Defining the chromatin-associated protein landscapes on Trypanosoma brucei repetitive elements using synthetic TALE proteins
- Centre for Cell Biology and Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, United Kingdom
- Institute of Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, United Kingdom
- Institute of Biotechnology, Technische Universität, Germany
Figures
Figure 1 with 3 supplements
T. brucei repetitive elements, TALE design, and target site number.
(A) Distinct repetitive elements are present at various locations on T. brucei chromosomes. (B) Construct designed to express the indicated TALE proteins that bind 15 bp target sequences fused to 3xTy1 and YFP tags when integrated at the β-tubulin locus. The Aldolase 5’UTR and PAD1 3’UTR regulate expression levels. A Bleomycin resistance marker gene provides Phleomycin selection (not shown). (C) Predicted number of target sequences for each TALE in the Lister 427 genome.
Figure 1—figure supplement 1
TALE-YFP construction and sequencing reveals rearranged TALE domain in TelR-TALE-YFP following integration in the T. brucei genome.
(A) Tetramer and trimer modules used to build plasmids designed to express each of six TALE-YFP when integrated in the T. brucei genome. (B) Each of the complete TALE plasmid constructs is expected to contain four modules comprising a complete TALE domain of the same size in all. (C) Sequencing of five assembled TALE-YFP constructs revealed that they had the expected layout following integration at the β-tubulin locus in T. brucei. (D) PCR of genomic DNA extracted from 427 control cells and 427 cells with the TelR-TALE, 177R-TALE, 70R-TALE, ingiR-TALE, or NonR-TALE constructs integrated at the β-tubulin locus confirms correct size (2 kb) (Figure 1—figure supplement 1—source data 1). PCR product for TelR-TALE is shorter than expected (1.5 kb). Position of left (LP) and right (RP) primer pair, common to all TALE-YFP constructs, is indicated. (E) Following integration at the β-tubulin locus, sequencing revealed that the TALE DNA binding domain of TelR-TALE had rearranged, explaining the shorter TelR-TALE protein made by T. brucei 427 TelR-TALE expressing cells.
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Figure 1—figure supplement 1—source data 1
Original DNA-stained agarose gel for Figure 1—figure supplement 1D indicating the relevant PCR bands from T. brucei cells containing the indicated synthetic TALE-YFP protein expression constructs.
- https://cdn.elifesciences.org/articles/109950/elife-109950-fig1-figsupp1-data1-v1.zip
Figure 1—figure supplement 2
Synthetic TALE proteins are expressed in Lister 427 T. brucei bloodstream-form cells, but the TelR-TALE protein is shorter than expected.
Protein extracted from 427 control cells and 427 cells with constructs designed to express 177R-TALE, 70R-TALE, 147R-TALE, ingiR-TALE, NonR-TALE, and TelR-TALE fused to Ty and YFP tags integrated at the β-tubulin locus was subject to western analysis using either: (A) monoclonal mouse anti-GFP (anti-YFP, Figure 1—figure supplement 2—source data 1 and 2) or (B) anti-BB2 (anti-Ty, Figure 1—figure supplement 2—source data 3). The TelR-TALE protein is smaller than the 177R-TALE, 70R-TALE, 147R-TALE, ingiR-TALE, and NonR-TALE proteins.
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Figure 1—figure supplement 2—source data 1
Original anti-YFP western for Figure 1—figure supplement 2A (left), indicating the relevant bands in T. brucei cells expressing the indicated synthetic TALE-YFP proteins.
- https://cdn.elifesciences.org/articles/109950/elife-109950-fig1-figsupp2-data1-v1.zip
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Figure 1—figure supplement 2—source data 2
Original anti-YFP western for Figure 1—figure supplement 2A (left), indicating the relevant bands in T. brucei cells expressing the indicated synthetic TALE-YFP proteins.
