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.
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.
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.
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 ...α-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.
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.
A series of heteroleptic quadruple-decker bislanthanide(III) complexes based on a flexible spacer-linked phthalocyanine ligand of clamshell-type, denoted sandwich-clamshell complexes, have been ...prepared. Complexation of 1,1’-benzene-1,2-diylbis(methanediyloxy)-bis9(10),16(17),23(24)-tri-tert-butylphthalocyanine (clam,tBuPc2H4, 1) with 2(3),9(10),16(17),23(24)-tetra-tert-butylphthalocyaninato lanthanide(III) acetates tBuPcLnOAc, 2a–d; Ln = Eu (a), Gd (b), Tb (c), Lu(d) afforded quadruple-deckers clam, tBuPc4Ln2 (3a–d) with good to excellent yields. The complexes reveal intrinsic UV–Vis–NIR absorption by analogy to classical double-decker phthalocyaninates, while demonstrating high thermal stability up to 350 °C. At the same time, a combination of 1H NMR, electrochemistry and SQUID magnetometry supported by DFT theoretical calculations indicates that bis(phthalocyanine) subunits in 3 are in close contact mainly governed by the spacer group and, in some extent, by the presence of bulky tert-butyl peripheral substituents in the four phthalocyanine decks.
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•The family of sandwich-clamshell phthalocyaninato quadruple-deckers was extended by Eu, Gd and Tb compounds.•Conditions for the rational synthesis were found with a multiple increase in the yield compared to literature.•A complex physicochemical and theoretical study of sandwich-clamshell quadruple-deckers was first conducted.•Electrochemistry reveals specific intramolecular π-system interactions•NMR and SQUID data show the existence of intramolecular magnetic interactions.
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.
Genetically marked P elements inserted into the subtelomeric satellites of Drosophila show repression and variegation of the reporter gene. One such white+ reporter, inserted between the subtelomeric ...satellite and the terminal HeT-A array in the left arm of chromosome 2 (2L), is sensitive to its context; changes in the structure of the telomere region can be identified by changes in eye color. Addition of HeT-A or TART elements to the 2L terminus increases w+ expression, and loss of sequence from the end decreases expression. This indicates that the telomeric retrotransposons in Drosophila have an activating influence on the repressed subterminal reporter gene. Changes in eye color due to altered expression of the transgene also allow the detection of interactions between homologous telomeres. The 2L arms that terminate in long HeT-A/TART arrays showed increased expression of the subterminal w+ transgene when the terminal repeats on the homologue are absent or markedly shorter. We propose that the chromatin structure of the terminal HeT-A/TART array and the activity of a putative promoter/enhancer element on HeT-A are affected by telomeric interactions. Such trans-activation may reflect control over HeT-A transcription and, thus, transposition activity.
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.