590 δidr sharp amino acids  (Addgene inc)

Journal: bioRxiv

Article Title: Xist spatially amplifies SHARP recruitment to balance chromosome-wide silencing and specificity to the X chromosome

doi: 10.1101/2021.10.27.466149

Figure Lengend Snippet: (A) Disordered scores across the SHARP protein using IUPred2 software predictions. Dotted line represents 0.5 probability value for a given structure to be ordered. Bottom visualization demarcates position of known SHARP domains – RNA Recognition motif (RRM; bright green), Spen Paralog and Ortholog C-terminal (SPOC, dark green). (B) Schematic representation of molecules within a nucleus organized in a diffused or non-diffused (focal) manner. (C) Images across four time-points from a live-cell movie of eGFP-tagged FL-SHARP in transiently transfected HEK293T cells showing non-diffused, focal organization of SHARP molecules. Top panel: 3D reconstructions of the fluorescent intensity signal; Bottom panel: 3D volume reconstructions color-coded based on the size of the condensate; Fluorescent Intensity (FI) (D) Images representing localization patterns of eGFP-tagged FL-SHARP in transiently transfected HEK293T cells across increasing expression levels of SHARP (SHARP under dox inducible promoter; dox 1x = 2 µg/mL). Images shown as max. projections; scale bars 10 µm; Fluorescent Intensity (FI). (E) Quantification of images (Fig. 1D) plotting the dispersion of SHARP signal across the nucleus versus average SHARP fluorescent intensity per nucleus. (F ) Representative images of FL-SHARP and ΔIDR-SHARP localization patterns in transiently transfected HEK293T cells. Images shown as max. projections; scale bars 10 µm; Fluorescent Intensity (FI). (G) Quantification of images (Fig. 1F) plotting the dispersion of SHARP signal across the nucleus versus average SHARP fluorescent intensity per nucleus. Dashed line represents range of fluorescent intensity that is similar for both groups. (H) Images representing localization patterns of mCherry-tagged FUS-ΔIDR-SHARP and eGFP-tagged ΔIDR-SHARP in transiently transfected HEK293T cells. Images shown as max. projections; scale bars 10 µm; Fluorescent Intensity (FI). (I) Schematic depicting formation of concentration-dependent SHARP assemblies.

Article Snippet: ΔRRM-SHARP: amino acids 2-590 ΔIDR-SHARP: amino acids 639-3460 These entry clones (FL-SHARP, ΔRRM-SHARP, ΔIDR-SHARP) were then recombined into two different modified versions of the doxycycline inducible PiggyBac destination vector PB-TAG-ERN (Addgene plasmid 80476) containing NGFR (truncated human nerve growth factor receptor) and HALO or eGFP.

Techniques: Software, Transfection, Expressing, Concentration Assay

Journal: bioRxiv

Article Title: Xist spatially amplifies SHARP recruitment to balance chromosome-wide silencing and specificity to the X chromosome

doi: 10.1101/2021.10.27.466149

Figure Lengend Snippet: (A) Schematic of the domains included in the eGFP-tagged FL-SHARP and ΔIDR-SHARP, and the mCherry-tagged FUS-ΔIDR-SHARP rescue construct used in and . (B) FRAP recovery curve of eGFP-tagged FL-SHARP (red), positive control PTBP1 (forms assemblies; light blue), and negative control EED (does not form assemblies; dark blue). Error bars represent standard deviation of at least five replicates. (C) Schematic depicting physical characteristics of concentration-dependent assemblies, including foci formation, fission and fusion, and rapid diffusion of proteins within an assembly (inset). (D) Images across nine time-points from a live-cell movie of eGFP-tagged FL-SHARP in transiently transfected HEK293T cells (Movie 1, Movie 2) showing non-diffused, focal organization of SHARP molecules. Top panel: 3D reconstructions of the fluorescent intensity signal; middle panel: 3D volume reconstructions color-coded based on the volume of the focus; bottom panel: zoom-in representing one region of the nucleus that changes volume; Fluorescent Intensity (FI). (E) Comparison of diffused or non-diffused localization patterns of FL-SHARP at different dox concentrations. Left: images representing FL-SHARP expressed with either 0.1x dox (diffused) or 1x dox (non-diffused) in transiently transfected HEK293T cells; images shown as max projections; scale bars show 10 μm.; Right: Histograms representing fluorescent intensities for two cells showing diffused and non-diffused localization patterns. The intensity at the 99th percentile of each distribution is shown with the dashed lines. (F) Images representing nuclear localization pattern of eGFP-tagged proteins in transiently transfected HEK293T cells. On the left: proteins that have not been reported to form assemblies (HALO and EED), on the right: an eGFP tagged protein that has been reported to form assemblies (Ptbp1) and ΔSPOC-SHARP that also forms assemblies. Images shown as max projections; scale bars show 10 μm.

