Cohesin controls X chromosome structure remodeling and X-reactivation during mouse iPSC-reprogramming

Abstract

Reactivation of the inactive X chromosome is a hallmark epigenetic event during reprogramming of mouse female somatic cells to induced pluripotent stem cells (iPSCs). This involves global structural remodeling from a condensed, heterochromatic into an open, euchromatic state, thereby changing a transcriptionally inactive into an active chromosome. Despite recent advances, very little is currently known about the molecular players mediating this process and how this relates to iPSC-reprogramming in general. To gain more insight, here we perform a RNAi-based knockdown screen during iPSC-reprogramming of mouse fibroblasts. We discover factors important for X chromosome reactivation (XCR) and iPSC-reprogramming. Among those, we identify the cohesin complex member SMC1a as a key molecule with a specific function in XCR, as its knockdown greatly affects XCR without interfering with iPSC-reprogramming. Using super-resolution microscopy, we find SMC1a to be preferentially enriched on the active compared with the inactive X chromosome and that SMC1a is critical for the decompacted state of the active X. Specifically, depletion of SMC1a leads to contraction of the active X both in differentiated and in pluripotent cells, where it normally is in its most open state. In summary, we reveal cohesin as a key factor for remodeling of the X chromosome from an inactive to an active structure and that this is a critical step for XCR during iPSC-reprogramming.

Keywords: X chromosome; X-inactivation; X-reactivation; cellular reprogramming; cohesin.

Fig. 1.

Functional shRNA-knockdown screen reveals that SMC1a knockdown affects X-reactivation. (A) Steps of the screening process. (B) Experimental design of the screen work-flow. (C) Characterization of the XGFP/OKSM-MEF iPSC-reprogramming system. Representative images of reprogramming time points (d0-d6-d10-d14) showing the colony formation in brightfield (BF) and the XGFP reactivation in green (Scale bars = 50 μm.) Reprogramming efficiency (Left quantification graph) was scored as the percent of primary colonies formed per input MEFs after 14 d of dox induction. X-Reactivation efficiency (Right graph) was scored as ratio between XGFP+ colonies and total colony number. Each graph depicts data from one experiment performed in triplicate (error bars = SEM). (D) Reprogramming efficiency upon shRNA knockdowns scored as the total colony number divided by the seeded cell number and normalized to LacZ shRNA as negative control (set as 1). Each bar is depicted in different colors according to Gene Ontology analysis. Black bars show shRNAs controls. (E) XCR efficiency scored as ratio between XGFP positive colony number and the total colony number and normalized to LacZ shRNA as a control (set as 1).

Fig. 2.

Impact of candidate knockdown on the reactivation of the endogenous X-linked gene Hprt and Xist de-repression (A) 6TG selection scheme upon knockdown and reprogramming to assess reactivation of the endogenous X-linked gene Hprt. On day 10 of reprogramming, iPSCs were dissociated and one-third of the cells has been reseeded on 24-well plate and cultured under 6TG drug selection for 6 d, which would eliminate iPSCs which have undergone XCR and reactivated Hprt. (B) Percentage of EpCAM1+ SSEA1+ iPSCs surviving 6 d of 6TG selection analyzed by FACS analysis. shRNAs used for knockdown are indicated in the X axis. Control hairpins against LacZ and GFP and for genes whose knockdown enhances XCR (Dnmt1Tcf3) are on the Left, while candidates with a potential role in XCR are on the Right. (C) Relative Xist expression upon XCR candidate knockdown in female ESCs (normalized to LacZ shRNA set as 1). Dark and light blue bars indicate two biological replicates. LacZ shRNA control and pluripotency genes Oct4 and Nanog with a known role in repressing Xist (–24), are shown on the Left as controls.

Fig. 3.

