SUMOylated SNF2PH promotes variant surface glycoprotein expression in bloodstream trypanosomes
2019; Springer Nature; Volume: 20; Issue: 12 Linguagem: Inglês
10.15252/embr.201948029
ISSN1469-3178
AutoresAndreu Saura, Paula Ana Iribarren, Domingo I. Rojas-Barros, Jean-Mathieu Bart, Diana López‐Farfán, Eduardo Andrés‐León, Isabel Vidal-Cobo, Cordula Boehm, Vanina E. Álvarez, Mark C. Field, Miguel Navarro,
Tópico(s)Parasitic Infections and Diagnostics
ResumoArticle6 November 2019Open Access Transparent process SUMOylated SNF2PH promotes variant surface glycoprotein expression in bloodstream trypanosomes Andreu Saura Andreu Saura Instituto de Parasitología y Biomedicina “López-Neyra”, CSIC (IPBLN-CSIC), Granada, Spain Search for more papers by this author Paula A Iribarren Paula A Iribarren IIB-UNSAM, Buenos Aires, Argentina Search for more papers by this author Domingo Rojas-Barros Domingo Rojas-Barros Instituto de Parasitología y Biomedicina “López-Neyra”, CSIC (IPBLN-CSIC), Granada, Spain Search for more papers by this author Jean M Bart Jean M Bart Instituto de Parasitología y Biomedicina “López-Neyra”, CSIC (IPBLN-CSIC), Granada, Spain Search for more papers by this author Diana López-Farfán Diana López-Farfán Instituto de Parasitología y Biomedicina “López-Neyra”, CSIC (IPBLN-CSIC), Granada, Spain Search for more papers by this author Eduardo Andrés-León Eduardo Andrés-León Instituto de Parasitología y Biomedicina “López-Neyra”, CSIC (IPBLN-CSIC), Granada, Spain Search for more papers by this author Isabel Vidal-Cobo Isabel Vidal-Cobo Instituto de Parasitología y Biomedicina “López-Neyra”, CSIC (IPBLN-CSIC), Granada, Spain Search for more papers by this author Cordula Boehm Cordula Boehm School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Vanina E Alvarez Vanina E Alvarez IIB-UNSAM, Buenos Aires, Argentina Search for more papers by this author Mark C Field Mark C Field School of Life Sciences, University of Dundee, Dundee, UK Biology Centre, Institute of Parasitology, Czech Academy of Sciences, Ceske Budejovice, Czech Republic Search for more papers by this author Miguel Navarro Corresponding Author Miguel Navarro [email protected] orcid.org/0000-0003-2301-2699 Instituto de Parasitología y Biomedicina “López-Neyra”, CSIC (IPBLN-CSIC), Granada, Spain Search for more papers by this author Andreu Saura Andreu Saura Instituto de Parasitología y Biomedicina “López-Neyra”, CSIC (IPBLN-CSIC), Granada, Spain Search for more papers by this author Paula A Iribarren Paula A Iribarren IIB-UNSAM, Buenos Aires, Argentina Search for more papers by this author Domingo Rojas-Barros Domingo Rojas-Barros Instituto de Parasitología y Biomedicina “López-Neyra”, CSIC (IPBLN-CSIC), Granada, Spain Search for more papers by this author Jean M Bart Jean M Bart Instituto de Parasitología y Biomedicina “López-Neyra”, CSIC (IPBLN-CSIC), Granada, Spain Search for more papers by this author Diana López-Farfán Diana López-Farfán Instituto de Parasitología y Biomedicina “López-Neyra”, CSIC (IPBLN-CSIC), Granada, Spain Search for more papers by this author Eduardo Andrés-León Eduardo Andrés-León Instituto de Parasitología y Biomedicina “López-Neyra”, CSIC (IPBLN-CSIC), Granada, Spain Search for more papers by this author Isabel Vidal-Cobo Isabel Vidal-Cobo Instituto de Parasitología y Biomedicina “López-Neyra”, CSIC (IPBLN-CSIC), Granada, Spain Search for more papers by this author Cordula Boehm Cordula Boehm School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Vanina E Alvarez Vanina E Alvarez IIB-UNSAM, Buenos Aires, Argentina Search for more papers by this author Mark C Field Mark C Field School of Life Sciences, University of Dundee, Dundee, UK Biology Centre, Institute of Parasitology, Czech