Journal: bioRxiv

Article Title: E-ChRPs: Engineered Chromatin Remodeling Proteins for Precise Nucleosome Positioning

doi: 10.1101/480913

Figure Lengend Snippet: (A) General architecture of the E-ChRP core where the yeast Chd1 catalytic domain is linked to a targeting domain with a flexible linker. (B) Summary of targeting methods employed in this work, including: sequence-specific DNA binding domain targeting to a recognition motif (top), SpyCatcher domain covalently attaching to a SpyTag-containing chromatin-bound protein (middle), and dCas9-bound gRNA interacting with a complementary sequence (bottom). (C) Predicted outcome from targeted E-ChRPs, indicating select nucleosomes are positioned by the E-ChRP onto the recruitment site.

Article Snippet: Fusions used in this study include S. cerevisiae Ume6 (residues 764-836, cloned from yeast genomic DNA), D. melanogaster Engrailed (residues 454-543, cloned from fly genomic DNA), S. pombe Res1 (residues 1-147, cloned from a gBlock), R. norvegicus Glucocorticoid Receptor (residues 428-513, cloned from a gBlock), E. coli AraC (residues 175-281, provided by Gregory Bowman), the SpyCatcher domain ( ) (a gift from Mark Howarth, Addgene 35044) and dCas9 (subcloned from Addgene 49013, a gift from Timothy Lu ( )).

Techniques: Sequencing, Binding Assay

Journal: bioRxiv

Article Title: E-ChRPs: Engineered Chromatin Remodeling Proteins for Precise Nucleosome Positioning

doi: 10.1101/480913

Figure Lengend Snippet: (A) Cartoon representation for introducing a Chd1-SpyCatcher E-ChRP into cells containing SpyTagged, chromatin-bound proteins. The SpyCatcher domain forms a covalent isopeptide bond with SpyTag, allowing for localization of E-ChRP activity to endogenously-bound chromatin proteins. (B) Nucleosome sliding assay demonstrating that a single SpyCatcher E-ChRP cannot position nucleosomes without a SpyTag-containing DBD (lanes 2-4) but can use a SpyTagged AraC DBD (lanes 5-7) or Engrailed DBD (lanes 8-10) to reposition nucleosomes containing respective DBD recognition motifs. (C) Representative motif in yeast where ADH1-driven SpyCatcher E-ChRP can reposition nucleosomes at a Ume6 binding site in the presence of Ume6-SpyTag (left) and genomic analysis of nucleosome positioning by SpyCatcher E-ChRP at 202 intergenic instances of the Ume6 recognition sequence in cells containing SpyTagged Ume6 (right). (D) Genomic analysis of nucleosome positions in Reb1-SpyTagged cells (left) or Ume6-SpyTagged cells (right) before and after 2-hour induction of galactose-inducible SpyCatcher E-ChRP. Heat maps show change in nucleosome dyad signal after induction of SpyCatcher E-ChRP while individual traces show average positions of nucleosomes in each cluster before and after SpyCatcher E-ChRP induction for each SpyTag-DBD strain. (See also )

Article Snippet: Fusions used in this study include S. cerevisiae Ume6 (residues 764-836, cloned from yeast genomic DNA), D. melanogaster Engrailed (residues 454-543, cloned from fly genomic DNA), S. pombe Res1 (residues 1-147, cloned from a gBlock), R. norvegicus Glucocorticoid Receptor (residues 428-513, cloned from a gBlock), E. coli AraC (residues 175-281, provided by Gregory Bowman), the SpyCatcher domain ( ) (a gift from Mark Howarth, Addgene 35044) and dCas9 (subcloned from Addgene 49013, a gift from Timothy Lu ( )).

