The domesticated transposase ALP2 mediates formation of a novel Polycomb protein complex by direct interaction with MSI1, a core subunit of Polycomb Repressive Complex 2 (PRC2).
ABSTRACT: A large fraction of plant genomes is composed of transposable elements (TE), which provide a potential source of novel genes through "domestication"-the process whereby the proteins encoded by TE diverge in sequence, lose their ability to catalyse transposition and instead acquire novel functions for their hosts. In Arabidopsis, ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN 1 (ALP1) arose by domestication of the nuclease component of Harbinger class TE and acquired a new function as a component of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a histone H3K27me3 methyltransferase involved in regulation of host genes and in some cases TE. It was not clear how ALP1 associated with PRC2, nor what the functional consequence was. Here, we identify ALP2 genetically as a suppressor of Polycomb-group (PcG) mutant phenotypes and show that it arose from the second, DNA binding component of Harbinger transposases. Molecular analysis of PcG compromised backgrounds reveals that ALP genes oppose silencing and H3K27me3 deposition at key PcG target genes. Proteomic analysis reveals that ALP1 and ALP2 are components of a variant PRC2 complex that contains the four core components but lacks plant-specific accessory components such as the H3K27me3 reader LIKE HETEROCHROMATION PROTEIN 1 (LHP1). We show that the N-terminus of ALP2 interacts directly with ALP1, whereas the C-terminus of ALP2 interacts with MULTICOPY SUPPRESSOR OF IRA1 (MSI1), a core component of PRC2. Proteomic analysis reveals that in alp2 mutant backgrounds ALP1 protein no longer associates with PRC2, consistent with a role for ALP2 in recruitment of ALP1. We suggest that the propensity of Harbinger TE to insert in gene-rich regions of the genome, together with the modular two component nature of their transposases, has predisposed them for domestication and incorporation into chromatin modifying complexes.
Project description:A large fraction of plant genomes is composed of transposable elements (TE), which provide a potential source of novel genes through “domestication” – the process whereby the proteins encoded by TE diverge in sequence, lose their ability to catalyse transposition and instead acquire novel functions for their hosts. In Arabidopsis, ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN 1 (ALP1) arose by domestication of the nuclease component of Harbinger class TE and acquired a new function as a component of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a histone H3K27me3 methyltransferase involved in regulation of host genes and in some cases TE. It was not clear how ALP1 associated with PRC2, nor what the functional consequence was. Here, we identify ALP2 genetically as a suppressor of Polycomb-group (PcG) mutant phenotypes and show that it arose from the second, DNA binding component of Harbinger transposases. Molecular analysis of PcG compromised backgrounds reveals that ALP genes oppose silencing and H3K27me3 deposition at key PcG target genes. Proteomic analysis reveals that ALP1 and ALP2 are components of a variant PRC2 complex that contains the four core components but lacks plant-specific accessory components such as the H3K27me3 reader LIKE HETEROCHROMATION PROTEIN 1 (LHP1). We show that the N-terminus of ALP2 interacts directly with ALP1, whereas the C-terminus of ALP2 interacts with MULTICOPY SUPPRESSOR OF IRA1 (MSI1), a core component of PRC2. Proteomic analysis reveals that in alp2 mutant backgrounds ALP1 protein no longer associates with PRC2, consistent with a role for ALP2 in recruitment of ALP1. We suggest that the propensity of Harbinger TE to insert in gene-rich regions of the genome, together with the modular two component nature of their transposases, has predisposed them for domestication and incorporation into chromatin modifying complexes.
