LSH mediates gene repression through macroH2A deposition

The human Immunodeficiency Centromeric Instability Facial Anomalies (ICF) 4 syndrome is a severe disease with increased mortality caused by mutation in the LSH gene. Although LSH belongs to a family of chromatin remodeling proteins, it remains unknown how LSH mediates its function on chromatin in vivo. Here, we use chemical-induced proximity to rapidly recruit LSH to an engineered locus and find that LSH specifically induces macroH2A1.2 and macroH2A2 deposition in an ATP-dependent manner. Tethering of LSH induces transcriptional repression and silencing is dependent on macroH2A deposition. Loss of LSH decreases macroH2A enrichment at repeat sequences and results in transcriptional reactivation. Likewise, reduction of macroH2A by siRNA interference mimicks transcriptional reactivation. ChIP-seq analysis confirmed that LSH is a major regulator of genome-wide macroH2A distribution. Tethering of ICF4 mutations fails to induce macroH2A deposition and ICF4 patient cells display reduced macroH2A deposition and transcriptional reactivation supporting a pathogenic role for altered marcoH2A deposition. We propose that LSH is a major chromatin modulator of the histone variant macroH2A and that its ability to insert marcoH2A into chromatin and transcriptionally silence is disturbed in the ICF4 syndrome.


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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. Sample size was not predetermined by statistical method as this work did not involve animal models or human subjects. All high-throughput sequencing assays were performed in triplicates using independent biological (cell culture) samples derived from individual embryos. All mutants were selected in advance. For all other assays, sample size was decided in advance, and uniform.
No data was excluded.
Each experiment, including ChIP-seq samples, includes at least three independent replicates to conduct statistical analysis. ChIP-seq samples as well as their respective Input samples were derived as biologic replicates from three murine embryonic fibroblast cell lines for each genotype. Each cell line was derived from an individual embryo. All immunoblots were repeated at least three times with different biological samples and led to similar results. All attempts at replication for all other assays were successful.
No human participants or animal models were reported in this manuscript. Experimental groups were selected based on the individual genetic variant (WT, KO or Lsh mutants). Appropriate controls were included for each experiment, such as no rapamycin treatment and no modified Oct4 allele (tethering assay), normal IgG control (ChIPs), cells transfected with control shRNA or control siRNA, Input DNA and H2B (ChIp-seq).
Data sets were not analyzed blindly, but were all processed according to uniform and identical processing steps.