Techniques: Activity Assay, Binding Assay, Sequencing

Journal: bioRxiv

Article Title: E-ChRPs: Engineered Chromatin Remodeling Proteins for Precise Nucleosome Positioning

doi: 10.1101/480913

Figure Lengend Snippet: (A) Genome Browser image showing nucleosome positions before and after induction of SpyCatcher E-ChRP in cells containing Ume6-SpyTag (top) or Reb1-SpyTag (bottom). Location of a proximal Reb1 binding motif or Ume6 binding motif is denoted by a dashed line while directional nucleosome positioning is indicated by blue and red arrows. At this locus, Reb1-SpyTag and Ume6-SpyTag cause different nucleosomes to be selectively moved by SpyCatcher E-ChRP as directed by the location of bound Ume6 or Reb1. (B) Same as (A) showing a locus where Reb1 and Ume6 motifs are adjacent to each other. In this case, both Reb1-SpyTag and Ume6-SpyTag allow the SpyCatcher E-ChRP to select the same nucleosome but the nucleosome is moved to different final locations based on the location of the individual bound factors. Reb1-SpyTag leads to further positioning than Ume6-SpyTag because the Reb1 motif is distal to the Ume6 motif. (C) Heat map (left) showing the difference in nucleosome dyad signal +/− 1000 bp from 943 Reb1 motifs after SpyCatcher E-ChRP induction in Reb1-SpyTag cells. Average change in nucleosome signal after SpyCatcher E-ChRP induction at Reb1 motifs where nucleosomes are moved (mobile cluster) or not moved (immobile cluster) are provided (right).

Article Snippet: Fusions used in this study include S. cerevisiae Ume6 (residues 764-836, cloned from yeast genomic DNA), D. melanogaster Engrailed (residues 454-543, cloned from fly genomic DNA), S. pombe Res1 (residues 1-147, cloned from a gBlock), R. norvegicus Glucocorticoid Receptor (residues 428-513, cloned from a gBlock), E. coli AraC (residues 175-281, provided by Gregory Bowman), the SpyCatcher domain ( ) (a gift from Mark Howarth, Addgene 35044) and dCas9 (subcloned from Addgene 49013, a gift from Timothy Lu ( )).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: E-ChRPs: Engineered Chromatin Remodeling Proteins for Precise Nucleosome Positioning

doi: 10.1101/480913

Figure Lengend Snippet: (A) Nucleosome dyad signal at 943 intergenic Reb1 binding motifs in Reb1-SpyTag strains before (left) and after (right) 2-hour induction of SpyCatcher E-ChRP. Rows are ordered by change in nucleosome positioning after galactose induction. Purple shading highlights the region to which nucleosomes are moved by SpyCatcher E-ChRP in the Reb1-SpyTag strain. (B) The purple mobile fraction from (A) was split into deciles (∼50 motifs per decile) showing average positioning by SpyCatcher E-ChRP for each decile. Dashed lines indicate the pre-induction, unremodeled position (red) or post-induction, remodeled position (black). Ume6-SpyTag control traces are provided for the top and bottom deciles demonstrating that SpyCatcher E-ChRP cannot function at Reb1 sites in the presence of Ume6-SpyTag instead of Reb1-SpyTag. (C) Genome Browser images for representative loci showing positioning by SpyCatcher E-ChRP in a Reb1-SpyTag strain for the top, middle and bottom deciles. Purple shading indicates the motif-proximal, repositioned nucleosomes. Dashed lines indicate the location of Reb1 motif. (See also )

Article Snippet: Fusions used in this study include S. cerevisiae Ume6 (residues 764-836, cloned from yeast genomic DNA), D. melanogaster Engrailed (residues 454-543, cloned from fly genomic DNA), S. pombe Res1 (residues 1-147, cloned from a gBlock), R. norvegicus Glucocorticoid Receptor (residues 428-513, cloned from a gBlock), E. coli AraC (residues 175-281, provided by Gregory Bowman), the SpyCatcher domain ( ) (a gift from Mark Howarth, Addgene 35044) and dCas9 (subcloned from Addgene 49013, a gift from Timothy Lu ( )).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: E-ChRPs: Engineered Chromatin Remodeling Proteins for Precise Nucleosome Positioning