Article Snippet: ΔRRM-SHARP: amino acids 2-590 ΔIDR-SHARP: amino acids 639-3460 These entry clones (FL-SHARP, ΔRRM-SHARP, ΔIDR-SHARP) were then recombined into two different modified versions of the doxycycline inducible PiggyBac destination vector PB-TAG-ERN (Addgene plasmid 80476) containing NGFR (truncated human nerve growth factor receptor) and HALO or eGFP.

Techniques: Construct, Positive Control, Negative Control, Standard Deviation, Concentration Assay, Diffusion-based Assay, Transfection

Journal: bioRxiv

Article Title: Xist spatially amplifies SHARP recruitment to balance chromosome-wide silencing and specificity to the X chromosome

doi: 10.1101/2021.10.27.466149

Figure Lengend Snippet: (A) Generation of SHARP-KO cell line in TX mESCs. Top: schematic of CRISPR cut sites used to generate SHARP-KO mESCs and PCR primers used to screen for KO clones; bottom: agarose gel confirming homozygous deletion of SHARP in SHARP-KO clone H8 mESCs. (B) Schematics of constructs used to generate rescue cell lines in TX SHARP-KO or TX SHARP-HALO-AID backgrounds. Grey arrow represents dox-inducible promoter; blue box represents HALO (or eGFP) tags used; light green boxes represent RNA Recognition Motifs (RRM); wavy green line represents the Intrinsically Disordered Regions (IDRs); dark green box represents the Spen Paralog and Ortholog C-terminal (SPOC) domain. Full-length SHARP (FL-SHARP), deletion of RRM domain (ΔRRM-SHARP), deletion of IDR domain (ΔIDR-SHARP), deletion of IDR domain and insertion of alternative IDR domain from FUS protein (FUS-ΔIDR-SHARP). (C) Schematic showing experimental workflow for generating and enriching stable SHARP rescue mESCs (FL-SHARP, ΔRRM-SHARP, ΔIDR-SHARP, FUS-ΔIDR-SHARP) using constructs from . (D) Representative images of SHARP enrichment (HALO, green) over the Xi (anti-Ezh2 immunofluorescence, magenta) in female mESCs containing dox-inducible Xist, genetic deletion of SHARP, and stable integrations of HALO-tagged FL-SHARP, ΔRRM-SHARP, or ΔIDR-SHARP. Xist and SHARP rescue constructs induced with doxycycline for 72 hours; images shown as Z-sections; scale bars show 10 μm. (E) Diagram of image analysis workflow for quantifying SHARP enrichment over the Xi .

Article Snippet: ΔRRM-SHARP: amino acids 2-590 ΔIDR-SHARP: amino acids 639-3460 These entry clones (FL-SHARP, ΔRRM-SHARP, ΔIDR-SHARP) were then recombined into two different modified versions of the doxycycline inducible PiggyBac destination vector PB-TAG-ERN (Addgene plasmid 80476) containing NGFR (truncated human nerve growth factor receptor) and HALO or eGFP.