Cohesin complex members have a preferential role in XCR during iPSC reprogramming. (A) Total colony numbers upon cohesin members knockdowns in male cells. (B) Flow cytometry analysis of iPSC colonies w/wo cohesin knockdown stained for EpCAM1 and SSEA1 at day 11 of reprogramming in male cells. (C) Quantification of double-positive EpCAM1/SSEA1 cells upon cohesin knockdown. (D) Representative images of female iPS colonies at day 10 showing the colony formation in brightfield (BF) and the XGFP reactivation upon knockdown in green (Scale bars = 50 μm.) (E) Total colony number upon cohesin members' knockdown in female cells. All samples below the horizontal line are not significantly different from lacZ control. (F) XCR efficiency scored as ratio between XGFP positive colony number and the total colony number at day 10 upon cohesin members' knockdown in female cells. (G) Flow cytometry analysis of iPSC colonies w/wo cohesin knockdown stained by EpCAM1 and SSEA1 at day 11 of the reprogramming in female cells. (H) Quantification of double-positive EpCAM1/SSEA1 population in female cells. (I) XGFP percentage scored within the pluripotent population (EpCAM1 and SSEA1 double-positive cells). All samples below the horizontal line are significantly different from lacZ control with ****P ≤ 0.0001. In all panels Oct4 shRNA is shown as reprogramming control. In panels (A, C, E, F, H, and I): Mean ± SD is displayed. Statistical comparisons with control LacZ shRNA controls are indicated. ns P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 for one-way ANOVA with Dunnett's multiple comparisons test, n = 3 replicates.

Fig. 4.

Differential localization of SMC1a between active and inactive X chromosomes shapes X chromosome territory. (A) Representative images of Immuno-DNA FISH experiments in combination with super resolution microscopy (STORM). MEFs with Smc1a or LacZ control shRNA knockdown were stained with X-paint (green), H3K27me3 (orange), and SMC1A (magenta). The dashed areas outline the two X chromosome territories (Xi = inactive X; Xa = active X) (scale bars = 1 µm). Diffraction-limited images for X-paint, H3K27me3 and SMC1a are shown in the left three columns. STORM images of SMC1a are shown in the right column. (B) Average number of SMC1a localizations per chromosome in LacZ shRNA control and Smc1a knockdown MEFs for both active and inactive X chromosomes. (Mean ± SD is displayed. **P ≤ 0.01 for two-tailed paired t test). Numbers indicate the absolute number of counted cells. (C) Density of SMC1a localizations in LacZ shRNA control and Smc1a knockdown MEFs for both active and inactive X chromosomes. Density was quantified by dividing the number of SMC1a localizations identified in the active and inactive chromosome shown in (B) by the area of the corresponding chromosome (Mean ± SD is displayed. ns P > 0.05 for two-tailed paired t test). Numbers indicate the absolute number of counted cells. (D) Ratio of SMC1a localizations in Xa/Xi per nucleus. (Mean ± SD is displayed. **P ≤ 0.01 for one sample t test). Numbers indicate the absolute number of counted cells. (E) Quantification of the active and inactive X chromosome areas after control LacZ (Left) and Smc1a (Right) shRNA knockdown in MEFs. (Mean ± SD is displayed. ***P ≤ 0.001 for two-tailed paired t test). Numbers indicate the absolute number of counted cells. (F) Representative confocal images of H3K27me3 mark upon LacZ and Smc1a shRNA knockdown in MEFs. The dashes areas outline the H3K27me3 spot (Scale bars = 9 μm.) (G) Comparison of inactive X chromosomal H3K27me3 territory after LacZ and Smc1a shRNA knockdown. (****P ≤ 0.0001 for unpaired t test). Numbers indicate the absolute number of counted cells.

Fig. 5.

