Fig. 1: Cbp1 recruits Cren7 to chromatinise CRISPR arrays. | Nature Communications

Fig. 1: Cbp1 recruits Cren7 to chromatinise CRISPR arrays.

From: Cbp1 and Cren7 form chromatin-like structures that ensure efficient transcription of long CRISPR arrays

Fig. 1

a Genome-wide overview of Cbp1 and Cren7 occupancy on the S. solfataricus P2 genome. The location of the six CRISPR arrays is indicated. ChIP-seq data represent the mean of two biological replicates with 2000 bp bin size. b Sequence alignment of the different repeat sequences in the S. solfataricus P2 genome and WebLogo of the motif identified in non-canonical Cbp1 binding sites (n = 92). c Scatter plot depicting correlated occupancy of Cbp1 and Cren7 on CRISPR arrays. Average occupancy (normalised against input) for two biological replicates was calculated over 30 bp consecutive bins. d Cren7 recruitment to CRISPR arrays is reduced in a cbp1 deletion strain. Volcano plot showing the differential binding analysis of Cren7 ChIP-seq data for S. islandicus E233S (WT) and \(\Delta\)cbp1. Read counts were calculated for 30 bp consecutive bins for two biological replicates. e Cbp1 facilitates recruitment of Cren7. EMSA testing Cbp1 and Cren7 binding to three different CRISPR repeats and a non-canoncial binding site of Cbp1. 12.5 nM Cbp1 and 3.125 to 25 nM Cren7 (2x dilution series) were incubated with 5’-radiolabelled dsDNA template encompassing the CRISPR repeats and 20 bp flanking spacer DNA on either side or the corresponding region for a non-canonical binding site derived from a ISC1229 transposon. Representative gels of three technical replicates are shown. f Cbp1 and Cren7 directly interact with each other in a DNA-dependent manner. Cbp1:Cren7:CRISPR A repeat DNA complexes were assembled with 2.5 µM Cbp1, 2.5 or 5 µM Cren7, and 2.5 µM dsDNA template. The complexes were then subjected to protein:protein cross-linking with 1 mM BS3. Cross-linked samples were resolved by SDS-PAGE with Coomassie staining. A Cbp1:Cren7 cross-linked species of ~25 kDa (labelled in red) was formed strictly in the presence of BS3, Cbp1, Cren7 and the CRISPR A repeat 1 template. Replacement of the CRISPR DNA template with a non-specific control DNA did not yield any detectable Cbp1:Cren7 cross-linked species. Some background signal at ~37 kDa can be attributed to a small fraction of Cbp1 dimers. A representative gel from three replicate experiments is shown.

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