The linker histone H1.0 generates epigenetic and functional intratumor heterogeneity

Abstract

Tumors comprise functionally diverse subpopulations of cells with distinct proliferative potential. Here, we show that dynamic epigenetic states defined by the linker histone H1.0 determine which cells within a tumor can sustain the long-term cancer growth. Numerous cancer types exhibit high inter- and intratumor heterogeneity of H1.0, with H1.0 levels correlating with tumor differentiation status, patient survival, and, at the single-cell level, cancer stem cell markers. Silencing of H1.0 promotes maintenance of self-renewing cells by inducing derepression of megabase-sized gene domains harboring downstream effectors of oncogenic pathways. Self-renewing epigenetic states are not stable, and reexpression of H1.0 in subsets of tumor cells establishes transcriptional programs that restrict cancer cells' long-term proliferative potential and drive their differentiation. Our results uncover epigenetic determinants of tumor-maintaining cells.

Figure 1. Intratumor heterogeneity of H1.0 levels.

A. qRT-PCR analysis of H1F0 mRNA levels in SSEA1⁺ and SSEA1^- cells isolated from 5 tumors induced by in vitro-transformed fibroblasts (15). Values indicate average ± SEM of three technical replicates. B-C. Quantitative immunodetection of H1.0 and H1.4 (red) by immunofluorescence microscopy in the indicated sorted tumor cells and telomerase-immortalized parental cells (hTERT). Scale bar: 20 μm. Two asterisks indicate p < 0.01 (Student t-test). 37 < N < 60 (C). D-E. Quantitative immunodetection of H1.0 by imaging flow cytometry in unsorted tumor cells. SSEA1⁺: self-renewing cells, CD166^low: highly differentiated cells, 7’AAD: nuclei, live/dead⁺: dead cells, H2Kd⁺: host mouse cells excluded from analysis. Scale bar: 10 μm. Scatter plot of H1.0 and SSEA1 levels in individual cells (E). F-G. Quantitative immunodetection of H1.0 and SSEA1 in GBM samples by immunofluorescence microscopy. Scale bar 10 μm. Quantification of H1.0 levels in 3 normal (N1-N3) and 4 tumor (Tum1-Tum4) samples (G). One and two asterisks indicate p < 0.05 and p < 0.01, respectively (Student t-test). 15 < N < 910. H. Quantification of H1F0 mRNA levels in cells from three GBM samples by RNA-seq. CSC: Cells grown as neurospheres FCS: cells differentiated in vitro by FCS addition, Tumor: whole tumor population. I-K. Quantitative immunodetection of H1.0 and ITGA6 in breast cancer samples by immunofluorescence microscopy. Scale bar 10 μm. Quantification of H1.0 levels in 2 normal breast tissues (N) and tumor samples of different histological grade. One and two asterisks indicate p < 0.05 and p < 0.01, respectively, compared to N2 (Studen t-test). 38 < N <134 (J). Quantification of H1.0 levels in 2 normal breast tissues (N1, N2) and 5 tumor samples (Tum 1-5) (K). One and two asterisks indicate p < 0.05 and p < 0.01, respectively (Student t-test). 7 < N <131 except ITGA6+ Tum 4 for which N =2 (K).

Figure 2. Dynamic methylation of an enhancer element regulates H1F0 expression in cancer.

A. Representation of the H1F0 regions probed by bisulfite sequencing analysis (Arrows indicate PCR-amplified regions) and 450K Infinium arrays. B. Bisulfite sequencing analysis comparing the H1F0 CGI shore methylation status in the indicated subsets of sorted tumor cells. Lines represent individual sequenced molecules. White and black circles represent unmethylated and methylated CpGs, respectively. The percentage of methylation of selected CpGs is indicated. Two asterisks indicate p <0.001 (Two-way ANOVA). C. qRT-PCR of SSEA1⁺ tumor cells treated with 5-Aza-2'-deoxycytidine (5-Aza) or with DMSO as control. Values represent average ± SEM from three technical replicates. Two independent experiments gave similar results. One and two asterisks indicate p <0.05 and p < 0.01, respectively, compared to DMSO (One-way ANOVA and Tukey Kramer test). D. Luciferase reporter assay. Normalized luciferase activity comparing the transactivation potential of two negative control DNA fragments (Neg. 1 and 2 (21, 43)) and the H1F0 CGI shore. Values are average ± SEM from three experiments. Two asterisks indicate p < 0.01 (Student t-test) E. qRT-PCR comparing H1F0 expression levels in cells expressing H1F0-targeting sgRNAs and Cas9 fused to either wild type (WT) or a catalytically dead (ANV) DNMT3A. Values are average ± SEM from three biological replicates. One asterisk indicates p < 0.05 (Student t-test). F. Normalized luciferase activity comparing the transactivation potential of untreated (NT) or in vitro methylated (Meth) H1F0 CGI shore, and a negative control fragment (Neg. 1). Values are average ± SEM from five biological replicates. Two asterisks indicate p < 0.01 (Student t-test). G. Analysis of H1F0 methylation in TCGA samples. Patients are sorted based on H1F0 expression levels (RSEM) and the corresponding DNA methylation levels are visualized as a heatmap. Each row corresponds to a patient and the number of patients for each cohort is indicated. P-values of the Spearman’s rank correlation between H1F0 mRNA levels and methylation of CpG 7 and 8 is indicated (see Fig. S6). Expression levels are normalized across tissues.

Figure 3. H1.0 inhibits cancer cell self-renewal and drives differentiation.