- https://cdn.elifesciences.org/articles/109950/elife-109950-fig1-figsupp2-data2-v1.zip
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Figure 1—figure supplement 2—source data 3
Original anti-Ty western for Figure 1—figure supplement 2B, indicating the relevant bands in T. brucei cells expressing the indicated synthetic TALE-YFP proteins.
- https://cdn.elifesciences.org/articles/109950/elife-109950-fig1-figsupp2-data3-v1.zip
Figure 1—figure supplement 3
Growth assays of cells expressing TelR-TALE-GFP, 177R-TALE-GFP, or ingi-TALE-GFP, and their cellular localisation.
(A) Indicated cell cultures were seeded, cell number monitored and diluted every 2 days. (B) DAPI and anti-GFP staining of fixed cells expressing indicated synthetic TALE-GFP fusion proteins. Bar = 10 μm.
Figure 2 with 1 supplement
Localisation and specific target sequence association of five synthetic TALE-YFP fusion proteins expressed in T. brucei compared to YFP-TRF and YFP-KKT.
(A) Bloodstream-form Lister 427 T. brucei cells expressing the indicated TALE-YFP fusion proteins fixed and TALE-YFP protein localisation detected with anti-GFP primary antibody and Alexa Fluor 568-labelled secondary antibody (red). Nuclear and kinetoplast (mitochondrial) DNA were stained with DAPI (green). Control cells expressing telomeric YFP-TRF, centromeric YFP-KKT2 kinetochore protein, or wild-type Lister 427 cells expressing no YFP are also shown. Scale bar, 10 μm. (B) Anti-GFP ChIP-seq analysis for 147R-TALE, 177R-TALE, 70R-TALE, TelR-TALE, and ingiR-TALE, demonstrating that each protein is enriched on the repeat elements they were designed to recognise: CIR147 repeats, 177 bp repeats, 70 bp repeats, telomeric (TTAGGG)n repeats and ingi retrotransposons. Enrichments obtained for the YFP-KKT2 kinetochore protein, the TRF telomere repeat binding protein, and with a No-Tag control are shown for comparison. Data are from two biological replicates.
Figure 2—figure supplement 1
Fields of T. brucei cells showing the cellular localisation of six expressed synthetic TALE-YFP fusion proteins compared to YFP-TRF and YFP-KKT.
Bloodstream-form Lister 427 T. brucei cells expressing the indicated TALE-YFP fusion proteins fixed, and TALE-YFP protein localisation detected with anti-GFP primary antibody and Alexa Fluor 568-labelled secondary antibody (red). Nuclear and kinetoplastid (mitochondrial) DNA were stained with DAPI (green). Control cells expressing telomeric YFP-TRF, centromeric YFP-KKT2 kinetochore protein, or wild-type Lister 427 cells expressing no YFP are also shown. Scale bar, 10 μm except the NonR-TALE-TFP field was captured at a quarter the size of the others.
Figure 3 with 1 supplement
TelR-TALE-YFP and 70R-TALE-YFP are enriched at or near telomeric T. brucei bloodstream expression sites.
(A) Telomeric repeat (TTAGGG)n sequence (top) and 70 bp repeat consensus sequence (bottom). Sequences that TelR-TALE and 70R-TALE were designed to bind are indicated. Deletion of TelR-TALE recognition modules following integration in T. brucei results in recognition of AGGGTTAG rather than the full 15 bp target sequence. (B) Anti-GFP ChIP-seq for cells expressing TelR-TALE-YFP, YFP-TRF, or 70R-TALE–YFP proteins, or 427 cells expressing no YFP-tagged protein. Anti-GFP ChIP-seq enrichment profiles are shown for telomeric bloodstream expression sites (BES) BES1 (top) and BES5 (bottom). Diagrams show the position of telomeric (TTAGGG)n repeats (black chevrons), VSG genes (blue), and upstream 70 bp repeats (green bars). Data are from two biological replicates. Y axis: log2 values, X axis: base pairs.
Figure 3—figure supplement 1
IngiR-TALE is enriched at matching binding sites located in retrotransposons.