doi: 10.1101/480913

Figure Lengend Snippet: (A) Analysis of Reb1 binding at 943 intergenic Reb1 motifs (TTACCCK) using indicated methods for Reb1 mapping. All data are ordered based on the ranked change in nucleosome positioning after SpyCatcher E-ChRP induction in a Reb1-SpyTag strain (left). All data are centered at the Reb1 motif and display +/− 250 base pairs from each motif. (B) Genome Browser image showing Reb1 binding across ChrIX for indicated Reb1 mapping strategies. Highlighted regions of interest are displayed in (C). (C) Zoomed-in Genome Browser images showing nucleosome repositioning by SpyCatcher E-ChRP at Reb1-SpyTag sites (blue versus red) and relative Reb1 signal from indicated methods. All regions show nucleosome shifts by SpyCatcher E-ChRP (black circles), ChEC-seq signal and ORGANIC signal. Regions 2 and 4 show Reb1 binding using all methods. Regions 1,3 and 5 lack ChIP signal. Regions 3 and 5 lack CUT&RUN signal. Regions 3 and 5 have very low but detectable ORGANIC signal despite significant nucleosome shifts by SpyCatcher E-ChRP and high ChEC-seq signal. Numbering corresponds to (B).

Article Snippet: Fusions used in this study include S. cerevisiae Ume6 (residues 764-836, cloned from yeast genomic DNA), D. melanogaster Engrailed (residues 454-543, cloned from fly genomic DNA), S. pombe Res1 (residues 1-147, cloned from a gBlock), R. norvegicus Glucocorticoid Receptor (residues 428-513, cloned from a gBlock), E. coli AraC (residues 175-281, provided by Gregory Bowman), the SpyCatcher domain ( ) (a gift from Mark Howarth, Addgene 35044) and dCas9 (subcloned from Addgene 49013, a gift from Timothy Lu ( )).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: E-ChRPs: Engineered Chromatin Remodeling Proteins for Precise Nucleosome Positioning

doi: 10.1101/480913

Figure Lengend Snippet: (A) Cartoon representation of Chd1-SpyCatcher combining with SpyTag-dCas9 to form a functional, gRNA-targeted E-ChRP and predicted nucleosome positioning at a target nucleosome. (B) SDS-PAGE demonstrating full conversion of SpyTag-dCas9 to Chd1-SpyCatcher-SpyTag-dCas9 in the presence of excess Chd1-SpyCatcher prior to remodeling assays. (C) Comparison of Chd1-SpyCatcher-SpyTag-dCas9 remodeling activity (top) on target nucleosomes to Chd1-dCas9 (direct fusion) activity (bottom) using indicated gRNAs. A catalytically active Chd1-Ume6 protein was used as a positive control (ctrl Chd1) for nucleosome positioning. Lanes 1 and 14 contain unremodeled nucleosome (25nM). Lanes 2-4 and 15-17 include 1.5, 15 and 150nM Chd1-Ume6. All other lanes contain the indicated Chd1-dCas9 (either direct fusion or SpyCatcher/SpyTag pair) with 1.5, 15 and 150nM remodeler for each gRNA condition. For “off-target gRNA” conditions, a gRNA with no complementarity to the nucleosome substrate was included in the reaction.

Article Snippet: Fusions used in this study include S. cerevisiae Ume6 (residues 764-836, cloned from yeast genomic DNA), D. melanogaster Engrailed (residues 454-543, cloned from fly genomic DNA), S. pombe Res1 (residues 1-147, cloned from a gBlock), R. norvegicus Glucocorticoid Receptor (residues 428-513, cloned from a gBlock), E. coli AraC (residues 175-281, provided by Gregory Bowman), the SpyCatcher domain ( ) (a gift from Mark Howarth, Addgene 35044) and dCas9 (subcloned from Addgene 49013, a gift from Timothy Lu ( )).

Techniques: Functional Assay, SDS Page, Activity Assay, Positive Control