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1.
Biophys J ; 112(3): 460-472, 2017 Feb 07.
Article in English | MEDLINE | ID: mdl-28038734

ABSTRACT

The evidence is now overwhelming that partially assembled nucleosome states (PANS) are as important as the canonical nucleosome structure for the understanding of how accessibility to genomic DNA is regulated in cells. We use a combination of molecular dynamics simulation and atomic force microscopy to deliver, in atomic detail, structural models of three key PANS: the hexasome (H2A·H2B)·(H3·H4)2, the tetrasome (H3·H4)2, and the disome (H3·H4). Despite fluctuations of the conformation of the free DNA in these structures, regions of protected DNA in close contact with the histone core remain stable, thus establishing the basis for the understanding of the role of PANS in DNA accessibility regulation. On average, the length of protected DNA in each structure is roughly 18 basepairs per histone protein. Atomistically detailed PANS are used to explain experimental observations; specifically, we discuss interpretation of atomic force microscopy, Förster resonance energy transfer, and small-angle x-ray scattering data obtained under conditions when PANS are expected to exist. Further, we suggest an alternative interpretation of a recent genome-wide study of DNA protection in active chromatin of fruit fly, leading to a conclusion that the three PANS are present in actively transcribing regions in a substantial amount. The presence of PANS may not only be a consequence, but also a prerequisite for fast transcription in vivo.


Subject(s)
Microscopy, Atomic Force , Molecular Dynamics Simulation , Nucleosomes/chemistry , Nucleosomes/metabolism , DNA/chemistry , DNA/genetics , DNA/metabolism , Genomics , Nucleic Acid Conformation , Nucleosomes/genetics
2.
Genes Dev ; 26(6): 603-14, 2012 Mar 15.
Article in English | MEDLINE | ID: mdl-22426536

ABSTRACT

SNF2-like motor proteins, such as ISWI, cooperate with histone chaperones in the assembly and remodeling of chromatin. Here we describe a novel, evolutionarily conserved, ISWI-containing complex termed ToRC (Toutatis-containing chromatin remodeling complex). ToRC comprises ISWI, Toutatis/TIP5 (TTF-I-interacting protein 5), and the transcriptional corepressor CtBP (C-terminal-binding protein). ToRC facilitates ATP-dependent nucleosome assembly in vitro. All three subunits are required for its maximal biochemical activity. The toutatis gene exhibits strong synthetic lethal interactions with CtBP. Thus, ToRC mediates, at least in part, biological activities of CtBP and Toutatis. ToRC subunits colocalize in euchromatic arms of polytene chromosomes. Furthermore, nuclear localization and precise distribution of ToRC in chromosomes are dependent on CtBP. ToRC is involved in CtBP-mediated regulation of transcription by RNA polymerase II in vivo. For instance, both Toutatis and CtBP are required for repression of genes of a proneural gene cluster, achaete-scute complex (AS-C), in Drosophila larvae. Intriguingly, native C-terminally truncated Toutatis isoforms do not associate with CtBP and localize predominantly to the nucleolus. Thus, Toutatis forms two alternative complexes that have differential distribution and can participate in distinct aspects of nuclear DNA metabolism.


Subject(s)
Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Alcohol Oxidoreductases/metabolism , Chromatin Assembly and Disassembly , Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Transcription Factors/metabolism , Adenosine Triphosphatases/genetics , Alcohol Oxidoreductases/genetics , Animals , Cell Nucleus/metabolism , Chromosomal Proteins, Non-Histone/genetics , DNA/metabolism , DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Transcription Factors/genetics
3.
J Biol Chem ; 285(20): 15027-15037, 2010 May 14.
Article in English | MEDLINE | ID: mdl-20154359

ABSTRACT

ATRX belongs to the family of SWI2/SNF2-like ATP-dependent nucleosome remodeling molecular motor proteins. Mutations of the human ATRX gene result in a severe genetic disorder termed X-linked alpha-thalassemia mental retardation (ATR-X) syndrome. Here we perform biochemical and genetic analyses of the Drosophila melanogaster ortholog of ATRX. The loss of function allele of the Drosophila ATRX/XNP gene is semilethal. Drosophila ATRX is expressed throughout development in two isoforms, p185 and p125. ATRX185 and ATRX125 form distinct multisubunit complexes in fly embryo. The ATRX185 complex comprises p185 and heterochromatin protein HP1a. Consistently, ATRX185 but not ATRX125 is highly concentrated in pericentric beta-heterochromatin of the X chromosome in larval cells. HP1a strongly stimulates biochemical activities of ATRX185 in vitro. Conversely, ATRX185 is required for HP1a deposition in pericentric beta-heterochromatin of the X chromosome. The loss of function allele of the ATRX/XNP gene and mutant allele that does not express p185 are strong suppressors of position effect variegation. These results provide evidence for essential biological functions of Drosophila ATRX in vivo and establish ATRX as a major determinant of pericentric beta-heterochromatin identity.


Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , DNA Helicases/metabolism , Drosophila Proteins/metabolism , Heterochromatin/metabolism , Animals , Blotting, Western , Chromobox Protein Homolog 5 , DNA Helicases/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Electrophoresis, Polyacrylamide Gel , Mutation , Protein Binding , X Chromosome
4.
Genes Dev ; 23(4): 452-65, 2009 Feb 15.
Article in English | MEDLINE | ID: mdl-19196654

ABSTRACT

We generated mutant alleles of Drosophila melanogaster in which expression of the linker histone H1 can be down-regulated over a wide range by RNAi. When the H1 protein level is reduced to approximately 20% of the level in wild-type larvae, lethality occurs in the late larval - pupal stages of development. Here we show that H1 has an important function in gene regulation within or near heterochromatin. It is a strong dominant suppressor of position effect variegation (PEV). Similar to other suppressors of PEV, H1 is simultaneously involved in both the repression of euchromatic genes brought to the vicinity of pericentric heterochromatin and the activation of heterochromatic genes that depend on their pericentric localization for maximal transcriptional activity. Studies of H1-depleted salivary gland polytene chromosomes show that H1 participates in several fundamental aspects of chromosome structure and function. First, H1 is required for heterochromatin structural integrity and the deposition or maintenance of major pericentric heterochromatin-associated histone marks, including H3K9Me(2) and H4K20Me(2). Second, H1 also plays an unexpected role in the alignment of endoreplicated sister chromatids. Finally, H1 is essential for organization of pericentric regions of all polytene chromosomes into a single chromocenter. Thus, linker histone H1 is essential in Drosophila and plays a fundamental role in the architecture and activity of chromosomes in vivo.


Subject(s)
Chromosomes/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Heterochromatin/genetics , Histones/metabolism , Animals , Centromere/genetics , Chromatids/genetics , Chromosomal Position Effects/physiology , Drosophila Proteins/genetics , Drosophila melanogaster/growth & development , Gene Expression Regulation, Developmental , Histones/genetics , RNA Interference
5.
Science ; 317(5841): 1087-90, 2007 Aug 24.
Article in English | MEDLINE | ID: mdl-17717186

ABSTRACT

The organization of chromatin affects all aspects of nuclear DNA metabolism in eukaryotes. H3.3 is an evolutionarily conserved histone variant and a key substrate for replication-independent chromatin assembly. Elimination of chromatin remodeling factor CHD1 in Drosophila embryos abolishes incorporation of H3.3 into the male pronucleus, renders the paternal genome unable to participate in zygotic mitoses, and leads to the development of haploid embryos. Furthermore, CHD1, but not ISWI, interacts with HIRA in cytoplasmic extracts. Our findings establish CHD1 as a major factor in replacement histone metabolism in the nucleus and reveal a critical role for CHD1 in the earliest developmental instances of genome-scale, replication-independent nucleosome assembly. Furthermore, our results point to the general requirement of adenosine triphosphate (ATP)-utilizing motor proteins for histone deposition in vivo.


Subject(s)
Chromatin Assembly and Disassembly , Chromatin/metabolism , DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila/physiology , Histones/metabolism , Transcription Factors/metabolism , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Animals , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/genetics , Drosophila/embryology , Drosophila/genetics , Drosophila/metabolism , Drosophila Proteins/genetics , Embryo, Nonmammalian/physiology , Embryonic Development , Female , Haploidy , Histone Chaperones , Male , Mutation , Nucleosomes/metabolism , Protamines/metabolism , Spermatozoa/physiology , Transcription Factors/genetics , Transgenes
6.
Mol Cell Biol ; 27(13): 4991-5001, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17470550

