Characterization of genetic loss-of-function of Fus in zebrafish

<p>The RNA-binding protein FUS is implicated in transcription, alternative splicing of neuronal genes and DNA repair. Mutations in FUS have been linked to human neurodegenerative diseases such as ALS (amyotrophic lateral sclerosis). We genetically disrupted <i>fus</i> in zebrafish (<i>Danio rerio</i>) using the CRISPR-Cas9 system. The <i>fus</i> knockout animals are fertile and did not show any distinctive phenotype. Mutation of <i>fus</i> induces mild changes in gene expression on the transcriptome and proteome level in the adult brain. We observed a significant influence of genetic background on gene expression and 3′UTR usage, which could mask the effects of loss of Fus. Unlike published <i>fus</i> morphants, maternal zygotic <i>fus</i> mutants do not show motoneuronal degeneration and exhibit normal locomotor activity.</p

S1 Table -

A Table. Identification of a new complex associated with HAT2. 10 proteins were identified via mass spectrometry in two Co-IP experiments. The initial Co-IP was performed with Tb927.11.11530, the reciprocal Co-IP with the protein Tb927.11.10070 was performed to confirm the Tb927.4.2000 co-IP data. Only Tb927.3.4140 could be identified in the initial but not in the reciprocal Co-IP experiment. The “Annotation” column indicates the curated annotation that was found for the corresponding accession number in the TriTyp database. The” identified domains” column displays the domains that were found by BLAST search using the NCBI database. The Phyre2 modelling column indicates proteins that were identified by homology modelling. Coverage (Cov.) indicates the coverage in percent between query and template. The confidence (Conf.) represents the relative probability in percent (from 0 to 100) that the match between query and template is a true homology. The nuclear enrichment score (NES) indicates a nuclear localization if positive. The last column shows in which of the two Co-IPs the protein could be identified. B Table. Identification of a new complex associated with HAT1. 11 proteins were identified via mass spectrometry in two co-IP experiments. The initial Co-IP was performed with Tb927.9.2910, the reciprocal Co-IP with the protein Tb927.1.650 was performed to confirm the Tb927.9.2910 co-IP data. Tb927.6.1240, Tb927.10.8310 and Tb927.10.9930 could only be identified in the initial but not in the reciprocal co-IP experiments. The “Annotation” column indicates the curated annotation that was found for the corresponding accession number in the TriTyp database. Proteins labelled in green exhibit a homologue in the S. cerevisiae NuA4 complex. The nuclear enrichment score (NES) indicates a nuclear localization if positive. The “ident. in co-IP” column shows in which of the two Co-IPs the protein could be identified. The”identified domains” column displays the domains that were found by BLAST search using the NCBI / Interpro database. The Phyre2 modelling column indicates proteins that were identified by homology modelling. Coverage (Cov.) indicates the coverage in percent between query and template. The confidence (Conf.) represents the relative probability in percent (from 0 to 100) that the match between query and template is a true homology. The “Yeast NuA4 subunit”column states the NuA4 complex subunit with its corresponding domain (“domain(s)” column) to which the identified trypanosome protein is homologous to. C Table. Primer list. Primer sequences that were used for cloning of RNAi and knock out constructs and PCR amplification for in situ tagging. (DOCX)</p

Depletion of the histone variant H2A.Z leads to a decreased luciferase activity within a PTU.

A luciferase reporter construct was integrated into the tubulin array of a H2A.Z (Tb927.7.6360) RNAi cell line. Samples for the luciferase assay were normalised to cell numbers. (A) Luciferase activity was monitored for 48 h after induction of RNAi in two independent clones. Values of non-induced cells were set to 1; (N = 3); (B) Live/dead staining of each RNAi cell line was performed in triplicates at the same time points. (C) Growth of parasites was monitored for 96 hours (N = 3) after RNAi-mediated depletion of H2A.Z using tetracycline (tet) induction. Growth of the parental 2T1 cell line was measured for 96h as a control. (N = 3 for all depicted experiments; *** = p-value <0.001; ** = p-value 0.001–0.01; * = p-value 0.01–0.05).</p

Summary of all mass spectrometry data.

Excel file with original mass spectrometry data of TbRuvB IP (sheet 1), Tb4040 IP (sheet 2), TbYEATS IP (sheet 3), TbYL1 IP (sheet 4), TbEaf6 IP (sheet 5), TbEaf3 IP (sheet 6), TbHAT2 IP (sheet 7), and TbBdf3 IP (sheet 8). (XLSX)</p

Loss of Bdf3 leads to cell death and a reduction of chromatin-associated H2A.Z.