Techniques: CRISPR, Clone Assay, Agarose Gel Electrophoresis, Construct, Immunofluorescence

Journal: bioRxiv

Article Title: Xist spatially amplifies SHARP recruitment to balance chromosome-wide silencing and specificity to the X chromosome

doi: 10.1101/2021.10.27.466149

Figure Lengend Snippet: (A) Representative images of SHARP enrichment (eGFP, green) over the Xi (anti-Ezh2 immunofluorescence, magenta) in TX SHARP-KO mESCs containing dox-inducible Xist, genetic deletion of SHARP, and stable integrations of: eGFP-FL-SHARP, eGFP-ΔRRM-SHARP, eGFP-ΔIDR-SHARP, or FUS-mCherry-ΔIDR-SHARP constructs (see for cell lines details). Xist induction for 72h, images shown as z-sections; scale bars show 10 μm. (B) Quantification of images (Fig. 3A) plotting (top panel) SHARP fluorescent intensity over the Xi (denoted by Ezh2) normalized to the fluorescent intensity of a random nuclear region of the same size or (bottom panel) Ezh2 fluorescent intensity over the same area normalized to random nuclear region (see for quantification details). Values for individual nuclei (n>10) are shown; red lines represent median values; 0 represents enrichment not higher than measured over a random nuclear region. (C) SHARP enrichment across the first exon of Xist after UV-crosslinking and purification using the HALO tag in female TX SHARP-AID mESCs treated with auxin. Halo-tags were fused to FL-SHARP (top), ΔIDR-SHARP (middle), or ΔRRM-SHARP (bottom, see for cell line details). Two replicates are shown for each cell line; magenta square represents beginning of the first Xist exon, pink square demarcates A-repeat (SHARP binding site). (D) Crosslink-induced truncation sites are shown for a zoom-in on the A-repeat region from demarcated by pink square across three conditions.

Article Snippet: ΔRRM-SHARP: amino acids 2-590 ΔIDR-SHARP: amino acids 639-3460 These entry clones (FL-SHARP, ΔRRM-SHARP, ΔIDR-SHARP) were then recombined into two different modified versions of the doxycycline inducible PiggyBac destination vector PB-TAG-ERN (Addgene plasmid 80476) containing NGFR (truncated human nerve growth factor receptor) and HALO or eGFP.

Techniques: Immunofluorescence, Construct, Purification, Binding Assay

Journal: bioRxiv

Article Title: Xist spatially amplifies SHARP recruitment to balance chromosome-wide silencing and specificity to the X chromosome

doi: 10.1101/2021.10.27.466149

Figure Lengend Snippet: (A) Illustration of our RNA FISH measurements in dox-inducible female mESCs. Green: genes that are silenced upon Xist induction; yellow: genes that escape XCI (remain active after Xist induction), magenta: Xist. (B) RNA FISH images representing (left to right): wildtype (no dox); wildtype (with dox); SHARP-KO (with dox); auxin-treated SHARP-AID (with dox). Cells were stained for DAPI (blue) and probed for Xist (magenta), escape gene Kdm5c (yellow), and silenced genes Atrx or Pgk1 (green). Images shown as max. projections; scale bars show 10 μm. (C) Quantification of multiple RNA FISH images (Fig. 1B) representing the frequency of cells containing two actively transcribed alleles (left to right): wildtype (no dox); wildtype (with dox); SHARP-KO (with dox); auxin-treated SHARP-AID (with dox). (D ) RNA FISH images for SHARP-KO female mESCs containing stable integrations of (left to right): FL-SHARP, ΔRRM-SHARP, ΔIDR-SHARP, or FUS-ΔIDR-SHARP after >72 hours of dox induction. Cells were stained for DAPI (blue) and probed for Xist (magenta), escape gene Kdm5c (yellow), and silenced genes Atrx or Pgk1 (green). Images shown as max projections; scale bars show 10 μm. (E) Quantification of RNA-FISH images representing the frequency of cells containing two actively transcribed alleles for the various SHARP rescue constructs in SHARP-KO female mESCs. (F) RNA FISH images for SHARP-AID female mESCs containing stable integrations of (left to right): FL-SHARP, ΔRRM-SHARP, ΔIDR-SHARP, or FUS-ΔIDR-SHARP after >72 hours of dox induction and auxin treatment. Cells were stained for DAPI (blue) and probed for Xist (magenta), escape gene Kdm5c (yellow), and silenced genes Atrx or Pgk1 (green). Images shown as max projections; scale bars show 10 μm. (G) Quantification of RNA-FISH images representing the frequency of cells containing two actively transcribed alleles for the various SHARP rescue constructs in SHARP-AID female mESCs. In all panels, only cells containing two escape gene spots (Kdm5c, Kdm6a) and Xist (for dox-induced conditions) were scored for the number of silenced gene spots. Asterisks represent p-values calculated for two proportion Z-test, distributions compared to FL group, * 0.05, ** 0.01, *** 0.001. (H) Schematic illustration of the spatial amplification mechanism by which Xist RNA (magenta) can act to amplify SHARP (green) recruitment and gene silencing across the X chromosome.

