Abstract:Lsh is a chromatin remodeling factor that regulates DNA methylation and chromatin function in mammals. The dynamics of these chromatin changes and whether they are directly controlled by Lsh remain unclear. To understand the molecular mechanisms of Lsh chromatin controlled regulation of gene expression, we established a tethering system that recruits a Gal4-Lsh fusion protein to an engineered Oct4 locus through Gal4 binding sites in murine embryonic stem (ES) cells. We examined the molecular epigenetic events … Show more
“…In addition, CpG methylation, a mark appearing upon long-term repression 39 was not detected at the locus (Fig. 2e ), which was consistent with the previous observations that H3K9 methylation precedes DNA methylation at the Oct4 locus, and that it occurs only in embryonic stem cells upon differentiation 29 , 39 . Fig.…”
Section: Resultssupporting
confidence: 92%
“…LSH can perform nucleosome sliding in vitro and its activity is enhanced by CDCA7, a protein that causes a related syndrome, known as ICF2, when it is mutated [26][27][28] . LSH recruitment results in reduced chromatin accessibility in vivo and LSH function is associated with transcriptional silencing [27][28][29][30][31] . LSH exerts its effects on repeat sequences embedded in heterochromatin and is associated with high DNA methylation levels 30,32 .…”
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.
“…In addition, CpG methylation, a mark appearing upon long-term repression 39 was not detected at the locus (Fig. 2e ), which was consistent with the previous observations that H3K9 methylation precedes DNA methylation at the Oct4 locus, and that it occurs only in embryonic stem cells upon differentiation 29 , 39 . Fig.…”
Section: Resultssupporting
confidence: 92%
“…LSH can perform nucleosome sliding in vitro and its activity is enhanced by CDCA7, a protein that causes a related syndrome, known as ICF2, when it is mutated [26][27][28] . LSH recruitment results in reduced chromatin accessibility in vivo and LSH function is associated with transcriptional silencing [27][28][29][30][31] . LSH exerts its effects on repeat sequences embedded in heterochromatin and is associated with high DNA methylation levels 30,32 .…”
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.
“…A more complete understanding of the epigenetic underpinnings of medulloblastoma could provide valuable avenues of research for therapeutic interventions that have the potential to be more effective and less harmful than our current non-specific DNA damaging radiation and chemotherapeutics. As a member of a family of chromatin remodelers with putative roles in senescence 41,44,69,70 , repression of tumor suppressor genes 41,44 , stem cell control 38,45,48,51 , Hox gene control 71 , and DNA repair 72 , our identification of HELLS upregulation in medulloblastoma could be an important finding. That this upregulation is a result of SHH pathway activation and HELLS is also upregulated in normal murine cerebellar development adds weight to this theory.…”
Medulloblastoma is a malignant pediatric tumor that arises from neural progenitors in the cerebellum. Despite a five-year survival rate of ~70%, nearly all patients incur adverse side effects from current treatment strategies that drastically impact quality of life. Roughly one-third of medulloblastoma are driven by aberrant activation of the Sonic Hedgehog (SHH) signaling pathway. However, the scarcity of genetic mutations in medulloblastoma has led to investigation of other mechanisms contributing to cancer pathogenicity including epigenetic regulation of gene expression. Here, we show that Helicase, Lymphoid Specific (HELLS), a chromatin remodeler with epigenetic functions including DNA methylation and histone modification, is induced by Sonic Hedgehog (SHH) in SHH-dependent cerebellar progenitor cells and the developing murine cerebella. HELLS is also up-regulated in mouse and human SHH medulloblastoma. Others have shown that HELLS activity generally results in a repressive chromatin state. Our results demonstrate that increased expression of HELLS in our experimental systems is regulated by the oncogenic transcriptional regulator YAP1 downstream of Smoothened, the positive transducer of SHH signaling. Elucidation of HELLS as one of the downstream effectors of the SHH pathway may lead to novel targets for precision therapeutics with the promise of better outcomes for SHH medulloblastoma patients.
“…Previous work has shown that Lsh is an in vitro chromatin remodeler [28], that Lsh regulates nucleosome occupancy at specific loci in vivo [13,20] and that artificial recruitment of Lsh reduces chromatin accessibility at the tethered site in vivo [53]. Using MNase digestion followed by high-throughput sequencing, we characterized genome wide changes in MNase digestibility mediated by Lsh.…”
Dynamic regulation of chromatin accessibility is a key feature of cellular differentiation during embryogenesis, but the precise factors that control access to chromatin remain largely unknown. Lsh/HELLS is critical for normal development and mutations of Lsh in human cause the ICF (Immune deficiency, Centromeric instability, Facial anomalies) syndrome, a severe immune disorder with multiple organ deficiencies. We report here that Lsh, previously known to regulate DNA methylation level, has a genome wide chromatin remodeling function. Using micrococcal nuclease (MNase)-seq analysis, we demonstrate that Lsh protects MNase accessibility at transcriptional regulatory regions characterized by DNase I hypersensitivity and certain histone 3 (H3) tail modifications associated with enhancers. Using an auxin-inducible degron system, allowing proteolytical degradation of Lsh, we show that Lsh mediated changes in nucleosome occupancy are independent of DNA methylation level and are characterized by reduced H3 occupancy. While Lsh mediated nucleosome occupancy prevents binding sites for transcription factors in wild type cells, depletion of Lsh leads to an increase in binding of ectopically expressed tissue specific transcription factors to their respective binding sites. Our data suggests that Lsh mediated chromatin remodeling can modulate nucleosome positioning at a subset of putative enhancers contributing to the preservation of cellular identity through regulation of accessibility.
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