Academy of Sciences, Ceske Budejovice, Czech Republic Search for more papers by this author Miguel Navarro Corresponding Author Miguel Navarro [email protected] orcid.org/0000-0003-2301-2699 Instituto de Parasitología y Biomedicina “López-Neyra”, CSIC (IPBLN-CSIC), Granada, Spain Search for more papers by this author Author Information Andreu Saura1, Paula A Iribarren2, Domingo Rojas-Barros1, Jean M Bart1, Diana López-Farfán1, Eduardo Andrés-León1, Isabel Vidal-Cobo1, Cordula Boehm3, Vanina E Alvarez2, Mark C Field3,4 and Miguel Navarro *,1 1Instituto de Parasitología y Biomedicina “López-Neyra”, CSIC (IPBLN-CSIC), Granada, Spain 2IIB-UNSAM, Buenos Aires, Argentina 3School of Life Sciences, University of Dundee, Dundee, UK 4Biology Centre, Institute of Parasitology, Czech Academy of Sciences, Ceske Budejovice, Czech Republic *Corresponding author. Tel: +34 958181651; Fax: +34 958181633; E-mail: [email protected] EMBO Reports (2019)20:e48029https://doi.org/10.15252/embr.201948029 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract SUMOylation is a post-translational modification that positively regulates monoallelic expression of the trypanosome variant surface glycoprotein (VSG). The presence of a highly SUMOylated focus associated with the nuclear body, where the VSG gene is transcribed, further suggests an important role of SUMOylation in regulating VSG expression. Here, we show that SNF2PH, a SUMOylated plant homeodomain (PH)-transcription factor, is upregulated in the bloodstream form of the parasite and enriched at the active VSG telomere. SUMOylation promotes the recruitment of SNF2PH to the VSG promoter, where it is required to maintain RNA polymerase I and thus to regulate VSG transcript levels. Further, ectopic overexpression of SNF2PH in insect forms, but not of a mutant lacking the PH domain, induces the expression of bloodstream stage-specific surface proteins. These data suggest that SNF2PH SUMOylation positively regulates VSG monoallelic transcription, while the PH domain is required for the expression of bloodstream-specific surface proteins. Thus, SNF2PH functions as a positive activator, linking expression of infective form surface proteins and VSG regulation, thereby acting as a major regulator of pathogenicity. Synopsis The trypanosome homeodomain protein SNF2PH functions as an epigenetic activator of bloodstream stage-specific surface proteins. SUMOylated SNF2PH positively regulates monoallelic variant surface glycoprotein expression. SNF2PH contains a chromatin remodeling (SNF_N) domain and a plant homeodomain (PH). SUMOylated SNF2PH is enriched at the active VSG-ES promoter, and is required for RNA Pol I recruitment. SNF2PH is also detected at silent VSG gene promoters, but to a lesser extent. The plant homeodomain is required to direct SNF2PH to bloodstream stage-specific surface protein genes. Introduction Antigenic variation, the major mechanism by which African trypanosomes evade the host immune response, is mediated by switching expression between immunologically distinct variant surface glycoprotein (VSG) genes 1. The active VSG gene is transcribed polycistronically by RNA polymerase I, together with several expression site-associated genes (ESAGs), from a large telomeric locus (40–60 kb), known as a VSG expression site (VSG-ES), currently named bloodstream ES (BESs) 2. In the mammalian bloodstream form (BF), where antigenic variation occurs, only one of ~15 VSG-ES genes is transcribed at a given time, resulting in monoallelic expression and a dense surface coat comprised of a single VSG type 3-5. The active VSG-ES is recruited to a nuclear compartment, the expression site body (ESB), which facilitates monoallelic transcription 