Previous studies suggested that MeCP2 competes with linker histone H1, but this hypothesis has never been tested in vivo. Here, we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) of Flag-tagged-H1.0 in mouse forebrain excitatory neurons. Unexpectedly, Flag-H1.0 and MeCP2 occupied similar genomic regions and the Flag-H1.0 binding was not changed upon MeCP2 depletion. Furthermore, mild overexpression of H1.0 did not alter MeCP2 binding, suggesting that the functional binding of MeCP2 and H1.0 are largely independent.
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This research was supported by NIH/NINDS 5R01NS057819 (H.Y.Z.), the Japan Society for the Promotion of Science (A.I.-I.), Genetically Engineered Mouse Core and Genomic and RNA Profiling Core at BCM, Neurovisualization Core at the BCM Intellectual and Developmental Disabilities Research Center (NIH/1U54HD083092), and NSF DMS#1263932 and CPRIT RP170387 (Z.L.). H.Y.Z. is an investigator with the Howard Hughes Medical Institute.
The authors declare no competing interests.
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Integrated Supplementary Information
Supplementary Figure 1 Expression of endogenous H1s in neuronal nuclei of Mecp2–/y and Mecp2Tg3/y mice.
a) Neuronal nuclei were isolated from mouse forebrains by fluorescence-assisted cell sorting (FACS) using anti-NeuN antibody conjugated with Alexa 555. Histogram of the compensated Alexa 555 intensity showed two clear peaks (top left). NeuN-positive fraction was isolated by sorting 70-75% of the nuclei from the right peak to increase specificity. NeuN-negative fraction was isolated as nuclei from the left peak. Forward scatter area (FSC-A) and back scatter area (BSC-A) indicated that NeuN-positive nuclei are significantly larger than NeuN-negative nuclei (top right). Anti-mouse IgG conjugated with Alexa 555 was used as a negative control (bottom). Positive fraction was almost negligible when the same threshold was used to sort nuclei labeled with anti-NeuN antibody (bottom left). The proportion of the sorted NeuN-positive nuclei to the total nuclei was comparable between Mecp2-/y, Mecp2Tg3/y and their controls: 54.6 ± 0.7% (Mecp2+/y in d), 55.7 ± 0.7% (Mecp2-/y in d), 52.1 ± 1.1% (Mecp2+/y in e) and 51.5 ± 1.1% (Mecp2Tg3/y in e). b, c) NeuN-positive and negative nuclei were sorted from Mecp2-/y and Mecp2Tg3/y mice together with their Mecp2+/y littermates, and were subjected to western blot to quantify MeCP2 protein levels. The samples from NeuN-positive and negative nuclei were run in a same gel, the images were adjusted with the same settings and were realigned to show the two groups side by side. MeCP2 level in NeuN-negative nuclei was significantly lower than that in NeuN-positive nuclei in both Mecp2+/y and Mecp2Tg3/y mice. d, e) Quantified results for b and c. MeCP2 levels were first normalized to the H3 levels. Relative MeCP2 levels, which were further normalized to the data from NeuN-positive nuclei of Mecp2+/y mice, are shown. MeCP2 was significantly enriched in NeuN-positive nuclei. N = 5 mice for each genotype from at least two litters. Paired t-test: p = 0.02, t4 = 3.71 (Mecp2+/y in d), p = 0.03, t4 = 3.26 (Mecp2+/y in e), and p = 0.001, t4 = 8.49 (Mecp2Tg3/y in e). f, g) Endogenous levels of H1.0, H1.c, and H1.x were quantified in cortical NeuN + neurons from Mecp2+/y and Mecp2-/y mice (8-10 weeks). The levels of H1s were normalized to H3 levels. Although neither of three H1 subtypes showed significant difference between Mecp2+/y and Mecp2-/y mice, H1.0 showed a small trend toward increase in Mecp2-/y mice. N = 6 (H1.0), 5 (H1.c), and 6 mice (H1.x) from at least three litters. Two-tailed Wilcoxon signed rank test: p = 0.22 (H1.0), 0.81 (H1.c), and 0.84 (H1.x). h, i) Endogenous levels of three H1 subtypes were quantified in sorted cortical NeuN + neurons of Mecp2+/y and Mecp2Tg3/y mice (8-10 weeks). Neither H1 showed significant difference between Mecp2+/y and Mecp2Tg3/y mice. N = 5 (H1.0), 7 (H1.c), and 6 mice (H1.x) from at least three litters. Two-tailed Wilcoxon signed rank test: p = 0.81 (H1.0), 0.69 (H1.c), and 0.84 (H1.x).