ABSTRACT

Drosophila melanogaster telomeres have two DNA domains: a terminal array of retrotransposons and a subterminal repetitive telomere-associated sequence (TAS), a source of telomere position effect (TPE). We reported previously that deletion of the 2L TAS array leads to dominant suppression of TPE by stimulating in trans expression of a telomeric transgene. Here, we compared the transcript activities of a w transgene inserted between the retrotransposon and TAS arrays at the 2L telomere in genotypes with different lengths of the 2L TAS. In contrast to individuals bearing a wild-type 2L homologue, flies with a TAS deficiency showed a significant increase in the level of telomeric w transcript during development, especially in pupae. Moreover, we identified a read-through w transcript initiated from a retrotransposon promoter in the terminal array. Read-through transcript levels also significantly increased with the presence of a 2L TAS deficiency in trans, indicating a stimulating force of the TAS deficiency on retrotransposon promoter activity. The read-through transcript contributes to total w transcript, although most w transcript originates at the w promoter. While silencing of transgenes in nonhomologous telomeres is suppressed by 2L TAS deficiencies, suggesting a global effect, the overall level of HeT-A transcripts is not increased under similar conditions.


Subject(s)
Drosophila melanogaster/genetics , Retroelements/genetics , Telomere/genetics , Transcription, Genetic , Animals , Base Sequence , Gene Expression Regulation , Molecular Sequence Data , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , Sequence Homology , Transgenes
7.
Genetics ; 168(3): 1353-70, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15579690

ABSTRACT

Heterochromatin is a specialized chromatin structure in chromosomal regions associated with repeated DNA sequences and low concentrations of genes. Formation of heterochromatin is determined in large part by enzymes that modify histones and structural proteins that bind to these modified histones in a cooperative fashion. In Drosophila, mutations in genes that encode heterochromatic proteins are often dominant and increase expression of genes placed into heterochromatic positions. To find components of telomeric heterochromatin in Drosophila, we screened a collection of autosomal deficiencies for dominant suppressors of silencing of a transgene at the telomere of chromosome 2L. While many deficiency chromosomes are associated with dominant suppressors, in the cases tested on chromosome 2 the suppressor mapped to the 2L telomere, rather than the deficiency. We infer that background effects may hamper the search for genes that play a role in telomeric heterochromatin formation and that either very few genes participate in this pathway or mutations in these genes are not dominant suppressors of telomeric position effect. The data also suggest that the 2L telomere region plays a major role in telomeric silencing.


Subject(s)
Drosophila/genetics , Gene Silencing , Telomere , Animals , Chromosome Mapping , Drosophila/metabolism , Eye/metabolism , Genetic Markers , Meiosis , Pigmentation/genetics , Pigmentation/physiology , Recombination, Genetic
8.
Genetica ; 117(2-3): 319-25, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12723711

ABSTRACT

The terminal DNA arrays on chromosomes of Drosophila melanogaster are composed of two families of non-LTR retrotransposons, HeT-A and TART. Available evidence suggests that chromosome length in this species and its close relatives is maintained by targeted transposition of these elements, with attachment of the elements to the chromosome end by their 3' oligo(A) tails. However, the regulation of transposition of these elements and the control of telomere length are poorly understood. Here we present the hypothesis that the forces involved in telomere length regulation in Drosophila are the underlying forces that manifest themselves as telomeric position effect (TPE). Based on recent studies of TPE, which found that expression of a reporter gene is influenced by telomere structure in cis and trans, we propose that the subtelomeric satellite (TAS) in D. melanogaster plays an important role in controlling telomere elongation. Transcription of a HeT-A element is probably initiated at a promoter in the 3' UTR of an upstream element, and TAS may repress this transcriptional activity in cis and trans. A region of HeT-A not at the extreme 3' end of the element may act as a transcriptional enhancer that may be modulated by TAS.


Subject(s)
Drosophila melanogaster/genetics , Telomere/genetics , Terminal Repeat Sequences/genetics , Animals , Chromosome Mapping , DNA Transposable Elements , Gene Expression Regulation/genetics , Gene Silencing , Retroelements/genetics , Transcriptional Activation , Transgenes
9.
Genetics ; 163(3): 917-30, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12663532

ABSTRACT

One model of telomeric position effect (TPE) in Drosophila melanogaster proposes that reporter genes in the vicinity of telomeres are repressed by subterminal telomere-associated sequences (TAS) and that variegation of these genes is the result of competition between the repressive effects of TAS and the stimulating effects of promoters in the terminal HeT-A transposon array. The data presented here support this model, but also suggest that TPE is more complex. Activity of a telomeric white reporter gene increases in response to deletion of some or all of the TAS on the homolog. Only transgenes next to fairly long HeT-A arrays respond to this trans-interaction. HeT-A arrays of 6-18 kb respond by increasing the number of dark spots on the eye, while longer arrays increase the background eye color or increase the number of spots sufficiently to cause them to merge. Thus, expression of a subtelomeric reporter gene is influenced by the telomere structure in cis and trans. We propose that the forces involved in telomere length regulation in Drosophila are the underlying forces that manifest themselves as TPE. In the wild-type telomere TAS may play an important role in controlling telomere elongation by repressing HeT-A promoter activity. Modulation of this repression by the homolog may thus regulate telomere elongation.


