Transposon control is a critical process during reproduction. The PIWI family proteins can play a key role, using a piRNA-mediated slicing mechanism to suppress transposon activity ...posttranscriptionally. In Drosophila melanogaster, Piwi is predominantly localized in the nucleus and has been implicated in heterochromatin formation. Here, we use female germ-line–specific depletion to study Piwi function. This depletion of Piwi leads to infertility and to axis specification defects in the developing egg chambers; correspondingly, widespread loss of transposon silencing is observed. Germ-line Piwi does not appear to be required for piRNA production. Instead, Piwi requires Aubergine (and presumably secondary piRNA) for proper localization. A subset of transposons that show significant overexpression in germ-line Piwi-depleted ovaries exhibit a corresponding loss of HP1a and H3K9me2. Germ-line HP1a depletion also leads to a loss of transposon silencing, demonstrating the functional requirement for HP1a enrichment at these loci. Considering our results and those of others, we infer that germ-line Piwi functions downstream of piRNA production to promote silencing of some transposons via recruitment of HP1a. Thus, in addition to its better-known function in posttranscriptional silencing, piRNA also appears to function in a targeting mechanism for heterochromatin formation mediated by Piwi.
Position-effect variegation (PEV) results when a gene normally in euchromatin is juxtaposed with heterochromatin by rearrangement or transposition. When heterochromatin packaging spreads across the ...heterochromatin/euchromatin border, it causes transcriptional silencing in a stochastic pattern. PEV is intensely studied in Drosophila using the white gene. Screens for dominant mutations that suppress or enhance white variegation have identified many conserved epigenetic factors, including the histone H3 lysine 9 methyltransferase SU(VAR)3-9. Heterochromatin protein HP1a binds H3K9me2/3 and interacts with SU(VAR)3-9, creating a core memory system. Genetic, molecular, and biochemical analysis of PEV in Drosophila has contributed many key findings concerning establishment and maintenance of heterochromatin with concomitant gene silencing.
Highlights • We propose a structure-based definition of HP1 family proteins. • We review evidence that shows HP1 to be a context-dependent modifier of gene expression. • Post-translational ...modifications of HP1 family proteins alter functionality.
A persistent question in epigenetics is how heterochromatin is targeted for assembly at specific domains, and how that chromatin state is faithfully transmitted. Stable heterochromatin is necessary ...to silence transposable elements (TEs) and maintain genome integrity. Both the RNAi system and heterochromatin components HP1 (Swi6) and H3K9me2/3 are required for initial establishment of heterochromatin structures in S. pombe. Here we utilize both loss of function alleles and the newly developed Drosophila melanogaster transgenic shRNA lines to deplete proteins of interest at specific development stages to dissect their roles in heterochromatin assembly in early zygotes and in maintenance of the silencing chromatin state during development. Using reporters subject to Position Effect Variegation (PEV), we find that depletion of key proteins in the early embryo can lead to loss of silencing assayed at adult stages. The piRNA component Piwi is required in the early embryo for reporter silencing in non-gonadal somatic cells, but knock-down during larval stages has no impact. This implies that Piwi is involved in targeting HP1a when heterochromatin is established at the late blastoderm stage and possibly also during embryogenesis, but that the silent chromatin state created is transmitted through cell division independent of the piRNA system. In contrast, heterochromatin structural protein HP1a is required for both initial heterochromatin assembly and the following mitotic inheritance. HP1a profiles in piwi mutant animals confirm that Piwi depletion leads to decreased HP1a levels in pericentric heterochromatin, particularly in TEs. The results suggest that the major role of the piRNA system in assembly of heterochromatin in non-gonadal somatic cells occurs in the early embryo during heterochromatin formation, and further demonstrate that failure of heterochromatin formation in the early embryo impacts the phenotype of the adult.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Transcription in heterochromatin seems to be an oxymoron--surely the 'silenced' form of chromatin should not be transcribed. But there have been frequent reports of low-level transcription in ...heterochromatic regions, and several hundred genes are found in these regions in Drosophila. Most strikingly, recent investigations implicate RNA interference mechanisms in targeting and maintaining heterochromatin, and these mechanisms are inherently dependent on transcription. Silencing of chromatin might involve trans-acting sources of the crucial small RNAs that carry out RNA interference, but in some cases, transcription of the region to be silenced seems to be required--an apparent contradiction.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The F element of the
karyotype (the fourth chromosome in
) is often referred to as the "dot chromosome" because of its appearance in a metaphase chromosome spread. This chromosome is distinct from ...other
autosomes in possessing both a high level of repetitious sequences (in particular, remnants of transposable elements) and a gene density similar to that found in the other chromosome arms, ∼80 genes distributed throughout its 1.3-Mb "long arm." The dot chromosome is notorious for its lack of recombination and is often neglected as a consequence. This and other features suggest that the F element is packaged as heterochromatin throughout. F element genes have distinct characteristics (
, low codon bias, and larger size due both to larger introns and an increased number of exons), but exhibit expression levels comparable to genes found in euchromatin. Mapping experiments show the presence of appropriate chromatin modifications for the formation of DNaseI hypersensitive sites and transcript initiation at the 5' ends of active genes, but, in most cases, high levels of heterochromatin proteins are observed over the body of these genes. These various features raise many interesting questions about the relationships of chromatin structures with gene and chromosome function. The apparent evolution of the F element as an autosome from an ancestral sex chromosome also raises intriguing questions. The findings argue that the F element is a unique chromosome that occupies its own space in the nucleus. Further study of the F element should provide new insights into chromosome structure and function.
