from pericentric and fourth chromosome inserts but not from telomeric inserts. Chromatin structure analysis revealed that the variegating inserts showed a reduction in accessibility to restriction enzyme digestion in the hsp26 regulatory region in isolated nuclei. Micrococcal nuclease digests showed that pericentric inserts were packaged in a more regular nucleosome array than that observed for euchromatic inserts. These data suggest that altered chromatin packaging plays a role in PEV.

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