Technical Comments

Response to Comment on “Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing”

+ See all authors and affiliations

Science  16 Jun 2017:
Vol. 356, Issue 6343, eaam5439
DOI: 10.1126/science.aam5439

Figures

  • Fig. 1 ∆LBS-Xist contains a deletion of the LBR binding site on the Xist RNA.

    (A) PCR of the genomic DNA of individual ΔLBS embryonic stem cell clones using primers flanking the LBS region. (B) The ΔLBS region defined by Sanger-sequenced PCR product of ΔLBS (top, red bar). A subset of sequence read pairs from RAP-DNA is shown. There are many read pairs that connect the LBS region and the rest of Xist in wild-type cells (top panel) and many read pairs that skip the entire LBS region in ΔLBS cells (bottom panel). (C) The number of read pairs connecting the LBS region and Xist as well as read pairs that skip the LBS junction in wild-type and ΔLBS cells, respectively. (D) Distribution plot of the genomic insert size for wild-type (black) and ΔLBS cells (red). Dashed line represents the average fragment size of the read pairs that skip the LBS region. (E) Quantitative RT-PCR of wild-type and ΔLBS cells using the primer set inside or outside of the LBS region, respectively. The graph represents the ratio of relative expression level of primer set 1 to primer set 2. The numbers represent the coordinates of the primers on the Xist cDNA. (F) Two-color single-molecule RNA FISH using probes against a region on Xist outside of the LBS region along with the probes against either the sense strand (top) or the antisense strand (bottom) of the LBS region. Although Xist RNA is colocalized with sense LBS RNA in wild-type cells, no sense LBS RNA signal was observed in ΔLBS cells. Neither wild-type cells nor ΔLBS cells show antisense LBS RNA signal in 80 single cells imaged for each.

  • Fig. 2 Discordant read pairs represent RAP-DNA probe sequences, not an inversion.

    (A) A schematic illustration of how we compute coverage of discordant read pairs across Xist. A window the size of the LBS region (784 base pairs) was slid base by base along Xist, and, for each window, the number of discordant read pairs that had one read fully contained within the window was counted. For all windows other than those overlapping the LBS, we did not count any read pair where one of the two reads mapped within the LBS region. (B) The numbers of discordant read pairs as described in (A) are plotted relative to the 5′ position of the window for ΔLBS and wild-type cells. (C) Coverage of proper and discordant read pairs in ΔLBS and wild-type cells plotted along with the probes used for RAP. (D) Distribution of insert size for proper (black) and discordant (red) read pairs for ΔLBS and wild-type cells. (E) Total number of discordant read pairs within Xist, Malat1(GSE55914), and Hdac2 (GSE55914) locus for RAP-DNA samples using DNA probes against Xist, Malat1, and Hdac2 RNA, respectively, and RNA probes against Xist RNA.

  • Fig. 3 Xist-LBR phenotypes observed by RAP-DNA are not affected by differences in read depth.

    (A) Total number of reads sorted in descending order and its corresponding observed phenotype. (B) Fold change of Xist enrichment across the X chromosome as measured by RAP-DNA averaged across all the actively transcribed genes and inactive genes on the X chromosome for ΔLBS, SHARP knockdown, LBR knockdown, and ΔLBS-BoxB + LMNB1-λN (ΔLBS rescue) and ΔA-HPRT, where each sample is randomly down-sampled to match the number of total reads of the ΔLBS sample.