Alexey V. Pindyurin

Interaction between the Drosophila heterochromatin proteins SUUR and HP1

SUUR (Suppressor of Under-Replication) protein is responsible for late replication and, as a consequence, for DNA underreplication of intercalary and pericentric heterochromatin in Drosophila melanogaster polytene chromosomes. However, the mechanism by which SUUR slows down the replication process is not clear. To identify possible partners for SUUR we performed a yeast two-hybrid screen using full-length SUUR as bait. This identified HP1, the well-studied heterochromatin protein, as a strong SUUR interactor. Furthermore, we have determined that the central region of SUUR is necessary and sufficient for interaction with the C-terminal part of HP1, which contains the hinge and chromoshadow domains. In addition, recruitment of SUUR to ectopic HP1 sites on chromosomes provides evidence for their association in vivo. Indeed, we found that the distributions of SUUR and HP1 on polytene chromosomes are interdependent: both absence and overexpression of HP1 prevent SUUR from chromosomal binding, whereas SUUR overexpression causes redistribution of HP1 to numerous sites occupied by SUUR. Finally, HP1 binds to intercalary heterochromatin when histone methyltransferase activity of SU(VAR)3-9 is increased. We propose that interaction with HP1 is crucial for the association of SUUR with chromatin.

DNA Copy-Number Control through Inhibition of Replication Fork Progression

Proper control of DNA replication is essential to ensure faithful transmission of genetic material and prevent chromosomal aberrations that can drive cancer progression and developmental disorders. DNA replication is regulated primarily at the level of initiation and is under strict cell-cycle regulation. Importantly, DNA replication is highly influenced by developmental cues. In Drosophila, specific regions of the genome are repressed for DNA replication during differentiation by the SNF2 domain-containing protein SUUR through an unknown mechanism. We demonstrate that SUUR is recruited to active replication forks and mediates the repression of DNA replication by directly inhibiting replication fork progression instead of functioning as a replication fork barrier. Mass spectrometry identification of SUUR-associated proteins identified the replicative helicase member CDC45 as a SUUR-associated protein, supporting a role for SUUR directly at replication forks. Our results reveal that control of eukaryotic DNA copy number can occur through the inhibition of replication fork progression.

Regulatory functions and chromatin loading dynamics of linker histone H1 during endoreplication in Drosophila

Eukaryotic DNA replicates asynchronously, with discrete genomic loci replicating during different stages of S phase. Drosophila larval tissues undergo endoreplication without cell division, and the latest replicating regions occasionally fail to complete endoreplication, resulting in underreplicated domains of polytene chromosomes. Here we show that linker histone H1 is required for the underreplication (UR) phenomenon in Drosophila salivary glands. H1 directly interacts with the Suppressor of UR (SUUR) protein and is required for SUUR binding to chromatin in vivo. These observations implicate H1 as a critical factor in the formation of underreplicated regions and an upstream effector of SUUR. We also demonstrate that the localization of H1 in chromatin changes profoundly during the endocycle. At the onset of endocycle S (endo-S) phase, H1 is heavily and specifically loaded into late replicating genomic regions and is then redistributed during the course of endoreplication. Our data suggest that cell cycle-dependent chromosome occupancy of H1 is governed by several independent processes. In addition to the ubiquitous replication-related disassembly and reassembly of chromatin, H1 is deposited into chromatin through a novel pathway that is replication-independent, rapid, and locus-specific. This cell cycle-directed dynamic localization of H1 in chromatin may play an important role in the regulation of DNA replication timing.

Piwi interacts with chromatin at nuclear pores and promiscuously binds nuclear transcripts in Drosophila ovarian somatic cells

Abstract Piwi in a complex with Piwi-interacting RNAs (piRNAs) triggers transcriptional silencing of transposable elements (TEs) in Drosophila ovaries, thus ensuring genome stability. To do this, Piwi must scan the nascent transcripts of genes and TEs for complementarity to piRNAs. The mechanism of this scanning is currently unknown. Here we report the DamID-seq mapping of multiple Piwi-interacting chromosomal domains in somatic cells of Drosophila ovaries. These domains significantly overlap with genomic regions tethered to Nuclear Pore Complexes (NPCs). Accordingly, Piwi was coimmunoprecipitated with the component of NPCs Elys and with the Xmas-2 subunit of RNA transcription and export complex, known to interact with NPCs. However, only a small Piwi fraction has transient access to DNA at nuclear pores. Importantly, although 36% of the protein-coding genes overlap with Piwi-interacting domains and RNA-immunoprecipitation results demonstrate promiscuous Piwi binding to numerous genic and TE nuclear transcripts, according to available data Piwi does not silence these genes, likely due to the absence of perfect base-pairing between piRNAs and their transcripts.