Project description:The Polycomb group (PcG) and trithorax group (trxG) genes play crucial roles in development by regulating expression of homeotic and other genes controlling cell fate. Both groups catalyse modifications of chromatin, particularly histone methylation, leading to epigenetic changes that affect gene activity. The trxG antagonizes the function of PcG genes by activating PcG target genes, and consequently trxG mutants suppress PcG mutant phenotypes. We previously identified the ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN1 (ALP1) gene as a genetic suppressor of mutants in the Arabidopsis PcG gene LIKE HETEROCHROMATIN PROTEIN1 (LHP1). Here, we show that ALP1 interacts genetically with several other PcG and trxG components and that it antagonizes PcG silencing. Transcriptional profiling reveals that when PcG activity is compromised numerous target genes are hyper-activated in seedlings and that in most cases this requires ALP1. Furthermore, when PcG activity is present ALP1 is needed for full activation of several floral homeotic genes that are repressed by the PcG. Strikingly, ALP1 does not encode a known chromatin protein but rather a protein related to PIF/Harbinger class transposases. Phylogenetic analysis indicates that ALP1 is broadly conserved in land plants and likely lost transposase activity and acquired a novel function during angiosperm evolution. Consistent with this, immunoprecipitation and mass spectrometry (IP-MS) show that ALP1 associates, in vivo, with core components of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a widely conserved PcG protein complex which functions as a H3K27me3 histone methyltransferase. Furthermore, in reciprocal pulldowns using the histone methyltransferase CURLY LEAF (CLF), we identify not only ALP1 and the core PRC2 components but also plant-specific accessory components including EMBRYONIC FLOWER 1 (EMF1), a transcriptional repressor previously associated with PRC1-like complexes. Taken together our data suggest that ALP1 inhibits PcG silencing by blocking the interaction of the core PRC2 with accessory components that promote its HMTase activity or its role in inhibiting transcription. ALP1 is the first example of a domesticated transposase acquiring a novel function as a PcG component. The antagonistic interaction of a modified transposase with the PcG machinery is novel and may have arisen as a means for the cognate transposon to evade host surveillance or for the host to exploit features of the transposition machinery beneficial for epigenetic regulation of gene activity.
Project description:Polycomb group (PcG) proteins maintain the silenced state of key developmental genes in animals, but how these proteins are recruited to specific regions of the genome is still poorly understood. In Drosophila, PcG proteins are recruited to Polycomb response elements (PREs) that include combinations of sites for sequence specific DNA binding "PcG recruiters," including Pho, Cg, and Spps. To understand their roles in PcG recruitment, we compared Pho-, Cg-, and Spps-binding sites against H3K27me3 and key PcG proteins by ChIP-seq in wild-type and mutant third instar larvae. H3K27me3 in canonical Polycomb domains is decreased after the reduction of any recruiter. Reduction of Spps and Pho, but not Cg, causes the redistribution of H3K27me3 to heterochromatin. Regions with dramatically depleted H3K27me3 after Spps knockout are usually accompanied by decreased Pho binding, suggesting their cooperative binding. PcG recruiters, the PRC2 component E(z), and the PRC1 components Psc and Ph cobind thousands of active genes outside of H3K27me3 domains. This study demonstrates the importance of distinct PcG recruiters for the establishment of unique Polycomb domains. Different PcG recruiters can act both cooperatively and independently at specific PcG target genes, highlighting the complexity and diversity of PcG recruitment mechanisms.
Project description:Most isolates of group B streptococci (GBS) express an alpha-like protein (Alp), Cα (encoded by bca), Alp1 (also called epsilon; alp1), Alp2 (alp2), Alp3 (alp3), Alp4 (alp4), or R4/Rib (rib). These proteins are chimeras with a mosaic structure and with antigenic determinants with variable immunological cross-reactivities between the Alps, including Alp1 and Cα cross-reactivity. This study focused on antigenic domains of Alp1, studied by using rabbit antisera in immunofluorescence, Western blotting, and enzyme-linked immunosorbent assay (ELISA)-based tests and whole cells of GBS or trypsin-extracted and partially purified antigens from the strains A909 (serotype Ia/Cα, Cβ) and 335 (Ia/Alp1). Alp1 and Cα shared an antigenic determinant, Alp1/Cα common, not harbored by other Alps, probably located in the Alp1 and Cα repeat units, as these units are nearly identical in genomic sequence. An antigenic Alp1 determinant was Alp1 specific and was most likely located in the N-terminal unit of Alp1 in which an Alp1-specific primer site for PCR is also located. In addition, Alp1 possessed a domain with low immunogenicity which cross-reacted immunologically with Alp2 and Alp3, with unknown location in Alp1. Alp1 was partially degraded by trypsin during antigen extraction but with the antigenic domains preserved. The results indicate that Cα and Alp1 are immunologically related in the same manner that R4 (Rib) and Alp3 are related. The domain called Alp1 specific should be important in GBS serotyping as a surface-anchored serosubtype marker. The Alp1/Cα common determinant may be of prime interest as an immunogenic domain in a GBS vaccine.