6-8. Interestingly, small ubiquitin-like modifier (SUMO) post-transcriptionally modified proteins are associated with the ESB within a highly SUMOylated focus (HSF) 9. However, in the insect or procyclic form, VSGs are not expressed and procyclin glycoproteins cover the parasite surface 10. SUMOylation is a large and reversible post-translational modification (PTM) that regulates many critical processes, including transcription, protein–protein interactions, protein stability, nuclear localization, DNA repair, and signaling 11. In Trypanosoma brucei, there is a single SUMO ortholog, which is essential for cell cycle progression of the procyclic form 12. Proteomic analyses of SUMO substrates in this life stage identified 45 proteins involved in multiple cellular processes, including epigenetic regulation of gene expression 13. Transcription factors are well known SUMO targets, whose activity can be modulated in both gene silencing and activation 14. In T. brucei BF, SUMO-conjugated proteins were detected highly enriched in the nucleus in a single focus (HSF) associated with the ES body (ESB) and in the active VSG-ES chromatin, suggesting chromatin SUMOylation acts as a positive epigenetic mark to regulate VSG expression 9. Chromatin SUMOylation to the active VSG-ES locus is required for efficient recruitment of RNA polymerase I in a SUMO E3 ligase (TbSIZ1/PIAS)-dependent manner, suggesting protein SUMOylation facilitates the accessibility of additional transcription factors 9. Thus, we sought to identify major SUMO-conjugated proteins in the mammalian infective form and found a novel protein annotated as a transcription activator in the database (Tb927.3.2140). Structural conserved domain predictions suggest that Tb927.3.2140 is a member of the Snf2 (Sucrose Nonfermenting Protein 2) SF2 helicase-like superfamily 2 of chromatin remodelers 15-17 and also contains a plant homeodomain (PHD). Thus, we designate the protein SNF2PH. Here, we show that SNF2PH is a developmentally regulated protein enriched at chromatin of the VSG-ES (BES) telomere, particularly at promoter regions when modified by SUMO. SNF2PH depletion leads to reduced VSG transcription and upregulation of developmental markers for the insect stage. ChIP-seq data suggest SNF2PH binds to selective regions in chromatin, in addition to the active VSG-ES, like developmentally regulated loci, rDNA, SL-RNA, and, interestingly, also to clusters of tRNA genes, which function as insulators for repressed and active chromatin domains in other eukaryotes. SNF2PH is strongly downregulated in quiescent (pre-adapted to host transition) trypanosomes generated in both pleomorphic (differentiation-competent) and monomorphic (by AMPKα1-activation) strains. Further, SNF2PH expression is negatively regulated in the insect procyclic form. Most importantly, overexpression of SNF2PH in the insect form triggers the expression of bloodstream stage-specific surface protein genes, suggesting a role as positive regulator of differentiation. Thus, SNF2PH links immune evasion with pathogenicity. Results Trypanosome SNF2PH is SNF2_N-related protein that contains an unusual plant homeodomain SUMOylation is a hallmark of epigenetic VSG regulation at the level of chromatin and nuclear architecture 9. The highly SUMOylated focus (HSF) detected by a specific mAb against TbSUMO in the nucleus of bloodstream form (BF) trypanosomes was recently associated with the nuclear body ESB 9, the site for VSG-ES monoallelic expression 6. Recognition of HSF together with the detection of highly SUMOylated proteins at the active VSG-ES chromatin by ChIP analysis suggests that a number of SUMOylated proteins are mechanistically involved in regulation of VSG expression 