(A) Growth of parasites was monitored for 96 hours after RNAi-mediated depletion of Bdf3 using tetracycline (tet) induction. The parental 2T1 cell line was used as a control (n = 3). (B) Quantification of live/dead staining with propidium iodide of Bdf3-depleted cells. Analysis was done by flow cytometry (n = 3). (C) Western blot analysis of the insoluble nuclear fraction with antibodies specific for histone H3 and the histone variant H2A.Z. Lysates from an equal number of cells (2x106 per lane) were analysed for each timepoint. (D) Quantification of chromatin-associated H3 (dark blue), Ty1-H2A.Z (turquoise) and H2AZ (grey); (N = 3 for all depicted experiments; *** = p-value <0.001; ** = p-value 0.001–0.01; * = p-value 0.01–0.05).</p

Summary of the RuvB2 co-IP.

15 proteins with a positive or unknown nuclear enrichment score were identified by MS in the RuvB2-HA co-IP. The nuclear enrichment score (NES; (52)) indicates the probability of a nuclear localisation based on cell fractionation combined with quantitative MS analysis. The “Annotation” column indicates the curated annotation that was found for the corresponding accession number on the TriTyp database. In the “p-value” column a probabilistic confidence measure (P-value) is assigned to each identified protein. The fold enrichment compared to the WT control is stated for every identified protein. The column "TrypTag localisation” indicates the cellular localisation of N- and C-terminally tagged proteins.</p

Interaction analysis for fine-mapped T1D SNPs.

<p>Two-dimensional interaction plots reveal the specific binding of transcription factors. A: CREB1 and TFAP4 are enriched on rs12722522*C while this allele resembles a perfect match to the CREB consensus binding motif. B: rs12722508 is bound by several transcription factors (upper panel) among them the RUNX1-CBFB heterodimer, although the sequence around rs12722508*A does not match perfectly the transcription factor consensus motif (lower panel); C: Quantitative SNP pull-down screen identifies LEF1 to bind around 8 times stronger to rs41295061*A.</p

The <i>Trypanosoma brucei</i> SNF2 ATPase complex exhibits characteristics of a SWR1 complex.

A comparison of the modular composition of the newly-identified SNF2 complex with the SWR1 and INO80 complexes of S. cerevisiae. INO80 complex S. cerevisiae: The Nhp-10 module forms a platform for nucleosome interaction. The Arp8 module is the nucleosome binding module, while the Arp5 module, which contains the YL1-domain protein Ies6 is responsible for the nucleosome remodelling step [14]. Ies2 with the PAPA-1-domain plays a structural role within the complex [14, 112]. INO80 specific domains are highlighted in bolt. SWR1 complex S. cerevisiae: Proteins listed under key features are essential for H2AZ incorporation [68]. The C-module of the SWR1 complex, which contains the Bromo-, YEATS- and SANT/DAMP1-domain mediates nucleosome affinity, while the N-module with the YL1- and Zinc finger ZNHIT1-domain is involved in the histone variant exchange reaction [49, 68]. SWR1 specific domains are highlighted in bolt letters [51, 68]. SWI2/SNF2 complex T. brucei: The identification of a BCNT- as well as a ZNHIT1 domain (highlighted in bold letters) hint toward the complex being a SWR1-like complex [51, 68]. Structure of the SWI2/SNF2 complex and its modules is only putative. The potential interaction interface of the species-specific proteins TbSWRC3 and TbARPx is unknown.</p

Loss of <i>Tb</i>SWR1 leads to a reduction of mRNA and rRNA.

(A) Representative Northern blot of two TbSWR1-depleted cell lines. RNAP II-depleted cells were used as a control. The samples were normalised to cell numbers. Upper and middle panel were hybridised with a probe specific for the spliced leader RNA (mini exon, red) the two lower panels with probes specific for ribosomal RNA (rRNA, green) (B) Quantification of results from three replicates showing total mRNA levels in red and 5.8S rRNA in green. Values of un-induced RNAP II RNAi cell lines were set to 100%.</p

Loss of <i>Tb</i>SWR1 leads to cell death and a reduction of chromatin-associated H2A.Z.

(A) Growth of parasites was monitored for 96 hours after RNAi-mediated depletion of TbSWR1 (Tb927.11.10730) using tetraycline (tet). The parental 2T1 cell line was used as a control (n = 3). (B) Quantification of live/dead staining with propidium iodide of TbSWR1-depleted cells at the indicated timepoints post-induction. Analysis was done by flow cytometry (n = 3). (C) Western blot analysis of the insoluble nuclear fraction with antibodies specific for histone H3 and the histone variant H2A.Z. Lysates from an equal number of cells (2x106 per lane) were analysed for each timepoint. (D) Quantification of chromatin-associated H3 (dark blue), Ty1-H2A.Z (turquoise) and H2AZ (grey) (N = 3 for all depicted experiments; *** = p-value <0.001; ** = p-value 0.001–0.01; * = p-value 0.01–0.05).</p