A persistent question in biology is how cis -acting sequence elements influence trans -acting factors and the local chromatin environment to modulate gene expression. We reported previously that the ...DNA transposon 1360 can enhance silencing of a reporter in a heterochromatic domain of Drosophila melanogaster . We have now generated a collection of variegating phiC31 landing-pad insertion lines containing 1360 and a heat-shock protein 70 (hsp70)-driven white reporter to explore the mechanism of 1360 -sensitive silencing. Many 1360 -sensitive sites were identified, some in apparently euchromatic domains, although all are close to heterochromatic masses. One such site (line 1198; insertion near the base of chromosome arm 2L) has been investigated in detail. ChIP analysis shows 1360 -dependent Heterochromatin Protein 1a (HP1a) accumulation at this otherwise euchromatic site. The phiC31 landing pad system allows different 1360 constructs to be swapped with the full-length element at the same genomic site to identify the sequences that mediate 1360 -sensitive silencing. Short deletions over sites with homology to PIWI-interacting RNAs (piRNAs) are sufficient to compromise 1360 -sensitive silencing. Similar results were obtained on replacing 1360 with Invader4 (a retrotransposon), suggesting that this phenomenon likely applies to a broader set of transposable elements. Our results suggest a model in which piRNA sequence elements behave as cis -acting targets for heterochromatin assembly, likely in the early embryo, where piRNA pathway components are abundant, with the heterochromatic state subsequently propagated by chromatin modifiers present in somatic tissue.
Genes normally resident in euchromatic domains are silenced when packaged into heterochromatin, as exemplified in Drosophila melanogaster by position effect variegation (PEV). Loss-of-function ...mutations resulting in suppression of PEV have identified critical components of heterochromatin, including proteins HP1, HP2, and histone H3 lysine 9 methyltransferase. Here, we demonstrate that this silencing is dependent on the RNA interference machinery, using tandem mini-white arrays and white transgenes in heterochromatin to show loss of silencing as a result of mutations in piwi, aubergine, or spindle-E (homeless), which encode RNAi components. These mutations result in reduction of H3 Lys9methylation and delocalization of HP1 and HP2, most dramatically in spindle-E mutants.
Scientists are sequencing new genomes at an increasing rate with the goal of associating genome contents with phenotypic traits. After a new genome is sequenced and assembled, structural gene ...annotation is often the first step in analysis. Despite advances in computational gene prediction algorithms, most eukaryotic genomes still benefit from manual gene annotation. This requires access to good genome browsers to enable annotators to visualize and evaluate multiple lines of evidence (e.g., sequence similarity, RNA sequencing RNA-Seq results, gene predictions, repeats) and necessitates many volunteers to participate in the work. To address the technical barriers to creating genome browsers, the Genomics Education Partnership (GEP; https://gep.wustl.edu/) has partnered with the Galaxy Project (https://galaxyproject.org) to develop G-OnRamp (http://g-onramp.org), a web-based platform for creating UCSC Genome Browser Assembly Hubs and JBrowse genome browsers. G-OnRamp also converts a JBrowse instance into an Apollo instance for collaborative genome annotations in research and educational settings. The genome browsers produced can be transferred to the CyVerse Data Store for long-term access. G-OnRamp enables researchers to easily visualize their experimental results, educators to create Course-based Undergraduate Research Experiences (CUREs) centered on genome annotation, and students to participate in genomics research. In the process, students learn about genes/genomes and about how to utilize large datasets. Development of G-OnRamp was guided by extensive user feedback. Sixty-five researchers/educators from >40 institutions participated through in-person workshops, which produced >20 genome browsers now available for research and education. Genome browsers generated for four parasitoid wasp species have been used in a CURE engaging students at 15 colleges and universities. Our assessment results in the classroom demonstrate that the genome browsers produced by G-OnRamp are effective tools for engaging undergraduates in research and in enabling their contributions to the scientific literature in genomics. Expansion of such genomics research/education partnerships will be beneficial to researchers, faculty, and students alike.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Eukaryotic genomes are packaged in two general varieties of chromatin: gene-rich euchromatin and gene-poor heterochromatin. Each type of chromatin has been defined by the presence of distinct ...chromosomal proteins and posttranslational histone modifications. This review addresses recent findings that appear to blur the definitions of euchromatin and heterochromatin by pointing to the presence of typically heterochromatic modifications (including H3K9me) in euchromatin and typically euchromatic enzymes (including RNA polymerases) in heterochromatin. We discuss the implications of these new findings for the current definition of heterochromatin.
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