9. Therefore, identifying these proteins is a novel approach for the discovery of factors involved in VSG regulation. To identify abundant SUMOylated proteins, we performed a non-exhaustive proteomic analysis utilizing BF protein extracts from a cell line expressing an 8His-HA-tagged SUMO (see Materials and Methods). LC-MS/MS analyses of His-HA-affinity-purified extracts robustly identified several proteins (see Appendix Table S1). Particularly, interesting was Tb927.3.2140 (length 948 aa), a protein annotated in the TriTrypDB database as a transcription activator, which contains a conserved SNF2 family N-terminal domain. Comparative analyses of Tb927.3.2140 at CDART 18 and the NCBI CDD domain database identified three conserved domains: DEXHc_Snf, e-value 9.4e−74, SF2_C_SNF, e-value 8.0e−50, PHD5_NSD, e-value 6.2e−14. Structural CD predictions suggest than Tb927.3.2140 is a member of the Snf2 family (Sucrose Nonfermenting Protein 2) from the SF2 helicase-like superfamily 2 of chromatin remodelers 15-17, which regulate DNA accessibility to facilitate central cellular processes as transcription, DNA repair, DNA replication and cell differentiation 15, 16. Next, searching for Tb927.3.2140 homologues using DELTA-BLAST against UniProtKB/SwissProt database, identified a protein member of the SWI/SNF family, SMARCA1 (e-value, 4e−157) (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 1) also known as the global transcription activator SNF2L1 (length, 1054 aa) (homonyms SWI; ISWI; SWI2; SNF2L; SNF2L1; SNF2LB; SNF2LT; hSNF2L; NURF140) all described to be involved in transcription for either gene activation or gene repression 16. In addition to the SNF2 N domain, a conserved helicase C-terminal domain was also detected, known to function as a chaperon-like in the assembly of protein complexes. Interestingly, Tb927.3.2140 also contains a plant homeodomain (PHD) that is absent from other known trypanosome chromatin remodelers. The PHD is a conserved homeodomain involved in development 19 that binds H3 tails and reads unmodified H3 tails 20 as well as H3 trimethylated at Lys4 (H3K4me3) 21 or acetylated at Lys8 and Lys14 22, 23. The PH domain is conserved in histone methyltransferases, including murine HMT3 and human NSD3 (Appendix Fig S1). Thus, we named this chromatin-remodeling factor trypanosome SNF2PH. SNF2PH is developmentally regulated and associated with the ESB nuclear body In order to investigate SNF2PH protein expression, we raised a monoclonal antibody (mAb) (11C10E4) against the recombinant protein expressed in bacteria. Western blot of total protein extracts and immunofluorescence (IF) showed that SNF2PH protein levels are developmentally regulated, with higher expression in the bloodstream compared to the insect form (Fig 1A and Appendix Fig S2). The specificity of mAb11C10E4 was demonstrated as SNF2PH levels in whole cell extracts were markedly reduced in RNAi cells (Fig 1B). Figure 1. SNF2PH is a developmentally regulated protein associated with the expression site body (ESB) and highly SUMOylated focus (HSF) SNF2PH is differentially expressed in T. brucei developmental stages. The mammalian infective form, bloodstream form (BF), and the insect form, procyclic form (PF). Knockdown of SNF2PH by inducible RNA interference in bloodstream form leads to protein depletion after 24 h. (5 × 106 cells/lane): parental, uninduced (dox−), and induced (dox+). Total cell extracts were analyzed by Western blotting using the anti-SNF2PH mAb. SNF2PH is diffusely distributed in the nucleoplasm with certain enrichment in the nucleolus. Panels show DAPI and green channels after IF with the anti-SNF2PH mAb (11C10E4). Scale bar, 1 μm. SNF2PH associates with the active VSG-ES. A cell line