(A) ‘Ingi’ repeat consensus sequence conserved in Ingi, RIME, SIDER, and DIRE elements. The sequence that ingiR-TALE was designed to bind is indicated. (B) Cross-hatched rectangle indicates the conserved region in Ingi, RIME, SIDER, and DIRE retrotransposons, which are predicted to provide approximately 295, 187, 101, and 21 binding sites for the ingiR-TALE, respectively. (C) Analysis of ChIP-seq data for cells expressing ingi-TALE shows enrichment of DNA residing at or near predicted ingiR-TALE binding sites.
Figure 4
The 147R-TALE-YFP protein is enriched at a subset of centromeres containing canonical CIR147 repeats.
(A) CIR147 repeat consensus sequence. Sequence that 147R-TALE-YFP was designed to bind is indicated. (B) Comparison of sequences enriched in YFP-KKT2 (purple) and 147R-TALE-YFP (blue) anti-GFP ChIP-seq for chromosomes 1, 3, 4, 5, and 8. DNA from all centromeres is enriched in YFP-KKT2 anti-GFP ChIP-seq, whereas only CIR147 repeats at centromeres on chromosomes 4, 5, and 8 are enriched in 147R-TALE-YFP anti-GFP ChIP-seq. (C) Split-Violin plot demonstrating the relative enrichment of YFP-KKT2 (purple) and 147R-TALE-YFP (blue) over the 11 main chromosome centromere regions. Data are from two biological replicates. Y axis: log2 values.
Figure 5
The 177R-TALE-YFP is enriched over 177 bp repeats located on intermediate-sized and mini-chromosomes.
(A) 177 repeat consensus sequence. Sequence that 177R-TALE-YFP was designed to bind is indicated. (B) Distribution of 177R-TALE-YFP, TelR-TALE-YFP, YFP-TRF, and 70R-TALE-YFP, at two intermediate/mini-chromosome telomeres determined by anti-GFP ChIP-seq. Anti-GFP ChIP-seq of 427 cells expressing no tagged protein is included as control. Diagrams below ChIP-seq profiles indicate the positions of 177 bp repeats (red chevrons), 70 bp repeats (green bars), VSG encoding genes (blue), and telomere (TTAGGG)n repeats (black chevrons) within Tb427VSG-671_unitig_Tb427v12:17,836–31,606 (31kb) and Tb427VSG-647_unitig_Tb427v12 (10 kb). Data are from two biological replicates. Y axis: log2 values, X axis: base pairs.
Figure 6 with 5 supplements
Affinity selection of TelR-TALE-YFP enriches for telomere-associated proteins and 177R-TALE-YFP protein enriches for kinetochore proteins.
Affinity selection was performed on control cells expressing YFP-TRF (A), YFP-RPA2 (C), YFP-KKT2 (E), or No-YFP-tagged protein, and cells expressing synthetic TelR-TALE-YFP (B), 70R-TALE-YFP (D), 177R-TALE-YFP (F). Enriched proteins were identified and quantified by LC-MS/MS analysis relative to the No-YFP tag control. The data for each plot is derived from three biological replicates. Cut-offs used for significance: p<0.05 (Student’s t-test). Enrichment scores for proteins identified in each affinity selection are presented in Supplementary file 1.
Figure 6—figure supplement 1
Overlap of proteins enriched in affinity purifications of both synthetic protein telomere binding protein TelR-TALE-YFP and YFP-TRF.
(A) The Telomere Repeat binding Factor (TRF) binds (TTAGGG)n repeats at the ends of T. brucei chromosomes. A Venn diagram is shown, comparing number of proteins enriched in YFP-TRF versus TelR-TALE-YFP affinity purifications. (B) List of known telomere-associated proteins and other proteins detected in YFP-TRF and/or TelR-TALE-YFP affinity purifications. + detected, – not detected, (–) weakly detected. Lists of all proteins detected in YFP-TRF and TelR-TALE-YFP affinity purifications are available in Supplementary file 1a and b, respectively. *See Reis et al., 2018; Leal et al., 2020; Weisert et al., 2024.
