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The linker histone H1.4 condenses chromatin in maturing postmitotic neurons

Andrew I. AldridgeiD, Martine W. TremblayiD, Vijyendra RameshiD, Xiaolin WeiiD, Yong-Hui JiangiD, Anne E. WestiD

Abstract

H1 histones are abundant nuclear proteins that bind to linker DNA at the entry and exit points of nucleosomes. Although individual H1 family members play partially redundant roles in chromatin organization, the discovery of disease-associated mutations in H1 genes has raised interest in their cell-type specific functions. Heterozygous, de novo frameshift mutations in H1-4 cause the neurodevelopmental disorder Rahman Syndrome, which is characterized by mild to severe intellectual disability along with other neurological and morphological features. H1.4 has mostly been studied in dividing cells, and its expression in the brain was poorly understood. Here we characterized the expression of H1f4 mRNA and H1.4 protein in the brains of male and female mice across postnatal development. Using an epitope-tagged H1f4 knockin mouse, we show that this linker histone is robustly expressed throughout the brain including in mature, post-mitotic neurons of adult mice. By chromatin immunoprecipitation, we observe that H1.4 binds broadly across the genome in neural progenitors and accumulates in heterochromatin over the course of neuronal maturation. Finally we show that developmental maturation of chromatin compaction in cerebellar granule neurons is disrupted in H1f2/H1f4 double knockout mice. These data raise the possibility that the neurological changes in Rahman Syndrome may arise from disrupted functions of histone H1.4 in neurons.

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Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (29 Jul 2026) · CC BY 4.0

Epitope-tagged knockin mice show histone H1.4 protein accumulates in postmitotic neurons, shifts toward B-compartment heterochromatin with maturation, and H1f2/H1f4 loss impairs cerebellar granule neuron chromatin compaction.

H1.4 is mutated in the neurodevelopmental disorder Rahman Syndrome, yet its brain expression and its role in chromatin organization of postmitotic cells were poorly understood. This work maps H1.4 expression and genomic binding, and tests how H1f2/H1f4 loss alters chromatin compartments in mouse cerebellum.

Key results

  1. ShownH1f4 mRNA declines as neurons leave the cell cycle but stays measurable in adult brain, while tagged H1.4 protein increased over time in cortex and hippocampus, suggesting post-transcriptional control or altered protein turnover.

    qRT-PCR: H1 mRNAs highest in embryo, declining with age; H1f4 and H1f0 highest in adult regions. Western blots at P7, P21, P60: H1.4 protein increased over time in cortex and hippocampus (n=3 knockin, 1 WT per timepoint; one-way ANOVA). · Histone H1f4 mRNA expression is reduced in…; H1.4 protein is widely expressed in neurons and…; Figure legends

  2. ShownH1.4 genomic distribution shifts with neuronal maturation: in progenitors it includes active H3K27ac regions and active TSSs, whereas in adult cerebellum it tracks heterochromatin like MeCP2 and aligns with B compartments.

    Anti-Myc ChIP-seq in P7 granule neuron progenitors (GNPs) vs P60 cerebellum, n=3 per group; H1.4 enriched at active TSSs in GNPs but de-enriched in adult; HiCAR compartments show A-like overlap in GNPs, B-like in adult. · The genomic distribution of H1.4 shifts toward…; Developmental chromatin compaction is impaired…; Figure legends

  3. ShownH1f2/H1f4 double knockout (dKO) adult cerebellum shows altered chromatin compartments, mostly loss of compaction, and the effect is largest in the strongest B-compartment regions.

    HiCAR (two WT and two dKO P60 cerebella): 1005 compartment bins significantly less compact in dKO vs 129 more compact; few B-to-A or A-to-B switches; limma with Benjamini-Hochberg FDR, adjusted P<0.05. · Developmental chromatin compaction is impaired…; HiCAR; HiCAR; Figure legends

  4. SuggestedRegions that fail to compact in dKO tend to be those that normally gain compaction between GNPs and mature granule neurons, pointing to a failure of developmental chromatin maturation.

    Comparison of 100kb genomic bins that gain or lose compaction in dKO versus WT and in GNPs versus mature neurons; no statistical test is stated for this comparison. · Developmental chromatin compaction is impaired…; Figure legends

  5. ShownGene expression changes in dKO cerebellum are small and do not map to the compaction changes; H1f0 is upregulated, consistent with compensation.

    Bulk RNA-seq, four dKO vs four C57BL/6J: 24 genes down, 44 up. Adjusted P>0.9 for every genotype comparison within compartment deciles. DE genes were mostly in weak A compartments, compaction changes in strong B. · Developmental chromatin compaction is impaired…; RNA-Seq; Supplemental figures

Methods, data and reproducibility

A 3xFLAG-Myc-H1f4 knockin mouse was used for western blot, immunofluorescence and anti-Myc ChIP-seq, alongside RNAScope FISH, qRT-PCR, HiCAR chromatin conformation and RNA-seq in H1f2/H1f4 dKO versus wild-type cerebellum.

  • qRT-PCR and RNAScope FISH of H1 mRNAs in cortex, hippocampus and cerebellum across development · mouse
  • Western blots of acid-extracted histones from heterozygous 3xFLAG-Myc-H1f4 knockin mice vs WT littermates at P7, P21, P60 · mouse · n = n=3 knockin and 1 WT per timepoint
  • Anti-Myc ChIP-seq from P7 purified GNPs and P60 cerebellum of knockin mice · mouse · n = n=3 per group · GSE333216
  • HiCAR from P7 CD1 GNPs and P60 cerebellum of dKO and C57BL/6J mice · mouse · n = two each · GSE333217
  • Bulk RNA-seq of adult cerebellum from H1f2/H1f4 dKO and C57BL/6J mice · mouse · n = four per genotype · GSE333219
Methods & tools
HiCAR, RNAScope FISH, nf-core/chipseq v2.0.0, BWA v0.7.17 (ChIP-seq), deepTools2.0 (bamCompare), featureCounts v2.0.3, jianhong/hicar ver2rc, cooltools v0.6.0, limma, DESeq2 v1.38.3, nf-core/rnaseq v3.20.0, TEtranscripts v2.2.3
Reagents & environment
Pipelines and versions: nf-core/chipseq v2.0.0, jianhong/hicar ver2rc, nf-core/rnaseq v3.20.0; reads aligned to mouse GRCm38; DESeq2 v1.38.3; STAR v2.7.11b; Salmon v1.10.3; cooltools v0.6.0; MACS2 v2.2.9.1.
Reproducibility gaps
No code repository is given; data are deposited in GEO (accessions only, no URLs). The dKO line was a gift from another laboratory; availability of the knockin line is not stated.
Limitations (5)
  • Upregulation of H1f0 in the dKO and general compensation among H1 family members may mask the effects of losing H1.2 and H1.4. · stated by the authors
  • The ChIP-seq signal for Myc-H1.4 had low signal-to-noise, so immunoprecipitated reads had to be normalized to input. · stated by the authors
  • Most altered compaction was in the most B-like heterochromatin, and how compartment changes influence gene expression remains unresolved; few expression changes were seen. · stated by the authors
  • The dKO removes both H1f2 and H1f4, so effects cannot be assigned to H1.4 alone; Rahman Syndrome frameshift mutants were not tested here. · noted in this summary
  • HiCAR used only two samples per genotype, western blots had one WT sample per timepoint, and the ChIP-seq age comparison contrasts purified GNPs with whole adult cerebellum of differing composition. · noted in this summary

Who should read:Read this if you study linker histone function, neuronal chromatin compartments, or the mechanisms that may underlie Rahman Syndrome.

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