with a GFP-LacI tag in the active VSG-ES 6 was subjected to double 3D-IF using anti-SNF2PH mAb (red), anti-GFP rabbit antiserum (green) and DAPI staining. Maximum intensity projections of deconvolved slices containing the GFP signal are shown (arrowhead). (D’) Inset shows a higher magnification of the nucleus including anti-SNFPH and anti-GFP fluorescence signals colocalization mask (white). Scale bar, 1 μm. Colocalization analysis of SNF2PH with the Highly SUMOylated focus (HSF). SNF2PH associates with the HSF (arrowhead) in bloodstream form nucleus. Indirect 3D-IF analyses were carried out using the rabbit anti-SNF2PH antiserum (red) and the anti-TbSUMO mAb 1C9H8 (green) 9. Scale bar, 1 μm. CSNF2PH partially colocalizes with YFP-tagged TbRPB5z in the ESB. A cell line expressing an N-terminal fusion of a Yellow Fluorescent Protein (YFG) with the RNA pol I-specific subunit RPB5z described previously 24 was used to analyze by double 3D-IF a possible association of SNF2PH with the ESB. The 3D-IF was performed using the anti-SNF2PH mAb (red) and rabbit anti-GFP antiserum (green) that recognizes the Yellow GFP variant. Deconvolved slices containing both SNF2PH and the extranucleolar ESB (arrowhead) are represented as maximum intensity projections. (F’) Inset shows a higher magnification of the nucleus including anti-SNF2PH and anti-GFP fluorescence signals colocalization mask (white). Scale bar, 1 μm. Download figure Download PowerPoint Subcellular localization of SNF2PH by 3D-deconvolution IF (3D-IF) microscopy with mAb11C10E4 showed a nuclear localization, with disperse distribution in the nucleoplasm, including puncta and enrichment at the nucleolar periphery (Fig 1C). To determine whether SNF2PH associates with the active VSG-ES locus, we used a GFP-lacI targeted VSG-promoter cell line 6. We detected 38.8% (n = 55) colocalization (Pearson's correlation coefficient) with GFP-tagged active VSG-ES using an anti-GFP rabbit antiserum and mAb11C10E4 anti-SNF2PH (Fig 1D). Statistical analysis showed even lower association in 2K1N cells (S-G2) (Fig EV1A), suggesting that SNF2PH association with the active VSG-ES locus occurs in a cell cycle-dependent manner. Click here to expand this figure. Figure EV1. SNF2PH statistical analysis of colocalization with the active VSG-ES, the ESB and the HSF Histogram representing the proportion of cells that colocalize with the GFP-tagged active VSG-ES (BES1) the VSG221-ES was 38.17% (32.72% in 1K1N cells, 5.45% of 2K1N) (n = 55). Colocalization statistical analysis of the SNF2PH with respect to the HSF. Histogram of statistical analysis shows a colocalization of SNF2PH with the HSF in 32.83% of 1K1N cells and 20.9% of 2K1N cells (n = 67). Statistical analysis of ESB (YFP:TbRPB5z) and SNF2PH colocalization. Histogram of shows colocalization in 60.47% of analyzed cells (n = 43). In addition, percentage colocalization determined in each cell cycle phase is shown (37.21% (1K1N) + 16.27% (2K1N)). Download figure Download PowerPoint To investigate the association with the HSF, we stained cells with anti-TbSUMO mAb 9 and anti-SNF2PH antiserum (Materials and Methods). 3D-IF showed colocalization between SNF2PH and HSF in 53.7% of the cells (n = 67)) (Figs 1E and EV1B), likely due to the highly dynamic nature of protein SUMOylation. Similar colocalization in 53.49% of the cells was observed between SNF2PH and the ESB (Figs 1F and EV1C), indicated by extranucleolar pol I signal visualized with a YFP-tagged RPB5z (specific RNA pol I subunit 5z 24) (Figs 1F and EV1C). SNF2PH is SUMO conjugated To investigate whether SNF2PH is a bona fide SUMO-modified protein, we carried out immunoprecipitation (IP) utilizing the anti-TbSUMO mAb 9 and anti-SNF2PH antiserum