Project description:Polycomb Group (PcG) proteins are epigenetic repressors essential for control of development and cell differentiation. They form multiple complexes of which PRC1 and PRC2 are evolutionary conserved and obligatory for repression. The targeting of PRC1 and PRC2 is poorly understood and was proposed to be hierarchical and involve tri-methylation of histone H3 (H3K27me3) and/or monoubiquitylation of histone H2A (H2AK118ub). Here, we present a strict test of this hypothesis using the Drosophila model. We discover that neither H3K27me3 nor H2AK118ub is required for targeting PRC complexes to Polycomb Response Elements (PREs). We find that PRC1 can bind PREs in the absence of PRC2 but at many PREs PRC2 requires PRC1 to be targeted. We show that one role of H3K27me3 is to allow PcG complexes anchored at PREs to interact with surrounding chromatin. In contrast, the bulk of H2AK118ub is unrelated to PcG repression. These findings radically change our view of how PcG repression is targeted and suggest that PRC1 and PRC2 can communicate independently of histone modifications.
Project description:HIGHLIGHTS The PRC2 interacting protein BLISTER likely acts downstream of PRC2 to silence Polycomb target genes and is a key regulator of specific stress responses in Arabidopsis. Polycomb group (PcG) proteins are key epigenetic regulators of development. The highly conserved Polycomb repressive complex 2 (PRC2) represses thousands of target genes by trimethylating H3K27 (H3K27me3). Plant specific PcG components and functions are largely unknown, however, we previously identified the plant-specific protein BLISTER (BLI) as a PRC2 interactor. BLI regulates PcG target genes and promotes cold stress resistance. To further understand the function of BLI, we analyzed the transcriptional profile of bli-1 mutants. Approximately 40% of the up-regulated genes in bli are PcG target genes, however, bli-1 mutants did not show changes in H3K27me3 levels at all tested genes, indicating that BLI regulates PcG target genes downstream of or in parallel to PRC2. Interestingly, a significant number of BLI regulated H3K27me3 target genes is regulated by the stress hormone absciscic acid (ABA). We further reveal an overrepresentation of genes responding to abiotic stresses such as drought, high salinity, or heat stress among the up-regulated genes in bli mutants. Consistently, bli mutants showed reduced desiccation stress tolerance. We conclude that the PRC2 associated protein BLI is a key regulator of stress-responsive genes in Arabidopsis: it represses ABA-responsive PcG target genes, likely downstream of PRC2, and promotes resistance to several stresses such as cold and drought.
Project description:Proteins encoded by transposable elements (TEs) play a vital role in their proliferation in host genomes. In recent years, an increasing number of studies have shown that TE proteins have contributed to the emergence of novel host proteins. In Drosophila, there are several proteins derived from the PIF/Harbinger transposase superfamily called Drosophila PIF Like Genes (DPLGs). Here, we investigated the function of four DPLGs (DPLG1-4) in D. melanogaster that are highly conserved and present across Drosophila but are deeply diverged from each other, and likely originated through independent domestication of the transposase from distinct PIF-like TE families. We have analyzed transcript localization of DPLG 1-4, tagged DPLG3 and DPLG4 and studied their protein localization and, produced null alleles for DPLG1 and DPLG4. RNA in situ hybridization reveals that DPLG1-4 have strikingly similar pattern of transcript localization in the gonads, and in the nervous system during embryogenesis, suggesting their domestication for related functions. DPLGs show co-expression enrichment with transcription factors in ovaries and during embryogenesis supporting that they may be domesticated as regulatory proteins in flies. DPLG3 and DPLG4 tagged proteins localize with DNA in the nucleus of the ovaries consistent with this hypothesis. Loss of DPLG1 or DPLG4 results in viability and/or fertility effects, while loss of both DPLG1 and DPLG4 resulted in no effect in viability or fertility suggesting genetic interaction between these two genes. Further, loss of DPLG4 showed increased survival in flies, and increased fertility in older females indicating its involvement in the process of ageing. Transcriptional analyses in the ovaries showed that the loss of either DPLG1 or DPLG4 protein leads to modest changes in transcription of a large and significantly overlapping set of genes, and also affects transposable elements activity. The observed functional overlap of independently domesticated transposases from the same TE family and genetic interaction between them suggests a stepping stone model in which domestication of a PIF/Harbinger transposase might promote the domestication of related transposases for gonad and nervous system regulatory functions. Overall design: Comparison of transcripts from ovaries of DPLG1 and DPLG4 mutants flies with control flies. All samples consisted of three biological replicates.