Contribution of the SuUR gene to the organization of epigenetically repressed regions of Drosophila melanogaster chromosomes

A significant portion of a eukaryotic genome is silent (epigenetically repressed). In Drosophila melanogaster, this portion includes mainly regions of pericentric and intercalary heterochromatin and euchromatin regions subject to position-effect variegation. Detailed study of the organization of intercalary heterochromatin regions of Drosophila melanogaster polytene chromosomes started from the discovery of the SuUR gene (Suppressor of UnderReplication). The ability of the SuUR mutation to suppress underreplication in intercalary heterochromatin regions was used for molecular tagging of these regions. We showed that underreplicated intercalary heterochromatin regions contained silent unique genes and retained the features of late replication and transcriptionally inactive chromatin state in various cell types. Over 50% of these regions contain unique genes clustered on the base of coordinated expression. The origin of clusters and putative mechanisms of their gene expression are discussed. Data on the SuUR gene, its expression, and effect on polytene chromosome structure and replication are summarized.

Maintenance of the patterns of expression of homeotic genes in the development of Drosophila melanogaster by proteins of the polycomb, trithorax, and ETP groups

Proteins encoded by genes of the Polycomb (PcG), trithorax (trxG), and the Enhancer of trithorax and Polycomb (ETP) groups are important regulators of expression of most developmental genes. Data concerning all currently described genes assigned to these groups are summarized in the review. Genetic interactions of these genes and phenotypic manifestation of their mutations are described. Data on the PcG, trxG, and ETP proteins are systematized. Questions are considered concerning the formation of multimeric complexes containing proteins of these groups, recruitment of these complexes to regulatory elements of target genes, and the mechanisms of activation/repression of gene expression.

DamID profiling of dynamic Polycomb-binding sites in Drosophila imaginal disc development and tumorigenesis

BackgroundTracking dynamic protein–chromatin interactions in vivo is key to unravel transcriptional and epigenetic transitions in development and disease. However, limited availability and heterogeneous tissue composition of in vivo source material impose challenges on many experimental approaches.ResultsHere we adapt cell-type-specific DamID-seq profiling for use in Drosophila imaginal discs and make FLP/FRT-based induction accessible to GAL driver-mediated targeting of specific cell lineages. In a proof-of-principle approach, we utilize ubiquitous DamID expression to describe dynamic transitions of Polycomb-binding sites during wing imaginal disc development and in a scrib tumorigenesis model. We identify Atf3 and Ets21C as novel Polycomb target genes involved in scrib tumorigenesis and suggest that target gene regulation by Atf3 and AP-1 transcription factors, as well as modulation of insulator function, plays crucial roles in dynamic Polycomb-binding at target sites. We establish these findings by DamID-seq analysis of wing imaginal disc samples derived from 10 larvae.ConclusionsOur study opens avenues for robust profiling of small cell population in imaginal discs in vivo and provides insights into epigenetic changes underlying transcriptional responses to tumorigenic transformation.

Correction to: DamID profiling of dynamic Polycomb-binding sites in Drosophila imaginal disc development and tumorigenesis

Unfortunately, the original version of this article contained a typographical error in one of the author names. The name of the author Alexey Pindyurin was incorrectly spelt as Alexey Pinduyrin. The correct spelling is included here and has been updated in the original article.

TRIP through the chromatin: a high throughput exploration of enhancer regulatory landscapes.

Enhancers are regulatory elements that promote gene expression in a spatio-temporal way and are involved in a wide range of developmental and disease processes. Both the identification and subsequent functional dissection of enhancers are key steps in understanding these processes. Several high-throughput approaches were recently developed for these purposes; however, in almost all cases enhancers are being tested outside their native chromatin context. Until recently, the analysis of enhancer activities at their native genomic locations was low throughput, laborious and time-consuming. Here, we discuss the potential of a powerful approach, TRIP, to study the functioning of enhancers in their native chromatin environments by introducing sensor constructs directly in the genome. TRIP allows for simultaneously analyzing the quantitative readout of numerous sensor constructs integrated at random locations in the genome. The high-throughput and flexible nature of TRIP opens up potential to study different aspects of enhancer biology at an unprecedented level.

Intercalary heterochromatin in the genome of Drosophila

The modern concept of intercalary heterochromatin as polytene chromosome regions exhibiting a number of specific characteristics is formulated. DNA constituting these regions is replicated late in the S period; therefore, some strands of polytene chromosomes are underrepresented; i.e., they are underreplicated. Late-replicating regions account for about 7% of the genome; genes are located there in clusters of as many as 40. In general, the gene density in the clusters is substantially lower than in the main part of the genome. Late-replicating regions have an inactivating capacity: genes incorporated into these regions as parts of transposons are inactivated with a higher probability. These regions contain a specific protein SUUR affecting the rate of replication completion.