Figure 6—figure supplement 2
A control TALE that binds no specific T. brucei sequence validates proteins enriched in TelR-TALE, 70R-TALE, and 177R-TALE affinity purifications.
A control NonR-TALE was designed to bind the sequence GGAAGTATACCTGGC that is not present in the T. brucei 427 genome. Affinity selection was performed on cells expressing the synthetic NonR-TALE-YFP, TelR-TALE, 70R-TALE-YFP, 177R-TALE-YFP, 147R-TALE-YFP, or ingiR-TALE proteins and control cells expressing no-YFP-tagged protein. Proteins enriched with the five repeat sequence targeted TALE-YFP proteins were identified and quantified by LC-MS/MS analysis relative to the NonR-TALE-YFP control rather than the No-YFP tag control. The data for each plot is derived from three biological replicates. Cut-offs used for significance: log2(tagged/untagged) p<0.05 (Student’s t-test). Enrichment scores for proteins identified in each affinity selection are presented in Supplementary file 1.
Figure 6—figure supplement 3
Affinity selection of TelR-TALE-YFP, 70R-TALE-YFP 177R-TALE-YFP relative to ingiR-TALE-YFP validates specificity.
Affinity selection was performed on cells expressing synthetic TelR-TALE-YFP (A), 70R-TALE-YFP (B), and 177R-TALE-YFP (C). Enriched proteins were identified and quantified by LC-MS/MS analysis relative to affinity-selected ingiR-TALE-YFP as a negative control. The data for each plot is derived from three biological replicates. Cut-offs used for significance: p<0.05 (Student’s t-test). Enrichment scores for proteins identified in each affinity selection are presented in Supplementary file 1.
Figure 6—figure supplement 4
No proteins of interest are detected following affinity selection of 147R-TALE or ingiR-TALE.
Affinity selection was performed on cells expressing synthetic (A) 147R-TALE-YFP or (B) ingiR-TALE-YFP proteins and control cells expressing no-YFP-tagged protein. Enriched proteins were identified and quantified by LC-MS/MS analysis relative to the No-YFP tag control. The data for each plot is derived from three biological replicates. Cut-offs used for significance: log2(tagged/untagged) p<0.05 (Student’s t-test). Enrichment scores for proteins identified in each affinity selection are available in Supplementary file 1.
Figure 6—figure supplement 5
Overlap of proteins enriched in affinity purifications of both kinetochore protein YFP-KKT2 and synthetic protein 177R-TALE.
Kinetoplastid KineTochore (KKT) proteins are known to be enriched at all centromeres on T. brucei main chromosomes (Akiyoshi and Gull, 2014). 177 bp repeats are confined to intermediate-sized or mini-chromosomes. (A) A Venn diagram comparing proteins enriched in YFP-KKT versus 177R-TALE versus affinity purifications. A high proportion of KKT proteins, in addition to cohesin (SCC1, SCC3, SMC1, and SMC3) and condensin (SMC2) subunits, are enriched on 177 bp repeats. (B) List of kinetochore, cohesin, and condensin proteins detected in YFP-KKT and/or 177R-TALE affinity purifications. Lists of all proteins detected in 177R-TALE-YFP and YFP-KKT2 affinity purifications are available in Supplementary file 1l and n, respectively.
Figure 7
Synthetic 177R-TALE-YFP and YFP-KKT2 kinetochore proteins co-localise over 177 bp repeats located on intermediate-sized and mini-chromosomes but not over centromeric CIR147 repeats where 147R-TALE-YFP binds.
