H1 variants are differentially enriched towards the nuclear periphery in T47D cells.

A) Confocal immunofluorescence of H1 variants (green) and DNA staining (blue). Bottom panels show the intensity profiles of H1 variants and DNA along the arrows depicted in the upper panel. Scale bar: 5µm. B) Example of one cell stained with H1.3 antibody in which four sections of an equivalent area and convergent to the nuclear center are shown. Sections are named A1 to A4, from the more peripheral section to the more central one. H1 variants immunofluorescence intensity were measured in each area and expressed as percentage. C) Quantifications of H1 variants using the segmentation illustrated in (B), where n=30 cells/condition were quantified, and data was represented in violin plots. Statistical differences between A1-A2, A2-A3 and A3-A4 for H1.0 and H1.4 are supported by paired t-test (***) p-value<0.001; (**) p-value<0.01; (ns/non-significant) p-value>0.05. D) H1 variants Input-subtracted ChIP-Seq median abundance per chromosome. Y-axis annotation indicated median %GC content per chromosome and their nuclear positions according to (Boyle et al., 2001; Girelli et al., 2020).

Super-resolution imaging shows that H1 variants occupy different regions in single-cells.

A) SRRF images of H1 variants (green) and DNA (red). Bottom-left Insets show the Reference confocal image in each case. Scale bar: 2µm. B) Percentage of co-localized pixels between H1 variants and DNA by SRRF imaging. n=20 cells/condition were quantified and values distribution were represented as violin plots. C) SRRF images of H1 variants (green) and H1.0 (red). Bottom-left Insets show the Reference confocal image in each case. In the bottom panel, the highlighted zoom-in insets at confocal (reference) or SRRF resolutions are shown. Scale bar: 2µm, scale bar in zoom-in insets: 200nm. D) Percentage of colocalized pixels of H1 variants with H1.0 by SRRF imaging. n=20 cells/condition were quantified and values distribution were represented as violin plots. E) Immunofluorescence of H1 variants (green) and DNA (blue) during metaphase. As H1.2 and H1.4 signal was not detected during metaphase (see main text and Figure 2-figure supplement 2 and 3), antibodies recognizing specific phosphorylations of these variants were used. To see H1 variants profiles along mitosis progression, see Figure 2-figure supplement 2 and 3. Scale bar: 5µm.

H1 variants presence within LADs and nucleoli.

A) Confocal (left) and super-resolution (right) images of a T47D cells stained for H1.2, H1.3 or H1.5 (in green) and Lamin A (in red) obtained using SRRF. Full nuclei (upper panel) and zoomed views of nuclear periphery (bottom panel) are shown. Scale bars: 2 µm (upper panel) and 200nm (bottom panel). Three representative cells are shown for each H1 variant. B) Pearson correlation coefficient (r) of H1 variants and H3K9me2 co-immunostaining signal. r values distribution in n=50 cells/condition are shown. Representative immunofluorescence images of H1 variants and H3K9me2 are shown in Figure 3-figure supplement 1A. Scale bar: 5µm. C) H1.3 and H3K9me2 immunofluorescence at confocal (reference) and super-resolution (SRRF) level. A zoom-in inset of the peripheral layer formed by both H1.3 and H3K9me3 is shown. Scaler bar: 2µm. D) Boxplots show the Input-subtracted H1 variants ChIP-Seq abundance within regions exclusively defined as NADs (NAD only) or LADs (LAD only) and those genomic segments defined as both NADs and LADs (NAD/LAD). A compartment regions are included as a reference. NADs coordinates were extracted from (Peng et al., 2023). E) Representative SRRF image of H1X, NPM1 and DNA. Zoom-in highlights the H1X nucleolar layer. Scale bar: 2µm. Scale bar in zoom-in: 0.2µm. F) Immunofluorescence of H1X, H1.2-pT165 or H1.4-pT146, Nucleophosmin (NPM1) and DNA. Insets show a zoom-in of a single nucleolus. Scale bar: 2µm. Three representative cells are shown for each H1.

Chromatin structural changes upon H1 depletion.

A) Representative SRRF images of DNA staining in the different H1 KD conditions indicated (multiH1, H1.2, H1.4 and H1X Dox-inducible shRNAs). In the bottom panels, a zoom-in inset is shown to appreciate DNA pattern in both Untreated and Dox conditions. Scale bar: 5µm (full nucleus) and 500nm (zoom-in). B) DNA-free areas percentage quantification in the different H1 KDs. n=20 cells/condition were quantified and the boxplot were constructed with the 20 average values in each condition. Statistical differences between Untreated and Dox-treated conditions are supported by paired-t-test. (***) p-value < 0.001; (ns/non-significant) p-value > 0.05. Additional representative images and full quantification are shown in Figure 4-figure supplement 1.

Nuclear distribution of H1 variants across multiple human cell lines.

A) Immunofluorescence analysis of H1 variants (green) with DNA staining (blue) in different cancer cell lines. Merged images are shown. H1.3 and H1.0 grids in HeLa cells are empty, as HeLa cells do not express these variants. Tumoral origin of the cell lines is indicated. Scale bar: 5µm. B) Immunofluorescence analysis of H1 variants (green) with DNA staining (blue) in cell lines lacking H1.3 and H1.5. Merged images are shown. Tumoral origin of the cell lines is indicated. Scale bar: 5µm. C) H1.4 and H1.0 show a more peripheral distribution in cell lines with a compromised H1 repertoire. Numbers correspond to Peripheral Index value in each cell line and color-coded as indicated. Each nucleus was divided into four equivalent sections A1-A4 and immunofluorescence signal of H1.4 or H1.0 was quantified. Peripheral index was defined as the ratio between Average value in A1 peripheral section and A4 central section. D) Immunofluorescence of H1X (green), nucleolar marker NPM1 (magenta) and DNA staining (blue). Merged images are shown. Insets show a zoom-in of a single nucleolus. Bottom panel includes cell lines lacking H1.3 and H1.5. Cell line origin is indicated. Scale bar: 5µm. E) Boxplots show the H1X Input-subtracted ChIP-Seq signal at eight groups of Giemsa bands in six different cancer cell lines. G-bands groups were defined according to (Serna-Pujol et al., 2021) (see Methods).