Extended Data Fig. 5 |. H1 density regulates higher order chromatin structure in vivo.

a, b, Freshly isolated spleens from WT and H1cTKO;Vav-iCre mice were prepared for transmission electron microscopy (TEM) to visualize (a) nuclear size and (b) chromatin density, as described. Nuclear area and nuclear density were quantified using ImageJ. WT nuclei (n = 22) and H1cTKO;Vav-iCre nuclei (n = 17). Images were selected at random using several sections. The unpaired t-test was used. Whiskers represent minima and maxima, boxes represent the 25%-to-75%, with the mean shown. c, The average C-scores of topologically associated domains (TADs) in each condition were calculated. The Hi-C A and B compartments are characterized by a positive and negative C-score, respectively. TADs with differences in C-score between H1cTKO;Vav-iCre and WT (dCS) >0.25 were considered decompacted, and those with dCS <-0.25 compacted. TADs which changed from chromatin compartments A-to-B or B-to-A were defined as 100 kb regions with a dCS >0.25 or dCS <-0.25 and a change in the C-score sign. The rolling average change was locally estimated with a scatterplot smoothing (loess) curve. d, Change in TAD compartment score as a function of TAD size. e, Meta-analysis of A-B compartment TAD borders which were altered upon H1 depletion demonstrating A compartment spreading into B TADs. f, A genome-wide meta-region visualization of the Hi-C contact frequencies for all regions which differed between wild-type and H1cTKO;Vav-iCre CD8⁺ T cells. Heatmaps of the Hi-C contact frequency for wild-type (top) and H1cTKO;Vav-iCre (bottom) meta-regions were prepared using coolpup.py. g, A 15-state chromatin-state model was established in CD8⁺ T cells using ChromHMM. Segmentation was performed with binarized bed files for H3K36me2, RNA-seq gene expression (GE), Pol II Ser2P, H3K4me3, ATAC-seq, H3K9me3, H3K27me3, CTCF, H3K4me1, H3K36me2 (accession numbers are available in Supplementary Table 2; also see Methods). Heat maps for model parameters are shown. h, ATAC-seq was performed on WT and H1cTKO;Vav-iCre splenic CD8⁺ T cells and random sampling of reads was performed as needed to ensure the same number of reads between samples. Read lengths were calculated for ATAC-seq sequencing reads that fell completely within each chromatin state and are plotted as the number of reads for a given read length divided by the size of each chromatin state in base pairs. The change in nucleosome repeat length (NRL; “left shift”) and the change in chromatin accessibility (“up shift”) within nucleosomal DNA were calculated using custom Python scripts, entitled “NRLfinder,” which we have made available as an executable file (see Methods). Both the left shift, in base-pairs, and the up shift, as a percent of wildtype, are indicated on each plot along with an arrow indicating the direction of change. Plots are grouped by “Chromatin Type”, and types are determined based on their behaviour upon H1 depletion: unaffected (Type 1), left shift (Type 2), left shift and up shift (Type 3). i, Contribution of Types 1, 2, and 3 chromatin in CD8⁺ T cells to the whole genome, and to the A and B Hi-C compartments. j, The Hi-C C-score for each genomic region is shown, grouped by chromatin state. Chromatin states are grouped in Type 1, 2, and 3 chromatin, shown below. k, l, 100 kb genomic segments composed purely (>95%) of (k) Type 3 and (l) Type 2 were analysed. In brief, the C-score was calculated for each 100 kb genomic segment in WT and H1cTKO;Vav-iCre CD8⁺ T cells using the CscoreTool and analysed as in c. m, Types 1, 2, and 3 chromatin were identified in mESCs using a previously reported 15-state ChromHMM model²⁵. The density distribution of normalized H1 ChIP signals from publicly available H1 ChIP-seq in mESCs²⁷ in Types 1, 2, and 3 chromatin are shown. Density was calculated using geom_density (ggplot2). n, Pooled MNase-digested chromatin isolated from wild-type mouse splenocytes (n = 9 mice) was subjected to sucrose gradient ultracentrifugation to isolate mono- and polynucleosomes. Left, agarose gel demonstrating DNA size distributions within each gradient fraction. Right, samples were subjected to mass spectrometry and H1 peptides corresponding to H1c, H1d and H1e were normalized to total peptide abundance. Shown are the Z-scores for each peptide (diamond, H1c; square, H1d; triangle, H1e) and average Z-score (blue/red bar) within each fraction. Note: ns, not statistically significant; * P ≤ 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.