(A) Distribution of 177R-TALE, YFP-KKT2, and 147R-TALE over two intermediate/mini-chromosome telomeres determined by anti-GFP ChIP-seq. Anti-GFP ChIP-seq of T. brucei 427 cells expressing no tagged protein is included as a control. The diagram below ChIP-seq profiles indicates the positions of 177 bp repeats (red chevrons), 70 bp repeats (green bars), VSG encoding genes (blue), and telomere (TTAGGG)n repeats (black chevrons) within Tb427VSG-671_unitig_Tb427v12:17,836–31,606 (31 kb) and Tb427VSG-647_untig_Tb427v12 (10 kb). (B) Comparison of distribution of 177R-TALE, 147R-TALE, and YFP-KKT2 over the chromosome 4 CIR147 centromere repeat array and adjacent unique sequences. Chr4:880,000–895,000 (15 kb) and Tb427VSG-671_unitig_Tb427v12:12,000–27,000 (31 kb). The diagram below ChIP-seq indicates the position of CIR147 repeats. (C) Comparison of YFP-KKT2 kinetochore protein enrichment on 177 bp and 147 bp repeats. Data are from two biological replicates. Y axis: log2 values, X axis: repeat types.
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (T. brucei) | TRF gene | TriTrypDB | Tb927.11.1000 | Telomere Repeat binding Factor |
| Gene (T. brucei) | KKT2 gene | TriTrypDB | Tb927.10.12850 | Kinetoplastid KineTochore protein 2 |
| Strain background (T. brucei Lister 427) | T. brucei Lister 427 | Standard | RRID:CVCL_K226 | Bloodstream form, monomorphic |
| Cell line (T. brucei) | T. brucei Lister 427 bloodstream form | Standard laboratory strain | RRID:CVCL_K226 | Monomorphic strain, used as parental line |
| Cell line (T. brucei) | YFP-TRF | Akiyoshi and Gull, 2014. Staneva et al., 2021 | N/A | T. brucei 427 expressing YFP-tagged TRF (Tb927.10.12850) |
| Cell line (T. brucei) | YFP-KKT2 | Akiyoshi and Gull, 2014. Staneva et al., 2021 | N/A | T. brucei 427 expressing YFP-tagged KKT2 (Tb927.10.12850) |
| Cell line (T. brucei) | TelR-TALE-YFP | This paper | N/A | T. brucei 427 expressing TelR-TALE-YFP |
| Cell line (T. brucei) | 70R-TALE-YFP | This paper | N/A | T. brucei 427 expressing 70R-TALE-YFP |
| Cell line (T. brucei) | 147R-TALE-YFP | This paper | N/A | T. brucei 427 expressing 147R-TALE-YFP |
| Cell line (T. brucei) | 177R-TALE-YFP | This paper | N/A | T. brucei 427 expressing 177R-TALE-YFP |
| Cell line (T. brucei) | ingiR-TALE-YFP | This paper | N/A | T. brucei 427 expressing ingiR-TALE-YFP |
| Cell line (T. brucei) | NonR-TALE-YFP | This paper | N/A | T. brucei 427 expressing NonR-TALE-YFP |
| Transfected construct (E. coli) | pTALE-TelR | This paper | De novo construct | Plasmid to generate TelR-TALE-YFP cell line |
| Transfected construct (E. coli) | pTALE-70R | This paper | De novo construct | Plasmid to generate 70R-TALE-YFP cell line |
| Transfected construct (E. coli) | pTALE-147R | This paper | De novo construct | Plasmid to generate 147R-TALE-YFP cell line |
| Transfected construct (E. coli) | pTALE-177R | This paper | De novo construct | Plasmid to generate 177R-TALE-YFP cell line |
| Transfected construct (E. coli) | pTALE-ingiR | This paper | De novo construct | Plasmid to generate ingiR-TALE-YFP cell line |
| Transfected construct (E. coli) | pTALE-NonR | This paper | De novo construct | Plasmid to generate NonR-TALE-YFP cell line |
| Recombinant DNA reagent | pPOTv4 TRF fusion PCR | As in Staneva et al., 2021 | N/A | For endogenous YFP-tagging of TRF |
| Recombinant DNA reagent | pPOTv4 KKT2 fusion PCR | As in Staneva et al., 2021 | N/A | For endogenous YFP-tagging of KKT2 |
