RNA binding proteins interact with an ∼7 kb domain within ASAR6-141 RNA.

A) RNA-DNA FISH images of ASAR6-141 expression in six individual HTD114 cells. ASAR6-141 RNA (green; green arrows) and chromosome 6 DNA (chromosome paint, red), and DNA was stained with DAPI. B) RNA-DNA FISH of ASAR6-141 RNA (red, red arrows), CHR6 DNA (chromosome paint, white), and XIST RNA (green, green arrows) visualized within individual GM12878 cells, top and bottom panels represent the same three cells with the nuclear outline drawn in white. C) RNA-protein interaction data (eCLIP from ENCODE) for 120 RBPs expressed in K562 cells for the genomic region that contains ASAR6-141, and ∼200 kb of upstream non-transcribed DNA (chromosome 6 140.8 mb-141.6 mb). Each point represents the FDR-corrected p-value of the z-score of log2-ratio between eCLiP vs control for each RBP within a 10kb sliding window. D) Number of 10kb regions with significant enrichments of eCLiP reads vs control within ASAR6-141. E) Histogram of eCLiP peaks count per RBP within ASAR6-141 7kb RBPD. F) Histogram of eCLiP peaks count per RBP within lncRNA XIST. G) Genome browser view of ASAR6-141 with RNA-seq expression and eCLiP peaks shown in K562 and HepG2. The zoomed in view shows the ∼7kb RBPD with the location of the peaks of eCLIP reads that map within the region (see Figure 1-source data 1). The eCLIP peaks for the RBPs used in the shRNA knockdown experiments are indicated and highlighted with arrows and red bars. The location of sgRNAs are shown with arrows (see Figure 1-source data 3), and the asterisks mark the sgRNAs from 5.

Replication timing in cells with ASAR6-141 ∼7kb RBPD deletion or ectopic integration.

A) Schematic illustration of the BrdU terminal label protocol 30. Cells were treated with BrdU (green) for 5 hours and then harvested for mitotic cells. B) BrdU incorporation in HTD114 cells containing a heterozygous deletion of the ∼7kb RBPD. Cells containing a deletion of the ∼7kb RBPD from the expressed allele of ASAR6-141 were exposed to BrdU, harvested for mitotic cells, and subjected to DNA FISH using a chromosome 6 centromeric probe (red; B). The larger centromere resides on the chromosome 6 with the silent ASAR6-141 allele (CHR6A), and the smaller centromere resides on the chromosome 6 with the expressed ASAR6-141 allele (CHR6B). DNA was stained with DAPI (blue). C and D) DAPI staining and BrdU incorporation were quantified by calculating the number of pixels (area x intensity) and displayed as a ratio of BrdU incorporation in CHR6B divided by the BrdU incorporation in CHR6A. E) Quantification of BrdU incorporation in multiple cells with heterozygous (Δ7kb-6B, expressed allele; or Δ7kb-6A, silent allele) or homozygous (Δ7kb-6B/6A) deletions of the ∼7kb RBPD. Also shown is the quantification of BrdU incorporation in heterozygous deletions of the entire ∼185 kb ASAR6-141 gene from chromosome 6B (Δ185-6B) or 6A (Δ185-6A) 5. Box plots indicate mean (solid line), standard deviation (dotted line), 25th, 75th percentile (box) and 5th and 95th percentile (whiskers) and individual cells (single points). P values were calculated using the Kruskal-Wallis test 43. Values for individual cells are shown as dots. **P = 0.00027 and *P = 0.03. F) Schematic view of the ∼7kb RBPD in both the sense and antisense orientation. The promoter used to drive expression is from ASAR6 9. G and H) Two color RNA FISH assay for expression of the ∼7kb RBPD transgenes. Individual clones were screened for RNA expressed from the ASAR6-141 (∼7kb RBPD) transgenes (red, arrows), and RNA hybridization using an Xist probe (green, arrows) was used as positive control. G and H represent examples of cells containing the sense and antisense transgenes, respectively. I) Quantitation of the size (pixels = area x intensity) of the RNA FISH signals expressed from the 7 kb sense (7 kb S) or antisense (7 kb AS) transgenes are shown. Box plots indicate mean (solid line), standard deviation (dotted line), 25th, 75th percentile (box) and 5th and 95th percentile (whiskers) and individual cells (single points). P value of 0.006 was calculated using the Kruskal-Wallis test 43. Values for individual cells are shown as dots. J) RNA from the ∼7kb RBPD remains localized to mouse chromosomes. RNA-DNA FISH using the ∼7kb RBPD as RNA FISH probe (red, arrows), plus a mouse chromosome 5 paint to detect chromosome 5 DNA (green, brackets). K) Cells containing the sense ∼7kb RBPD transgene integrated into mouse chromosome 5 were exposed to BrdU (green), harvested for mitotic cells, and subjected to DNA FISH using the ∼7kb RBPD (red, arrows). Note that the chromosome that contains the transgene shows delayed mitotic condensation and more BrdU incorporation than any other chromosome within the same cell. L) Cells containing the antisense ∼7kb RBPD transgene integrated into a mouse chromosome were exposed to BrdU (green), harvested for mitotic cells, and subjected to DNA FISH using the ∼7kb RBPD (red). Note that the chromosome that contains the transgene shows normal mitotic condensation and only a small amount of BrdU incorporation. Scale bars are 10 uM (B, G, H) 5 uM (K, L) and 2 uM (J).

