Tracking pairwise genomic loci by the ParB-ParS and Noc-NBS systems in living cells

Abstract

The dynamics of genomic loci pairs and their interactions are essential for transcriptional regulation and genome organization. However, a robust method for tracking pairwise genomic loci in living cells is lacking. Here we developed a multicolor DNA labeling system, mParSpot (multicolor ParSpot), to track pairs of genomic loci and their interactions in living cells. The mParSpot system is derived from the ParB/ParS in the parABS system and Noc/NBS in its paralogous nucleoid occlusion system. The insertion of 16 base-pair palindromic ParSs or NBSs into the genomic locus allows the cognate binding protein ParB or Noc to spread kilobases of DNA around ParSs or NBSs for loci-specific visualization. We tracked two loci with a genomic distance of 53 kilobases and measured their spatial distance over time. Using the mParSpot system, we labeled the promoter and terminator of the MSI2 gene span 423 kb and measured their spatial distance. We also tracked the promoter and terminator dynamics of the MUC4 gene in living cells. In sum, the mParSpot is a robust and sensitive DNA labeling system for tracking genomic interactions in space and time under physiological or pathological contexts.

Graphical Abstract

Figure 1.

Imaging site-specific genomic DNA by orthogonal ParB–ParS systems. (A) Schematic of DNA labeling by the ParB–ParS system. 8XParS (8 copies) was inserted into 36 kilobases downstream of the C3 repeat (600 copies) in U2OS cells. The genomic locus inserted with 8XParS was termed P1. ParB-HaloTag was used to label the P1 locus and dCas9-GFP/sgRNA-C3 was used to visualize C3 repeat. (B) ParS sequences from different bacterial species. BcParB1 and BcParS2 are from B. cenocepacia, BsParS from B. subtilis, PaParS from P. aeruginosa and TtParS from T. thermophilus, ParSc is the ParS consensus sequence. (C) Loci-specific labeling by orthogonal ParB–ParS. 8XBcParS1, 8XBcParS2, 8XBsParS, 8XParSc, 8XPaParS or 8XTtParS was integrated into 36 kilobases downstream of the C3 repeat in U2OS cells. The integration site (P1 locus, red) was visualized by their cognate ParB-HaloTag along with CRISPR-based labeling of C3 repeat (green). The scale bars are 5 μm for the whole cell and 1 μm for the zoom images. (D) The percentage of specific labeling cells by orthogonal ParB–ParS. The percentage of cells with specific P1 foci was shown in red. The percentage of cells with no foci was shown in blue and cells with non-specific foci was shown in grey. n = 36 cells for BcParS1/BcParB1, 16 for BcParS2/BcParB2, 42 for BsParS/BsParB, 20 for PaParS/PaParB, 29 for TtParS/TtParB, 25 for ParSc/HhParB. (E) The foci numbers of orthogonal ParB–ParS from single cell clones. n = 20 cells for each group. (F) The signal-to-noise ratio of labeling by orthogonal ParB–ParS. The signal-to-noise (S/N) ratio of each ParB–ParS pair was measured. Each data point represents the S/N of each P1 labeling. The red bar represents the average of S/N in U2OS cells. n = 28 for PaParS/PaParB group, 27 for TtParS/TtParB group.

Figure 2.

The specificity of ParS recognition by orthogonal ParBs. (A) Schematic of ParS labeling by the orthogonal ParBs. 8XPaParS or 8XTtParS was inserted into 36 kb downstream of the C3 repeat (600 copies) in U2OS cells. P1 locus was labeled by ParB-HaloTag and C3 repeat was visualized by dCas9-GFP/sgRNA-C3. (B) Labeling specificity of 8XPaParS and 8XTtParS by PaParB and TtParB. The 8XPaParS or 8XTtParS integration site (P1 locus, red) was visualized by orthogonal ParB-HaloTag along with CRISPR-based labeling of C3 repeat (green). The scale bars are 5 μm for the cells and 1 μm for the zoom images. (C) The percentage of specific labeling cells by orthogonal ParSs and ParBs. The percentage of cells with specific P1 foci was shown in red, non-specific foci in grey, and no foci in blue. n = 20 cells in each group.

Figure 3.

Imaging site-specific genomic DNA by the Noc-NBS system. (A) Phylogenetic tree of ParB and paralogous Noc proteins. Tree scale: 0.2 million years. (B) Schematic of DNA labeling by the Noc-NBS system. 8XNBSc (8 copies of NBS consensus sequence) was inserted into 36 kb downstream of the C3 repeat in U2OS cells. P1 locus (8XNBSc) was labeled by Noc-HaloTag and C3 repeat was visualized by dCas9-GFP/sgRNA-C3. (C) Labeling specificity of NBSc by orthogonal Noc proteins. The 8XNBSc integration site (P1 locus, red) was visualized by orthogonal Noc-HaloTag along with CRISPR-based labeling of C3 repeat (green). The scale bars are 5 μm for the cells and 1 μm for the zoom images. (D) The percentage of specific labeling cells by NBSc and orthogonal Noc. The percentage of cells with specific P1 foci was shown in red, no foci in blue, and non-specific foci in grey. n = 50 cells in each group. (E) The foci numbers of orthogonal Noc-NBS from single cell clones. n = 34 for LaNoc, n = 44 cells for SaNoc. (F) The signal-to-noise ratio of labeling by NBSc and orthogonal Noc. The signal-to-noise (S/N) ratio of each NBSc and Noc pair was measured. Each data point represents the S/N of each P1 labeling. The red bar represents the average of S/N in U2OS cells. n = 29 for LaNoc, n = 23 for SaNoc.