| Recombinant DNA reagent | pPOTv4 RPA2 fusion PCR | As in Staneva et al., 2021 | N/A | For endogenous YFP-tagging of RPA2 |
| Antibody | Mouse anti-Ty1 (BB2) (Monoclonal) | Thermo Fisher Scientific | Cat# MA5-23513; RRID:AB_2610643 | (1:5) for western blots |
| Antibody | Mouse anti-GFP (Monoclonal) | Roche | Cat# 11814460001; RRID:AB_390913 | Used for Affinity Purification (beads cross-linked) |
| Antibody | Mouse anti-GFP (Monoclonal) | Roche | Cat# 11814460001;RRID:AB_390913 | (1:1000) for western blots |
| Antibody | Goat anti-mouse IgG (H+L) Alexa Fluor 568 | Thermo Fisher Scientific | Cat# A-11004; RRID:AB_2534072 | (1:1000) for IF |
| Antibody | Goat anti-mouse IgG (H+L) HRP-conjugated | Thermo Fisher Scientific | Cat# 31430; RRID:AB_228307 | (1:5000) for western blot |
| Sequence-based reagent | TelR-TALE target sequence | This paper | N/A | Designed to bind AGGGTTAGGGTTAGG. TALE in vivo truncation recognises AGGGTTAG |
| Sequence-based reagent | 70R-TALE target sequence | This paper | N/A | AGGAGAGTGTTGTGA |
| Sequence-based reagent | 147R-TALE target sequence | This paper | N/A | GCAGCGTTGTGCATG |
| Sequence-based reagent | ingiR-TALE target sequence | This paper | N/A | GCCGGCCACCTCAAC |
| Sequence-based reagent | NonR-TALE target sequence | This paper | N/A | GGAAGTATACCTGGC (no genomic match) |
| Sequence-based reagent | TALE-PCR-LP (Primer) | This paper | N/A | Forward primer for integration check (Figure 1—figure supplement 1) |
| Sequence-based reagent | TALE-PCR-RP (Primer) | This paper | N/A | Reverse primer for integration check (see Figure 1—figure supplement 1) |
| Peptide, recombinant protein | Protein G Dynabeads | Thermo Fisher Scientific | Cat# 10004D | Used for ChIP |
| Peptide, recombinant protein | Trypsin Protease, MS Grade | Thermo Fisher Scientific (Pierce) | Cat# 90057 | Used for protein digestion |
| Commercial assay or kit | TALEN module kit | Ding et al., 2013. | N/A | Used for TALE assembly |
| Commercial assay or kit | NEXTflex barcoded adapters | Bio Scientific | N/A | Used for library preparation |
| Commercial assay or kit | NuPAGE Bis-Tris Mini Gels | Thermo Fisher Scientific | Cat# NP0321BOX | For protein separation |
| Commercial assay or kit | NuPAGE LDS Sample Buffer (4×) | Thermo Fisher Scientific | Cat# NP0007 | For western blot sample preparation |
| Commercial assay or kit | Amersham ECL Prime | GE Healthcare | Cat# RPN2232 | Western blot detection |
| Commercial assay or kit | Amersham Hyperfilm ECL | GE Healthcare | Cat# 28906839 | Film for western blot visualisation |
| Commercial assay or kit | Vivacon 500 (30 K MWCO) | Sartorius | Cat# VN01H22 | Spin filters used for FASP protocol |
| Chemical compound, drug | RapiGest SF Surfactant | Waters | Cat# 186001861 | Used for protein elution in AP-MS |
| Chemical compound, drug | HMI-9 medium | Standard | N/A | For T. brucei culture |
| Chemical compound, drug | Fetal Calf Serum (FCS) | Gibco (Thermo Fisher Scientific) | Cat# 10500064 | 10% supplement for HMI-9 |
| Chemical compound, drug | Ponceau S | Sigma-Aldrich | Cat# P3504 | Membrane staining |
| Chemical compound, drug | Dithiothreitol (DTT) | Sigma-Aldrich (or similar) | Cat# D0632 | Reducing agent |
| Chemical compound, drug | Iodoacetamide (IAA) | Sigma-Aldrich (or similar) | Cat# I1149 | Alkylating agent |
| Chemical compound, drug | Urea | Sigma-Aldrich (or similar) | Cat# U5378 | Denaturing agent (8 M) |
| Chemical compound, drug | Bovine Serum Albumin (BSA) | Sigma-Aldrich (or similar) | N/A | Blocking agent (2%) for IF |