under denaturing conditions to capture only proteins with covalent SUMO modifications. IP suggested that SNF2PH is SUMOylated when analyzed by Western blotting using the anti-TbSUMO mAb on a SNF2PH immunoprecipitate (Fig 2A). The reciprocal experiment, using anti-SNF2PH antiserum on a TbSUMO immunoprecipitate reproducibly detected SNF2PH conjugated to TbSUMO (Fig 2B). Figure 2. SNF2PH is SUMOylated in vivo in trypanosomes and in vitro using a SUMOylation heterologous system Immunoprecipitation (IP) of bloodstream SUMOylated proteins revealed that SNF2PH is SUMOylated. A nuclear fraction was lysed in urea-containing buffer, and proteins were immunoprecipitated with rabbit anti-SNF2PH antiserum or unspecific antiserum (prebleed) and probed with anti-TbSUMO mAb 1C9H8 (arrow). As a control, IP samples were reprobed with SNF2PH antiserum (below). A reciprocal IP experiment was performed using anti-TbSUMO mAb and probed with SNF2PH antiserum. As a control, the blot was reprobed with anti-TbSUMO mAb (lower panel). Inp: Input, IP (0.5%). Anti-Flag Western blot analysis of SNF2PHN performed on soluble cell extracts from induced cultures of E. coli transformed with pET28-SNF2PHN-3xFlag alone (lane 1), or in the background of an incomplete (lane 2, pACYCDuet-1-TbE1a-TbE1b; lane 3, pCDFDuet-1-TbSUMO-TbE2) or a complete (lane 4, pCDFDuet-1-TbSUMO-TbE2 plus pACYCDuet-1-TbE1a-TbE1b) SUMOylation system. Similar samples as described in (C) were analyzed for SNF2PHC. Cell lysates of E. coli heterologously expressing SNF2PH and the complete T. brucei SUMOylation system were incubated at 28°C in the absence (−) or presence (+) of recombinant TbSENP. The deconjugation activity of TbSENP was specifically inhibited by the addition of 20 mM NEM. Reaction mixtures were analyzed by Western blot using anti-Flag monoclonal antibodies. Western blot analysis of SUMOylated SNF2PHN pattern performed on soluble cell extracts from a complete bacterial SUMOylation system using a wild type version of SUMO (lane 1) or a Lys-deficient version of SUMO (TbSUMO K9R) unable to form chains (lane 2). Download figure Download PowerPoint While IP demonstrates that SNF2PH is SUMOylated, it is unknown whether nuclear conjugation with SUMO is associated with dispersed nuclear foci or localization to a specific subnuclear site. We performed Proximity Ligation assays (PLA) (O-link Bioscience), an IF method where a signal is produced only if two proteins, or a protein and its PTM, are within 40 nm. After a first IF experiment using anti-SUMO mAb and the SNF2PH antiserum, secondary species-specific antibodies conjugated with oligonucleotides are hybridized to the two PLA probes to produce a DNA by rolling circle replication. As the PLA assay detected positive amplification this suggests that SNF2PH is SUMOylated in situ in both the nucleolus and nuclear periphery in one (84.12% ± 0.25%) or two puncta (15.88% ± 0.25%) (Appendix Fig S3). The low signal of SNF2PH antibody in TbSUMO IP experiments is likely a consequence of the dynamic nature of SUMOylation, yielding a small population of SUMOylated SNF2PH form at any given time; similar behavior has been demonstrated for TbRPAI (RNA Polymerase I largest subunit) 9 and additional SUMO proteins in T. brucei 25. To determine which domains of SNF2PH are SUMOylated, we used an E. coli strain expressing the complete T. brucei SUMOylation system 13. We evaluated two different constructs encompassing the SNF2PH N-terminal or C-terminal domain (SNF2PH-N and SNF2PH-C, respectively), bearing a Flag tag. We co-expressed SNF2PH-N and SNF2PH-C in E. coli with TbSUMO (already exposing the diGly motif) and both activating enzyme subunits (TbE1a/TbE1b) plus the conjugating enzyme (TbE2). SNF2PHN