Project description:Polycomb-group (PcG)-mediated transcriptional repression of target genes can be delineated into two phases. First, following initial repression of target genes by gene-specific transcription factors, PcG proteins recognize the repressed state and assume control of the genes' repression. Second, once the silenced state is established, PcG proteins may maintain repression through an indefinite number of cell cycles. Little is understood about how PcG proteins initially recognize the repressed state of target genes and the steps leading to <i>de novo</i> establishment of PcG-mediated repression. We describe a genetic system in which a <i>Drosophila</i> PcG target gene, <i>giant</i> (<i>gt</i>), is ubiquitously repressed during early embryogenesis by a maternally expressed transcription factor, and show the temporal recruitment of components of three PcG protein complexes: PhoRC, PRC1 and PRC2. We show that <i>de novo</i> PcG recruitment follows a temporal hierarchy in which PhoRC stably localizes at the target gene at least 1 h before stable recruitment of PRC2 and concurrent trimethylation of histone H3 at lysine 27 (H3K27me3). The presence of PRC2 and increased levels of H3K27me3 are found to precede stable binding by PRC1.
Project description:Many developmental control genes contain paused RNA polymerase II (Pol II) and are thereby "poised" for rapid and synchronous activation in the early Drosophila embryo. Evidence is presented that Polycomb group (PcG) repressors can influence paused Pol II. ChIP-Seq and GRO-Seq assays were used to determine the genome-wide distributions of Pol II, H3K27me3, and H3K4me3 in extra sex combs (esc) mutant embryos. ESC is a key component of the Polycomb repressive complex 2 (PRC2), which mediates H3K27me3 modification. Enhanced Pol II occupancy is observed for thousands of genes in esc mutant embryos, including genes not directly regulated by PRC2. Thus, it would appear that silent genes lacking promoter-associated paused Pol II in wild-type embryos are converted into "poised" genes with paused Pol II in esc mutants. We suggest that this conversion of silent genes into poised genes might render differentiated cell types susceptible to switches in identity in PcG mutants.
Project description:Polycomb group (PcG) proteins form essential epigenetic memory systems for controlling gene expression during development in plants and animals. However, the mechanism of plant PcG protein functions remains poorly understood. Here, we probed the composition and function of plant Polycomb repressive complex 2 (PRC2). This work established the fact that all known plant PRC2 complexes contain MSI1, a homologue of Drosophila p55. While p55 is not essential for the in vitro enzymatic activity of PRC2, plant MSI1 was required for the functions of the EMBRYONIC FLOWER and the VERNALIZATION PRC2 complexes including trimethylation of histone H3 Lys27 (H3K27) at the target chromatin, as well as gene repression and establishment of competence to flower. We found that MSI1 serves to link PRC2 to LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), a protein that binds H3K27me3 in vitro and in vivo and is required for a functional plant PcG system. The LHP1-MSI1 interaction forms a positive feedback loop to recruit PRC2 to chromatin that carries H3K27me3. Consequently, this can provide a mechanism for the faithful inheritance of local epigenetic information through replication.