| Chemical compound, drug | Triton X-100 | Sigma-Aldrich (or similar) | N/A | Permeabilisation (0.1%) for IF |
| Chemical compound, drug | Paraformaldehyde | Sigma-Aldrich (or similar) | N/A | Fixation (4%) for IF |
| Chemical compound, drug | Blasticidin S | InvivoGen or similar | Cat# ant-bl-1 | (10 µg/ml) Used for TALE-YFP selection |
| Software, algorithm | MaxQuant | Cox and Mann, 2008 | RRID:SCR_014485 | v.2.0.3.0 used for proteomic analysis |
| Software, algorithm | Perseus | Tyanova et al., 2016 | RRID:SCR_015753 | v.1.6.15.0 used for proteomic statistical analysis |
| Software, algorithm | Bowtie2 | Langmead and Salzberg, 2012 | RRID:SCR_016368 | v.2.4.2 used for ChIP-seq alignment |
| Software, algorithm | MACS2 | Zhang et al., 2008 | v.2.2.7.1 used for peak calling | |
| Software, Algorithm | SAMtools | Danecek et al., 2021 | RRID:SCR_002105 | Used for removing duplicate reads |
| Software, algorithm | deepTools | Ramírez et al., 2016 | RRID:SCR_016366 | Used for genome overview |
| Other | Orbitrap Fusion Lumos | Thermo Fisher Scientific | N/A | Mass spectrometer used |
| Other | Zeiss Axio Imager | Zeiss | N/A | Microscope used for imaging |
| Other | Illumina NextSeq 500/550 | Illumina | N/A | Used for ChIP-seq library sequencing |
| Other (software, algorithm) | TriTrypDB | https://tritrypdb.org | RRID:SCR_007043 | Database used for T. brucei gene identifiers |
Additional files
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Supplementary file 1
Proteomics analyses comparing protein enrichments in the indicated affinity selections a-to-q.
(a) Affinity selection data for wild-type cells expressing No YFP versus cells expressing YFP-TRF (WT NoYFP vs. YFP-TRF). Proteins enriched in YFP-TRF affinity selections were identified and quantified by LC-MS/MS analysis relative to affinity selections from wild-type cells lacking any YFP as a negative control. (b) Affinity selection data for wild-type cells expressing No YFP versus cells expressing TelR-TALE (WT NoYFP vs. TelR-TALE). Proteins enriched in TelR-TALE affinity selections were identified and quantified by LC-MS/MS analysis relative to affinity selections from wild-type cells lacking any YFP as a negative control. (c) Affinity selection data for wild-type cells expressing No YFP versus cells expressing NonR-TALE (WT NoYFP vs. NonR-TALE). Proteins enriched in NonR-TALE affinity selections were identified and quantified by LC-MS/MS analysis relative to affinity selections from wild-type cells lacking any YFP as a negative control. (d) Affinity selection data for cells expressing NonR-TALE versus cells expressing TelR-TALE (NonR-TALE vs. TelR-TALE). Proteins enriched in TelR-TALE affinity selections were identified and quantified by LC-MS/MS analysis relative to affinity selections of NonR-TALE. (e) Affinity selection data for wild-type cells expressing No YFP versus cells expressing 147R-TALE (WT NoYFP vs. 147R-TALE). Proteins enriched in 147R-TALE affinity selections were identified and quantified by LC-MS/MS analysis relative to affinity selections from wild-type cells lacking any YFP as a negative control. (f) Affinity selection data for cells expressing NonR-TALE versus cells expressing 147R-TALE (NonR-TALE vs. TelR-TALE). Proteins enriched in 147R-TALE affinity selections were identified and quantified by LC-MS/MS analysis relative to affinity selections of NonR-TALE. (g) Affinity selection data for