appears as a single band migrating at the expected position when expressed alone in E. coli (Fig 2C, lane 1) or when co-expressed with a partially reconstituted system (lanes 2 and 3). However, when co-expressed with the complete SUMOylation system, additional slower-migrating bands can be detected (lane 4), suggesting that the N-terminal domain of SNF2PH can be SUMO conjugated. In contrast, the C-terminal domain is not a target of SUMOylation since it is only detected as a single protein band at the expected position of the unmodified protein (Fig 2D). To confirm heterologous SUMOylation of SNF2PHN, we performed in vitro deconjugation reactions using the specific T. brucei SUMO isopeptidase TbSENP. As shown in Fig 2E, the additional slowly migrating bands observed when SNF2PHN was co-expressed with the T. brucei SUMOylation bacterial system (lane 1) completely disappear upon treatment of cell lysates with TbSENP (lane 2), and the deconjugation ability of TbSENP was specifically inhibited by addition of 20 mM NEM (lane 3). To investigate the nature of SUMOylation of SNF2PHN, we compared the patterns obtained in the bacterial system when replacing wild-type SUMO with a variant unable to form SUMO chains (Fig 2F). In the latter case, a doublet near the 55 kDa marker can be detected, suggesting that there are at least two major sites for SUMOylation in the SNF2PH N-terminus. SNF2PH is highly enriched at active VSG-ES promoter chromatin To investigate SNF2PH occupancy at VSG-ES loci, we performed chromatin IP (ChIP) using anti-SNF2PH antiserum in a promoter-tagged cell line. To overcome the problem of highly homologous sequences at the promoter region among the 15 telomeric VSG-ESs, we developed a tagged cell line (Dual-reporter Renilla Active Luciferase Inactive or DRALI) (loci of interest schematic in Fig 3A). The reporter genes in the DRALI cell line allowed us to determine SNF2PH occupancy at the region downstream of the promoter in either active or inactive VSG-ESs. First, we analyzed occupancy of SNF2PH by ChIP and quantitative PCR (qPCR), which detect significant SNF2PH enrichment at the RLuc gene downstream of the active VSG-ES promoter (P < 0.001) as well as at the active VSG221 gene located in the telomere of BES1 (P < 0.01). However, FLuc located downstream of an inactive VSG-ES promoter (Fig 3A) was not significantly detected (Fig 3B). SNF2PH enrichment was also not detected at VSG genes known to be located at silent telomeric ES position in this strain 2, such as VSG121 (VSG in BES3), VSGVO2 (BES2) and VSGJS (BES13) (Fig 3B). Altogether, the active VSG221 gene immunoprecipitated more efficiently than all inactive VSG telomeric loci analyzed, suggesting SNF2PH associates preferentially with the active ES telomere. Additionally, SNF2PH was detected at other RNA pol I-transcribed loci, including rDNA and EP procyclin promoters. Occupancy of SNF2PH at the two promoters of the surface glycoprotein genes characteristic of mammalian and insect forms (VSG-ES and EP) implicates SNF2PH in regulation of developmental gene expression. Enrichment was also detected for the splice leader (SL) promoter (P < 0.05) and coding regions. However, SNF2PH was most enriched at the active VSG-ES chromatin compared with EP and rDNA promoters. Figure 3. SNF2PH is highly enriched upstream of the active VSG-ES chromatin while is detected to a lesser extent in silent promoters Schematic representation of loci of interest in DRALI, the dual-reporter cell line (not to scale). Two reporters were inserted, Renilla luciferase gene (RLuc) downstream of the Active VSG221-ES (BES1) promoter and firefly Luciferase gene (FLuc) downstream of an Inactive VSG-ES (DRALI). Few other inactive VSGs known to be