Figure 4.

The specificity of ParS or NBS recognition by ParB or Noc proteins. (A) Schematic of ParS or NBS labeling by ParB or Noc proteins. P1 locus (8XParSc or 8XNBSc) was labeled by TtParB-HaloTag or SaNoc-HaloTag and C3 repeat was visualized by dCas9-GFP/sgRNA-C3. (B) Sequence comparison between ParSc and NBSc. The different nucleotides between ParSc and NBSc were marked in red. (C) Labeling specificity of ParS or NBS labeling by ParB or Noc proteins. The 8XParSc or 8XNBSc integration site (P1 locus, red) was visualized by TtParB-HaloTag or SaNoc-HaloTag along with CRISPR-based labeling of C3 repeat (green). The scale bars are 5 μm for the cells and 1 μm for the zoom images. (D) The percentage of specific ParSc or NBSc labeling cells by TtParB or SaNoc. The percentage of cells with P1 foci labeled by TtParB was shown in green, and SaNoc in brown. n = 30 cells for each group.

Figure 5.

Dual color labeling of genomic loci pairs by the mParSpot system. (A) Schematic of dual color DNA labeling by mParSpot, the combinatory ParB–ParS and Noc-NBS system. 8XNBSc (green) or 8XParSc (purple) were inserted into 36 kb or 89 kb downstream of the C3 repeat in U2OS cells. SaNoc-TdStaygold or TtParB-HaloTag was used to label the P1 locus (8XNBSc) and P2 locus (8XParSc) respectively. dCas9-GFP/sgRNA-C3 was used to visualize C3 repeat. (B) Dual color labeling of genomic loci by ParB–ParS and Noc-NBS. The 8XNBSc (P1 locus, green) or 8XParSc (P2 locus, red) was visualized by SaNoc-TdStaygold or TtParB-HaloTag along with CRISPR-based labeling of C3 repeat (blue). The scale bars are 5 μm for the cells and 1 μm for the zoom images. (C) Spatial distance between genomic loci pairs. The spatial distance between C3 and P1, P1 and P2, C3 and P2 was measured. The red bar represents the average spatial distance in each group. n = 32 for C3-P1, 32 for P1-P2, 27 for C3-P2.

Figure 6.

Labeling of promoter and terminator of the MSI2 gene by mParSpot. (A) Schematic figure of labeling promoter and terminator of the MSI2 gene by mParSpot. 8XParSc (P3 locus) was integrated upstream and 8XNBSc (P4 locus) was downstream of the MSI2 gene. The genomic distances are shown below. The MSI2 gene was located on chromosome 17. (B) Representative images of the MSI2’s promoter and terminator labeling by mParSpot. Scale bars, 5 μm for the whole cell and 1 μm for the zoom images. (C) The labeling efficiency of promoter and terminator of the MSI2 gene by mParSpot. n = 25 cells. (D) The spatial distance of promoter and terminator of the MSI2 gene. Each dot represents one cell.

Figure 7.

mParSpot labeling the promoter and terminator of MUC4 along with Pol II clusters. (A) Schematic of mParSpot labeling the promoter and terminator of MUC4 along with Pol II. 8XNBSc with a hygromycin expression cassette (8XNBSc-P_EFS-Hygro) or 8XPaParS with a puromycin expression cassette (8XPaParS-P_CMV-Puro) was inserted into 11 kb upstream or 10 kb downstream of the MUC4 gene in U2OS cells. SaNoc-TdStaygold or PaParB-SNAP was used to label the P5 locus (8XNBSc) or P6 locus (8XPaParS) along with endogenous HaloTag tagged Pol II. (B) mParSpot labeling of the promoter and terminator of MUC4 along with Pol II. The 8XNBSc (P5 locus, green) or 8XPaParS (P6 locus, blue) was visualized by SaNoc-TdStaygold or PaParB-SNAP along with endogenous HaloTag tagged Pol II (red). The scale bars are 5 μm for the cells and 1 μm for the zoom images. (C) The percentage of cells with P5, P6 and Pol II association. The percentage of cells with P5, P6 and Pol II association was shown in purple, and without P5, P6 and Pol II association in grey. n = 37 cells. (D) Comparison of spatial distances between genomic loci pairs with and without Pol II association. The spatial distance between P5 and P6 with or without Pol II association was measured. The red bar represents the average spatial distance in each group. n = 58 for the distance of P5 or P6 with Pol II association, 95 for the distance of P5 and P6 without Pol II association.