Article Snippet: ΔRRM-SHARP: amino acids 2-590 ΔIDR-SHARP: amino acids 639-3460 These entry clones (FL-SHARP, ΔRRM-SHARP, ΔIDR-SHARP) were then recombined into two different modified versions of the doxycycline inducible PiggyBac destination vector PB-TAG-ERN (Addgene plasmid 80476) containing NGFR (truncated human nerve growth factor receptor) and HALO or eGFP.

Techniques: Staining, Construct, Amplification

Journal: bioRxiv

Article Title: Xist spatially amplifies SHARP recruitment to balance chromosome-wide silencing and specificity to the X chromosome

doi: 10.1101/2021.10.27.466149

Figure Lengend Snippet: (A) Schematic of mouse X chromosome showing the locations of the various genes probed in RNA-FISH experiments. (B) Frequency of Xist induction (left) and X chromosome ploidy (right) in wildtype and SHARP-KO mESCs based on quantification of RNA-FISH images. (C) Quantification of RNA-FISH images representing the frequency of cells containing two, one, or zero actively transcribed alleles. Left to right: wildtype (-dox); wildtype (+dox); dox-induced SHARP-KO; dox-induced, auxin-treated SHARP-AID female mESCs. Only cells containing two escape gene spots (Kdm5c, Kdm6a) and Xist (for dox-induced conditions) were scored for the number of silenced gene spots. (D) RNA-FISH images from SHARP-KO female mESCs containing stable integrations of (left to right): FL-SHARP, ΔRRM-SHARP, ΔIDR-SHARP, or FUS-ΔIDR-SHARP after >72 hours of dox induction. Cells were stained for DAPI (blue) and probed for Xist (magenta), escape gene Kdm5c (yellow), and silenced genes Gpc4 or MeCP2 (green). Images shown as max projections; scale bars show 10 μm. (E) Quantification of RNA-FISH images representing the frequency of cells containing two, one, or zero actively transcribed alleles for the various SHARP rescue constructs in SHARP-KO female mESCs. Only cells containing two escape gene spots (Kdm5c, Kdm6a) and Xist (for dox-induced conditions) were scored for the number of silenced gene spots . (F) RNA-FISH images from SHARP-AID female mESCs containing stable integrations of (left to right): FL-SHARP, ΔRRM-SHARP, ΔIDR-SHARP, or FUS-ΔIDR-SHARP after >72 hours of dox induction. Cells were stained for DAPI (blue) and probed for Xist (magenta), escape gene Kdm5c (yellow), and silenced genes Gpc4 or MeCP2 (green). Images shown as max projections; scale bars show 10 μm. (G) Quantification of RNA-FISH images representing the frequency of cells containing two, one, or zero actively transcribed alleles for the various SHARP rescue constructs in SHARP-KO female mESCs. Only cells containing two escape gene spots (Kdm5c, Kdm6a) and Xist (for dox-induced conditions) were scored for the number of silenced gene spots .

Article Snippet: ΔRRM-SHARP: amino acids 2-590 ΔIDR-SHARP: amino acids 639-3460 These entry clones (FL-SHARP, ΔRRM-SHARP, ΔIDR-SHARP) were then recombined into two different modified versions of the doxycycline inducible PiggyBac destination vector PB-TAG-ERN (Addgene plasmid 80476) containing NGFR (truncated human nerve growth factor receptor) and HALO or eGFP.

Techniques: Staining, Construct