Depletion of RBPs results in disruption of the chromosome territory localization of ASAR6-141 RNA.

A-J) shRNA mediated depletion of RBPs. K562 cells were transfected with empty vector (A, C, E, G, and I) or vectors expressing shRNAs directed against HNRNPU (B), HNRNPUL1 (D), HNRNPC (F), HNRNPM (H), or HLTF (J). K) Cells were stained with the appropriate antibodies and quantitation of each RBP was determined in >25 individual cells. Box plots indicate mean (solid line), standard deviation (dotted line), 25th, 75th percentile (box), 5th and 95th percentile (whiskers), and individual cells (single points). P values were calculated using the Kruskal-Wallis test 43. L-V) RNA-DNA FISH for ASAR6-141 (green) and XIST (red) RNA. K562 cells were transfected with empty vector (L) or vectors expressing shRNAs against HNRNPU (M), HNRNPUL1 (N), HNRNPC (O), HNRNPM (P), HLTF (Q), HNRNPA1 (R), HNRNPL (S), KHSRP (T), UCHL5 (U), or PTBP1 (V). The red arrows mark the RNA FISH signals for XIST. The brackets mark cytoplasmic regions that hybridized to both RNA FISH probes. DNA was stained with DAPI, and Bars are 10 uM (A-J and L) or 5 uM (M-V). W) Quantitation of the XIST RNA FISH signals. K562 cells transfected and processed for RNA FISH as in L-V were analyzed for quantitation of XIST RNA cloud size (pixels: area X intensity) in >25 individual cells. Box plots indicate mean (solid line), standard deviation (dotted line), 25th, 75th percentile (box), 5th and 95th percentile (whiskers) and individual cells (single points). P values were calculated using the Kruskal-Wallis test 43.

Ectopic integration of the ASAR6-141 ∼7kb RBPD transgene into an inactive X chromosome.

A) Two color RNA FISH assay for expression of the sense ∼7kb RBPD transgene plus Xist RNA. Examples of three different cells showing colocalization of the ASAR RNA and Xist RNA. Individual cells were processed for expression of the sense strand ∼7kb RBPD transgene (left panels, red, arrow) in combination with RNA FISH for Xist (middle panels, green, arrow). The right panels show the merged images. The white lines show the path for the pixel intensity profiles used for panel B. B) Pixel intensity profiles across the Xist RNA hybridization domain showing enhanced signal intensity over the ∼7kb RBPD RNA hybridization domain. C and D) Delayed mitotic chromosome condensation of the X chromosome that contains the ∼7kb RBPD transgene. An example of a mitotic spread processed for DNA FISH using the ∼7kb RBPD as probe (magenta, panel C) plus a mouse X chromosome paint (red, panel D). The arrows mark the location of the transgene hybridization signal. E-H) Cells containing the ∼7kb RBPD transgene integrated into the mouse X chromosome were exposed to BrdU (G, green), harvested for mitotic cells, and subjected to DNA FISH using the ∼7kb RBPD (E, magenta) plus a mouse X chromosome paint (F, red). The merged images are shown in panel H.