Supplementary Figure 2 Genome-wide distribution of MeCP2 in the frontal cortex of wild-type (WT) and Cam-tTA:Fl-H1.0 mice.
a) Representative tracks of normalized MeCP2 ChIP-seq reads obtained from the frontal cortex of WT and Cam-tTA:Fl-H1.0 mice. The tracks show data from the three biological replicates of each genotype (R1-R3) and the pooled data (P). b-d) MeCP2 ChIP-seq from both WT and Cam-tTA:Fl-H1.0 mice positively correlated with the density of methylated-CG (b) and methylated-CH (c) while they negatively correlated with the density of non-methylated cytosine (d). Methylation density was quantified as a ratio of the number of methylated-cytosine in either CG or CH to the number of total covered cytosine within 1 kb window. Bold lines indicate mean and light ribbons indicate 95% confidence intervals from the biological replicates. Note the overlapping in the confidence intervals. There was no significant difference between the genotypes. e) MeCP2 was depleted around CG-islands (CGIs) in both WT and Cam-tTA:Fl-H1.0 mice (top). The p-values were obtained from the biological replicates by running t-tests at 100-bp intervals and plotted around the CGIs (bottom). Dotted line indicates p = 0.05. There was no significant difference between the genotypes. f) Results from PCA analysis showing six MeCP2 ChIP-seq data sets. Note that the first PCA component (PC1) explained around 80% of variation (f2) and the PC1 values for six samples were comparable (f1).
a, b) Cam-tTA:Fl-H1.0:Mecp2-/y (fh-Mecp2-/y) mice and their littermate Cam-tTA:Fl-H1.0:Mecp2+/y (fh-Mecp2+/y) mice were generated in order to characterize H1.0 in vivo. Expression of Fl-H1.0 did not alter body weight (a) or phenotypic score (b) in Mecp2+/y or in Mecp2-/y mice (8-9 weeks). fh-Mecp2-/y mice showed increased body weight and phenotypic score, and the results were comparable to the results from Mecp2-/y mice. N = 11 (Mecp2+/y), 10 (fh-Mecp2+/y), 8 (Mecp2-/y), and 8 mice (fh-Mecp2-/y) from at least three litters. One-way ANOVA: p = 0.0003, F (3, 33) = 8.141 (body weight, a); p < 0.0001, F (3, 33) = 89.2 (phenotypic score, b). Tukey’s multiple comparison test for body weight (a): p = 0.7934 (Mecp2+/y vs. fh-Mecp2+/y); p = 0.0188 (Mecp2+/y vs. Mecp2-/y); p = 0.0256 (Mecp2+/y vs. fh-Mecp2-/y); p = 0.0024 (fh-Mecp2+/y vs. Mecp2-/y); p = 0.0034 (fh-Mecp2+/y vs. fh-Mecp2-/y); p = 0.9994 (Mecp2-/y vs. fh-Mecp2-/y). Tukey’s multiple comparison test for phenotypic score (b): p = 0.9603 (Mecp2+/y vs. fh-Mecp2+/y); p < 0.0001 (Mecp2+/y vs. Mecp2-/y); p < 0.0001 (Mecp2+/y vs. fh-Mecp2-/y); p < 0.0001 (fh-Mecp2+/y vs. Mecp2-/y); p < 0.0001 (fh-Mecp2+/y vs. fh-Mecp2-/y); p = 0.0620 (Mecp2-/y vs. fh-Mecp2-/y). c) RNA expression level of Fl-H1.0 in the cortex was similar in fh-Mecp2+/y and fh-Mecp2-/y mice (8-9 weeks). N = 5 mice per genotype from more than three litters. Two-tailed Mann Whitney U test: p > 0.9999. d) Protein level of Fl-H1.0 detected by anti-Flag antibody in fh-Mecp2-/y cortex was comparable to fh-Mecp2+/y mice (8-9 weeks). e) Quantification of (d). Flag intensity was normalized to H3 levels. N = 4 mice from more than three litters. Two-tailed t-test: p = 0.52, t6 = 0.69. Graphs show individual data and average ± sem. * p < 0.05, # p < 0.0001, n.s. not significant.