wild-type cells expressing No YFP versus cells expressing ingiR-TALE (WT NoYFP vs. ingiR-TALE). Proteins enriched in ingiR-TALE affinity selections were identified and quantified by LC-MS/MS analysis relative to affinity selections from wild-type cells lacking any YFP as a negative control. (h) Affinity selection data for cells expressing NonR-TALE versus cells expressing ingiR-TALE (NonR-TALE vs. ingiR-TALE). Proteins enriched in ingiR-TALE affinity selections were identified and quantified by LC-MS/MS analysis relative to affinity selections of NonR-TALE. (i) Affinity selection data for wild-type cells expressing No YFP versus cells expressing 70R-TALE (WT NoYFP vs. 70R-TALE). Proteins enriched in 70R-TALE affinity selections were identified and quantified by LC-MS/MS analysis relative to affinity selections from wild-type cells lacking any YFP as a negative control. (j) Affinity selection data for wild-type cells expressing No YFP versus cells expressing YFP-RPA2 (WT NoYFP vs. YFP-RPA2). Proteins enriched in YFP-RPA2 affinity selections were identified and quantified by LC-MS/MS analysis relative to affinity selections from wild-type cells lacking any YFP as a negative control. (k) Affinity selection data for cells expressing NonR-TALE versus cells expressing 70R-TALE (NonR-TALE vs. 70R-TALE). Proteins enriched in 70R-TALE affinity selections were identified and quantified by LC-MS/MS analysis relative to affinity selections of NonR-TALE. (l) Affinity selection data for wild-type cells expressing No YFP versus cells expressing 177R-TALE (WT NoYFP vs. 177R-TALE). Proteins enriched in 177R-TALE affinity selections were identified and quantified by LC-MS/MS analysis relative to affinity selections from wild-type cells lacking any YFP as a negative control. (m) Affinity selection data for cells expressing NonR-TALE versus cells expressing 177R-TALE (NonR-TALE vs. 177R-TALE). Proteins enriched in 177R-TALE affinity selections were identified and quantified by LC-MS/MS analysis relative to affinity selections of NonR-TALE. (n) Affinity selection data for wild-type cells expressing No YFP versus cells expressing YFP-RPA2 (WT NoYFP vs. YFP-KKT2). Proteins enriched in YFP-KKT2 affinity selections were identified and quantified by LC-MS/MS analysis relative to affinity selections from wild-type cells lacking any YFP as a negative control. (o) Affinity selection data for cells expressing ingiR-TALE versus cells expressing TelR-TALE (ingiR-TALE vs. TelR-TALE). Proteins enriched in TelR-TALE affinity selections were identified and quantified by LC-MS/MS analysis relative to affinity selections of ingiR-TALE. (p) Affinity selection data for cells expressing ingiR-TALE versus cells expressing 70R-TALE (ingiR-TALE vs. 70R-TALE) Proteins enriched in 70R-TALE affinity selections were identified and quantified by LC-MS/MS analysis relative to affinity selections of ingiR-TALE. SuppFile1p_ingiR-TALEv70R-TALE (q) Affinity selection data for cells expressing ingiR-TALE versus cells expressing 177R-TALE (ingiR-TALE vs. 70R-TALE) Proteins enriched in 177R-TALE affinity selections were identified and quantified by LC-MS/MS analysis relative to affinity selections of ingiR-TALE. SuppFile1q_ingiR-TALEv177R-TALE.
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Defining the chromatin-associated protein landscapes on Trypanosoma brucei repetitive elements using synthetic TALE proteins
eLife 14:RP109950.
https://doi.org/10.7554/eLife.109950.2
