Journal: Cell

Article Title: Parallel pathways for recruiting effector proteins determine centromere drive and suppression.

doi: 10.1016/j.cell.2021.07.037

Figure Lengend Snippet: Figure 2. Introducing rat CENP-C in mouse oocytes weakens the kinetochore pathway and makes centromeres functionally more symmetric (A) CENP-C divergence between Mus musculus (mouse), Rattus norvegicus (rat), and Rhabdomys pumilio (a model organism closely related to Rhabdomys dilectus, Figure 4B). (B and C) CF-1 oocytes were microinjected with cRNA for GFP-tagged mouse or rat CENP-C and fixed in prometaphase/metaphase I. Cells were stained for SGO2 (A) or analyzed for GFP fluorescence (B). 10 mm scale bars, 2.2 mm square insets. Plots show centromere signal intensities. Each dot represents a single centromere (n = 200 centromeres from 20 oocytes for each construct); red line, mean; *p < 0.05; ns, not significant. (D and E) CF-1 oocytes were microinjected with cRNA for GFP-tagged mouse or R. pumilio CENP-C and fixed in prometaphase/metaphase I. Cells were analyzed for GFP fluorescence (D) or stained for SGO2 (E). 10 mm scale bars, 2.2 mm square insets. Plots show centromere signal intensities. Each dot represents a single centromere (n R 170 centromeres from R22 oocytes for each construct); red line, mean. (F) Different CENP-C interfaces have changed to modulate effector recruitment. Schematics summarize the results of (B)–(E). Compared to mouse CENP-C, rat CENP-C is similarly recruited to mouse centromere chromatin, but downstream effector recruitment is reduced. In contrast, R. pumilio CENP-C is recruited at higher levels to mouse centromere chromatin, leading to increased effector recruitment.

Article Snippet: Reagent or resource Source Identifier GeneArt Precision gRNA Synthesis Kit Thermo Fisher Scientific A29377 MEGAclear Transcription Clean-Up Kit Thermo Fisher Scientific AM1908 Deposited data Genome assemblies NCBI BioProject database Accession Number PRJNA669840 Experimental models: Organisms/strains Mouse: C57BL/6J The Jackson Laboratory 664 Mouse: ZALENDE/EiJ (CHPO) The Jackson Laboratory 1392 Mouse: NSA (CF-1) Envigo 33 Oligonucleotides Forward primer for Cenpb genotyping: 50- CAGCTGACGTTCCGGGAGAA-30 This paper N/A Reverse primer for Cenpb genotyping: 50- GGGGACAGCTTGTTGGTCTT-30 This paper N/A gRNA target sequence for Cenpb null mice: 50-GAAGAACAAGCGCGCCA-30 This paper N/A gRNA target sequence for minor satellite repeats: 50- ACACTGAAAAACACATTCGT-30 This paper N/A Recombinant DNA H2B-EGFP Akera et al., 2017 N/A H2B-mCherry Akera et al., 2017 N/A dCas9-EGFP This paper N/A dCas9-mCherry This paper N/A EGFP-MmCENP-C This paper, cDNA from the Mus musculus liver N/A EGFP-RnCENP-C This paper, cDNA from the Rattus norvegicus liver N/A EGFP-RpCENP-C This paper, cDNA from the Rhabdomys pumilio liver N/A Software and algorithms GraphPad Prism v7 GraphPad https://www.graphpad.com/ FIJI/ImageJ v2.0.0-rc-61/1.51n Schindelin et al., 2012; Schneider et al., 2012 https://fiji.sc/ Geneious Prime v2020.1.2 Geneious https://www.geneious.com/ LongRanger v2.2.2 10x Genomics https://www.10xgenomics.com/ Supernova v2.1.1 10x Genomics https://www.10xgenomics.com/ ncbi-blast-2.10.1+ NCBI http://blast.ncbi.nlm.nih.gov//blast.ncbi. nlm.nih.gov/Blast.cgi anaconda 4.6.14 Anaconda https://www.anaconda.com/ paml 4.9 Yang, 2007 https://anaconda.org/bioconda/paml MAFFT 7.407 Katoh and Standley, 2013; Katoh et al., 2002 https://anaconda.org/bioconda/mafft RAxML 8.2.12 Stamatakis, 2014 https://anaconda.org/bioconda/raxml In-house Scripts and Pipelines This paper https://github.com/TomoKumon/ Kumon_Cell_2021 (DOI: 10.5281/ zenodo.5129774) ll e2 Cell 184, 4904–4918.e1–e5, September 16, 2021 Article ll

Techniques: Staining, Construct