Subject(s)
Drosophila melanogaster/genetics , Telomere/genetics , Animals , Animals, Genetically Modified , Base Sequence , Eye Color/genetics , Genes, Reporter , Models, Genetic , Molecular Sequence Data , Polymerase Chain Reaction/methods
10.
Genetics ; 165(4): 2039-53, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14704184

ABSTRACT

Heterochromatin is a major component of higher eukaryotic genomes, but progress in understanding the molecular structure and composition of heterochromatin has lagged behind the production of relatively complete euchromatic genome sequences. The introduction of single-copy molecular-genetic entry points can greatly facilitate structure and sequence analysis of heterochromatic regions that are rich in repeated DNA. In this study, we report the isolation of 502 new P-element insertions into Drosophila melanogaster centric heterochromatin, generated in nine different genetic screens that relied on mosaic silencing (position-effect variegation, or PEV) of the yellow gene present in the transposon. The highest frequencies of recovery of variegating insertions were observed when centric insertions were used as the source for mobilization. We propose that the increased recovery of variegating insertions from heterochromatic starting sites may result from the physical proximity of different heterochromatic regions in germline nuclei or from the association of mobilizing elements with heterochromatin proteins. High frequencies of variegating insertions were also recovered when a potent suppressor of PEV (an extra Y chromosome) was present in both the mobilization and selection generations, presumably due to the effects of chromatin structure on P-element mobilization, insertion, and phenotypic selection. Finally, fewer variegating insertions were recovered after mobilization in females, in comparison to males, which may reflect differences in heterochromatin structure in the female and male germlines. FISH localization of a subset of the insertions confirmed that 98% of the variegating lines contain heterochromatic insertions and that these schemes produce a broader distribution of insertion sites. The results of these schemes have identified the most efficient methods for generating centric heterochromatin P insertions. In addition, the large collection of insertions produced by these screens provides molecular-genetic entry points for mapping, sequencing, and functional analysis of Drosophila heterochromatin.


Subject(s)
DNA Transposable Elements , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Heterochromatin/genetics , Animals , Chromosomes/genetics , Female , Germ Cells/cytology , In Situ Hybridization, Fluorescence , Male , Phenotype , Selection, Genetic
11.
Genetics ; 161(1): 217-29, 2002 May.
Article in English | MEDLINE | ID: mdl-12019236

ABSTRACT

Approximately one-third of the human and Drosophila melanogaster genomes are heterochromatic, yet we know very little about the structure and function of this enigmatic component of eukaryotic genomes. To facilitate molecular and cytological analysis of heterochromatin we introduced a yellow(+) (y(+))-marked P element into centric heterochromatin by screening for variegated phenotypes, that is, mosaic gene inactivation. We recovered >110 P insertions with variegated yellow expression from approximately 3500 total mobilization events. FISH analysis of 71 of these insertions showed that 69 (97%) were in the centric heterochromatin, rather than telomeres or euchromatin. High-resolution banding analysis showed a wide but nonuniform distribution of insertions within centric heterochromatin; variegated insertions were predominantly recovered near regions of satellite DNA. We successfully used inverse PCR to clone and sequence the flanking DNA for approximately 63% of the insertions. BLAST analysis of the flanks demonstrated that either most of the variegated insertions could not be placed on the genomic scaffold, and thus may be inserted within novel DNA sequence, or that the flanking DNA hit multiple sites on the scaffold, due to insertions within different transposons. Taken together these data suggest that screening for yellow variegation is a very efficient method for recovering centric insertions and that a large-scale screen for variegated yellow P insertions will provide important tools for detailed analysis of centric heterochromatin structure and function.


Subject(s)
DNA Transposable Elements , Drosophila Proteins , Drosophila melanogaster/genetics , Heterochromatin , Animals , DNA/isolation & purification , Female , In Situ Hybridization, Fluorescence , Insect Proteins/genetics , Male , Sequence Analysis, DNA
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