Supplementary Figure 4 Additional results from the analyses on Fl-H1.0 ChIP-seq showing little impact of MeCP2 depletion on Fl-H1.0 distribution.
a) Spearman correlation for the individual replicates of Fl-H1.0 and MeCP2 ChIP-seq reads. MeCP2 Chip-seq data set was pooled from three data-sets from the three Fl-H1.0 expressing mice (Cam-tTA:Fl-H1.0, i.e. fh-Mecp2+/y mice). The number of data points used to obtain the correlation between each pair is summarized in Supplementary Table 2. b) Results from PCA analysis showing six Fl-H1.0 ChIP-seq data sets from fh-Mecp2+/y mice and fh-Mecp2-/y mice (8-9 weeks). Note that the first PCA component (PC1) explained > 95% of variation (b2) and the PC1 values for six samples were similar (b1). c) Spearman correlation coefficients between the pooled Fl-H1.0 ChIP-seq reads and MeCP2 ChIP-seq reads (combined from three datasets from three mice per genotype) were analyzed using various window sizes from 0.2 kb to 10 Mb. The correlation for Fl-H1.0 ChIP-seq reads between fh-Mecp2+/y and fh-Mecp2-/y was consistently high regardless of the bin size. The number of data points used to obtain the correlation between each pair is summarized in Supplementary Table 3. d) Average enrichment of MeCP2 around the TSS (plus and minus 1 kb) of the genes grouped by their expression levels. The average enrichment was obtained from three biological replicates of fh-Mecp2+/y and fh-Mecp2-/y mice. There was no significant difference between the genotypes. N = 3 mice for each genotype. Two-way ANOVA; p = 0.74 (genotype factor). e) Average enrichment of MeCP2 around the TSS (plus and minus 1 kb) of the genes whose expression levels were either altered or unaltered in Mecp2-null mice. The average enrichment was obtained from biological replicates of fh-Mecp2+/y and fh-Mecp2-/y mice. There was no significant difference between the genotypes. N = 3 mice for each genotype. Two-way ANOVA; p = 0.68 (genotype factor). f) Fl-H1.0 density for each biological replicate was plotted along TSS of differentially expressed genes in layer 5 cortical pyramidal cells of Mecp2-/y mice. Although the results from the pooled data showed a small gap between fh-Mecp2+/y and fh-Mecp2-/y mice in the genes up in Mecp2-/y mice and in the genes unaltered (Fig. 3f), there was no consistent difference between three biological replicates.
Western-blot membranes were cut into pieces to probe for multiple antibodies. The scanned images were cropped around the molecular weight of the respective proteins and were presented in the figures. The images above show the full scans of the corresponding blots with the images of molecular size marker.
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Ito-Ishida, A., Yamalanchili, H.K., Shao, Y. et al. Genome-wide distribution of linker histone H1.0 is independent of MeCP2. Nat Neurosci 21, 794–798 (2018). https://doi.org/10.1038/s41593-018-0155-8
Nature Communications (2020)
Nature Communications (2020)