A. Protocol used to modulate H1.0 levels in established tumors. B. Quantification of in vivo induction efficiency of H1.0 cDNA by qRT-PCR. Every number indicates a tumor, either uninduced (NT) or induced (DOX) for either 4 weeks or 3 days. Tumors shown in C are marked by stars. P-value from Student t-test C. Immunodetection of H1.0 in the indicated tumors analyzed by qRT-PCR in B (stars). Scale bar: 50 μm. D. Flow cytometry analysis of one uninduced and one induced tumor generated by cells containing Dox-responsive H1.0 cDNA constructs. The gates used to measure the fraction of undifferentiated (SSEA1⁺/CD166^high) or differentiated (SSEA1^-/CD166^low) cells are indicated. E,G. Quantification of the indicated subsets of cells by FACS (undifferentiated and differentiated cells) or by soft agar assay (in vitro self-renewing cells) in tumors generated by cells containing the indicated constructs. . P-value from Student t-test. F. Representative images of clonogenic soft agar assay. H. Limiting dilution transplantation assay for secondary tumor formation using cells from uninduced or induced primary tumors containing the indicated constructs. See also Table S2 and Supplementary text. I. Growth of secondary tumors induced by 5000 cells from uninduced or induced primary tumors containing the indicated constructs. Tumor volume values represent mean ± SEM from four tumors each. The value indicated by a dagger corresponds to 2 tumors, due to earlier culling of other animals bearing large tumors. P-value from Student t-test based on the last time point. J-K. Growth of secondary tumors induced by 1(K) or 2 (J) million cells from the indicated breast cancer cell lines containing the indicated constructs. Tumor volume values represent mean ± SEM from five tumors. Significance of the differences between uninduced and induced H1.0 cDNA or shH1.0 at the last time point is indicated (Student t-test). Differences between induced and uninduced control tumors (contr.), which express TurboRFP and the mir30 cassette from empty pTRIPZ, are not significant.

Figure 4. Activation of transcriptional programs supporting oncogenic self-renewal via upregulation of large gene domains.

A. Venn diagrams showing the percentage of genes upregulated (Up) or downregulated (Down) in SSEA1⁺ cells compared to SSEA1^- cells that are affected by H1.0 knockdown (See also Table S4). The significance of the overlap is indicated (hypergeometric test). B. Oncogenic gene signatures positively correlating with DOX samples. NES: normalized enrichment score, NOM p-val: nominal p-value, FDR q-val: false discovery rate q-value. Blue: stem cell-related gene signatures. C. GSEA plots of positional gene sets positively (CHR4Q21) or negatively (CHR19P13) correlating with DOX samples. D-E. Smoothed log₂ fold change of gene expression between DOX and NT (D) or washDOX and DOX samples (E) (shH1.0-1) along the human genome. Similar plots were obtained with shH1.0-2. Numbers indicate the chromosomes delimited with vertical lines. Only expressed genes (TPM > 0) are plotted. Blue: upregulated domains, red: downregulated domains. F. Distribution of GC content in RefSeq genes (all) and in the subsets of upregulated (Up) or downregulated (Down) DEGs. P-value from Student t-test.

Figure 5. Destabilization of nucleosome-DNA interactions in AT-rich regions in the absence of H1.0.

A. Average peak density profiles of H3K4me3 and H3K27me3 centered on H1.0 binding sites. B. Correlation between H1.0 peak density and DNA GC content. The best fit line of the experimental values for GC content > 0.4, the correlation coefficient R and statistical accuracy of the fit are indicated. C. H1.0 peak density along chromosome 3. The approximate location of cytogenetic bands, upregulated (Up) or downregulated (Down) positional gene sets identified by GSEA and the corresponding DNA GC content are shown. D-F, H. Comparison between genes upregulated (Up) or downregulated (Down) in response to H1.0 knock-down, with respect to the indicated features. Smoothed average density profile of H1.0 peaks (F) and FAIRE peaks (H) show enrichment of H1.0 and increased FAIRE signal (decreased nucleosome occupancy) upon H1.0 knock-down around the transcriptional start site (TSS) of upregulated genes. RefSeq genes (All) are shown as reference. P-value from paired t-test, with Benjamini-Hochberg adjustment for F and H. G. Heatmap showing tag density of H1.0 ChIP-seq around the TSS of genes upregulated upon H1.0 knock-down. Each line represents a gene. I. Relative abundance of the indicated types of FAIRE peaks in GC-rich and AT-rich domains. Nucleosome occupancy corresponding to the different types of FAIRE peaks is schematized next to the legend. Black line: DNA, gray circles: nucleosomes. Results from shH1.0-1 are shown. Similar results were obtained with shH1.0-2. P-value from Fisher's exact test for constitutive peaks. J. Average DNA GC content centered on FAIRE peaks that appear or disappear upon H1.0 knock-down. See also Fig. S14D. P-value from paired t-test with Benjamini-Hochberg adjustment. Grey area: 95% confidence interval of the best fit after smoothing. K. Number of upregulated or downregulated H1.0-sensitive genes showing altered acetylated or methylated H3K27 at TSS upon H1.0 knockdown. Differences are not significant (n.s., Fisher's exact test).

Figure 6. Low H1F0 levels correlate with low patient survival in multiple cancer types.

A-C. Kaplan-Meier analysis of the indicated datasets showing significant correlation between H1F0 levels and patient survival. P-value from Log-rank test. Multivariate analysis is shown in Fig. S16.