Depletion of RBPs results in disruption of the chromosome territory localization of ASAR RNAs.

shRNA mediated depletion of RBPs in K562 cells (A-K) or HTD114 cells (L-Q). Cells were transfected with empty vector (A-C and L-N) or vectors expressing shRNAs directed against HNRNPA1 (D), HNRNPC (E), HNRNPL (F), HNRNPM (G), HNRNPU (H, and O-Q), HLTF (I), KHSRP (J), or UCHL5 (K). Cells were processed for RNA FISH with probes for ASAR6 (red, arrows) and ASAR1-187 (green, arrows). Cells were also processed for DNA FISH using BAC DNA to detect chromosome 1 (CHR1 BAC DNA, magenta) and chromosome 6 (CHR6 BAC DNA, orange; A-C and L-Q). Arrows mark the sites of RNA FISH hybridization and arrowheads mark the sites of DNA hybridization. RNA-DNA FISH for ASAR1-187 (RNA in green, arrows; DNA in magenta and arrowheads), ASAR6 (RNA in red, arrows; DNA in orange and arrowheads). The brackets mark the cytoplasmic regions that hybridized to both RNA FISH probes. DNA was stained with DAPI, and scale bars are 10 uM.

Depletion of RBPs results in asynchronous replication on autosome pairs.

A) BrdU incorporation in parental HTD114 cells. Cells were exposed to BrdU for five hours and processed for BrdU incorporation using an antibody against BrdU. This panel represents chromosomes from multiple mitotic spreads showing representative BrdU incorporation in chromosome pairs. Both homologs of autosome pair were captured from the same mitotic cell, and each pair displays a typical BrdU incorporation pattern that is consistent with synchronous replication timing. B) HTD114 cells were transfected with the HNRNPU shRNA expression vector, exposed to BrdU for five hours, and processed for BrdU incorporation. This panel represents chromosomes from multiple mitotic spreads showing representative BrdU incorporation in pairs of autosomes. Both homologs of autosome pairs were captured from the same mitotic cell, and each pair displays a differential BrdU incorporation pattern that is consistent with asynchronous replication timing. C) Shows two chromosome 4 homologs (CHR4A and CHR4B) side by side. D) Shows the BrdU incorporation in chromosome 18 homologs (CHR18A and CHR18B). For each chromosome pair the inverted DAPI staining (black and white), BrdU incorporation (green), and DAPI staining (blue) are shown. E and F) Quantification of BrdU incorporation in multiple cells depleted for HNRNPU, HNRNPUL1, HTLF, KHSRP, UCHL5, HNRNPA1, MATR3, PTBP1, PTBP2, SAFB and SAFB2. Box plots indicate mean (solid line), standard deviation (dotted line), 25th, 75th percentile (box), 5th and 95th percentile (whiskers) and individual cells (single points). P values were calculated using the Kruskal-Wallis test 43. G) DRT/DMC on chromosome 6 following HNRNPU depletion. HTD114 cells were transfected with the HNRNPU shRNA expression vector, exposed to BrdU for 5 hours, harvested for mitotic cells, and processed for BrdU incorporation (green) and DNA FISH using a chromosome 6 centromeric probe (red). H) DRT/DMC on chromosome 1 following UCHL5 depletion. HTD114 cells were transfected with the UCHL5 shRNA expression vector, exposed to BrdU for 5 hours, harvested for mitotic cells, and processed for BrdU incorporation (green) and DNA FISH using a chromosome 1 paint as probe (red). The arrow marks the chromosome 1 with DRT/DMC, and the inset shows only the DAPI staining of the chromosome 1 highlighting the DMC.

Human C0T-1 DNA detects ASAR6 RNA.

A and B) RNA-DNA FISH on mouse cells containing an ASAR6 BAC transgene integrated into mouse chromosome 3 8. A) RNA-DNA FISH using an ASAR6 fosmid probe to detect ASAR6 RNA (green; arrows) plus a mouse chromosome 3 paint probe to detect DNA (red; arrowheads). B) RNA-DNA FISH using human C0T-1 DNA to detect RNA (green; arrows) plus the ASAR6 BAC (red arrowheads) to detect DNA.