telomeric in this strain are also represented (VSG121 (BES3), VSGJS1 (BES13), and VSGVO2 (BES2)). Schematic representations for other chromosomal loci (ribosomal DNA and procyclin locus) are shown. Color code: gray (reporters), green (active VSG-ES), red (Inactive VSG-ESs), blue (procyclin locus). Arrow (promoters). Chromatin at the active VSG-ES is enriched for SNF2PH. Chromatin immunoprecipitation (ChIP) analysis by quantitative PCR of reporter sequences inserted downstream of the VSG-ES promoters indicates SNF2PH is highly enriched at the active VSG-ES (RLuc) (BES1) compared to an inactive VSG-ES promoter (FLuc) (***P < 0.001). SNF2PH enrichment on the active telomeric VSG221 (BES1) compared to inactive VSGs (VSG121 (BES3), VSGJS1 (BES13), and VSGVO2 (BES2)) was also significant (*P < 0.05–**P < 0.01). SNF2PH occupancy was detected at the splice leader promoter (SL promoter, pol II-transcribed) and EP procyclin (P < 0.05), as well as the rDNA promoter (P < 0.01) (Student's t-test) *P < 0.05 **P < 0.01, ***P < 0.001). ChIP analyses are shown as the average of at least three independent experiments with standard error of the mean (SEM). Data are represented as percent of input immunoprecipitated (% input). Distribution of SNF2PH across the genome. ChIP-seq analysis using the SNF2PH antiserum and T. b. brucei 427 genomic library (v4) excluding the telomeres. Histogram illustrates peak enrichment of representative genes expressed as log10 fold enrichment (FE). This global analysis confirmed previous ChIP data locating SNF2PH on developmentally regulated genes (EP and GPEET), RNA pol I driven rDNA (ribRNAs) and the SL cluster of small RNAs that are trans-spliced in every mRNA. Interestingly, beside those essential genes for cell growth, SNF2PH occupies few other coding genes; noteworthy is H3V protein recently linked to the regulation of VSG monoallelic expression 28. In addition, SNF2PH was consistently enriched at tRNA gene clusters in 7 chromosomes. Due to highly homologous sequences among ESAGs, all ES-related sequences as ESAGs genes, VSG basic copies located in chromosomal internal positions were excluded of this graph since we cannot rule out whether the ChIP-seq reads came from the active VSG-ES (all sequences are included in Dataset EV1). Schema of VSG-promoter region indicating the location of ESPM PCR fragments amplified (upper panel). Detailed schema of the promoter region showing both upstream and downstream (dw) BES from the tandem repeated promoters ESPM 1 and 4 (lower panel). Chromatin at the core promoter of the active VSG-ES is highly enriched in SNF2PH. ChIP-seq data using SNF2PH antiserum reveal a higher number of reads corresponding to the sequence polymorphism of the BES1 at the PCR fragment 1, (ESPM1) mapping at the VSG-ES promoter (Fig 3D) described before in 9. dw, downstream promoter. Download figure Download PowerPoint In eukaryotes, chromatin remodelers are detected at RNA pol II promoters and play important roles in their activity 26, 27. We investigated the presence of SNF2PH in chromatin across the genome, aside from the multiallelic VSG-ESs, to identify additional genes targeted by this protein. We compared quantitative enrichment profiles with ChIP-seq peak distribution and considered 0-mismatch error to avoid bias in polymorphic sequences within repetitive chromosomal loci, leading to defined peaks (q value < 0.05, Dataset EV1; Fig 3C). As demonstrated by quantitative ChIP, the site of enrichment corresponded to developmentally regulated loci EP and GPEET2 procyclin, and 18S ribosomal DNA and SL-RNA-related sequences. Interestingly, SNF2PH localizes at H3.V, a histone variant recently associated with VSG monoallelic expression
Referência(s)