Remodeling of intra- and intermegadomain distances on the active and inactive X chromosomes during iPSC reprogramming. (A) Diagram of the experimental setup using oligopaint probes for regions a, b (Left megadomain) and c (Right megadomain). Within each region, two subregions were labeled and detected (a1, a2, b1, b2, c1, and c2), the distances between subregions were scored and the shortest distances between probes of different subregions (bold black arrows) selected as a–b and b–c distances. (see SI Appendix, Fig. S5A for probe location in relation to TADs and compartments). (B) Violin plots of the shortest distances between a–b and b–c megadomains in LacZ control knockdown MEFs, day 8 of reprogramming and ESCs labeled with oligopaint probes. The shortest distances measured for each locus are plotted. Oligopaint signals belonging to Xa and Xi were classified based by the overlap with the H3K27me3-rich signal accumulated on the Xi (see SI Appendix, Fig. S5B for representative examples). Median ± IQR is displayed. ns P > 0.05, ****P ≤ 0.0001 for two-tailed unpaired t test. Numbers indicate the absolute number of counted cells. (C) Violin plots of the distances between a–b and b–c mega domains in LacZ-control and Smc1a-shRNA MEFs, labeled with oligopaint probes. The shortest distances measured for each loci are plotted. Oligopaints signals belonging to Xa and Xi were classified based on the overlap with the H3K27me3-rich signal accumulated on the Xi. Median ± IQR is displayed. ns P > 0.05, **P ≤ 0.01, for two-tailed unpaired t test. Numbers indicate the absolute number of counted cells. (D) Representative confocal images (maximum intensity projections) of LacZ- and Smc1a-shRNA MEFs, labeled with oligopaint probes for regions a (AF488, in green), b (AF647, in magenta) and c (AF488, pseudocolored in cyan). Nuclear areas are shown with a dashed line, Xa and Xi were classified based on the overlap with H3K27me3-rich signal accumulated on the Xi (AF568, in yellow) (Scale bars = 5 μm.) (E) Violin plots of the distances between a–b and b–c mega domains in LacZ-control cells at day 8 of reprogramming, labeled with oligopaint probes. The shortest distances measured for each loci are plotted. Oligopaints signals belonging to Xa and Xi were classified based on the overlap with the H3K27me3-rich signal accumulated on the Xi, which is present in XaXi prereactivation cells and absent in XaXa reactivated cells. Median ± IQR is displayed. ns P > 0.05, *P ≤ 0.05, for two-tailed unpaired t test. Numbers indicate the absolute number of counted cells. (F) Representative confocal images (maximum intensity projections) of LacZ-control cells at day 8 of reprogramming labeled with Oligopaint probes for regions A (AF488, in green), B (AF647, in magenta) and C (AF488, pseudocolored in cyan). Nuclear areas are shown with a dashed line, Xa and Xi were classified based on the overlap with H3K27me3-rich signal accumulated on the Xi (AF568, in yellow) (Scale bars = 5 μm.) (G) Violin plots of the distances between a–b and b–c mega domains in LacZ-control and Smc1a-shRNA ESCs, labeled with oligopaint probes. The shortest distances measured for each loci are plotted. Median ± IQR is displayed. ns P > 0.05, **P ≤ 0.01, for two-tailed unpaired t test. Numbers indicate the absolute number of counted cells. (H) Representative confocal images (maximum intensity projections) of LacZ-control and Smc1a-shRNA ESCs labeled with oligopaint probes for regions a (AF488, in green), b (AF647, in magenta), and c (AF488, pseudocolored in cyan). (Scale bars = 5 μm.)

Fig. 6.

Schematic representation of the proposed model for SMC1a function in the remodeling of the X chromosomes and XCR during iPSC-reprogramming. Under control conditions (Top), MEFs have an Xa, which is larger in volume and bound by more SMC1a protein (green circles), than the Xi, where Xist RNA coating repels SMC1a, leading to a more compact structure. Upon Smc1a knockdown (KD, Bottom), the Xi loses its megadomain structure (as shown in ref. 32), while the Xa becomes more compact (this study). On day 8 of iPSC-reprogramming, wt cells are undergoing XCR and reactivating XGFP. Some cells still contain an Xist-coated/H3K27me3 enriched Xi and an Xa (shaded, prereactivation), and some cells contain an Xa and an Xi on the path to becoming an Xa (Xi→ Xa) by having lost Xist coating/H3K27me3 enrichment (reactivated). Smc1a knockdown cells form iPSC colonies at similar rates to controls, but do not undergo XCR and reactivate XGFP as efficiently. The ? indicates that we did not assess the X chromosome structure in KD cells on day 8 and therefore can only speculate about it at this time point. At the end of reprogramming, wt iPSC colonies become mostly XGFP+, i.e., completion of XCR. In pluripotent stem cells (ESCs and iPSCS), the two Xa chromosomes reach their most relaxed state. Smc1a KD perturbs XCR/XGFP-reactivation in iPSCs and leads to restructuring of the Xa chromosomes in ESCs.