A versatile mouse model of epitope-tagged histone H3.3 to study epigenome dynamics

Bachu, Mahesh; Tamura, Tomohide; Chen, C.; Narain, Ankur S.; Nehru, Vishal; Sarai, Naoyuki; Ghosh, Subrata; Ghosh, Anu; Kavarthapu, Raghuveer; Dufau, Maria L.; Ozato, Keiko

doi:10.1074/jbc.ra118.005550

Cited by 12 publications

(12 citation statements)

References 36 publications

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“…We found that exogenously added H3.3 was preferentially incorporated into transcriptionally active genes, rather than inactive genes. This distribution pattern is consistent with many previous studies ( Bachu et al, 2019 ; Mito et al, 2005 ; Pchelintsev et al, 2013 ). The RhIP-ChIP-qPCR analysis also demonstrated the H3.3 enrichment at the transcriptionally active GAPDH, as compared to that at the inactive gene, LNC02199 ( Figure 1—figure supplement 1B and C ).…”

Section: Resultssupporting

confidence: 93%

Chromatin structure-dependent histone incorporation revealed by a genome-wide deposition assay

Tachiwana

Dacher

Maehara

et al. 2021

eLife

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In eukaryotes, histone variant distribution within the genome is the key epigenetic feature. To understand how each histone variant is targeted to the genome, we developed a new method, the RhIP (Reconstituted histone complex Incorporation into chromatin of Permeabilized cell) assay, in which epitope-tagged histone complexes are introduced into permeabilized cells and incorporated into their chromatin. Using this method, we found that H3.1 and H3.3 were incorporated into chromatin in replication-dependent and -independent manners, respectively. We further found that the incorporation of histones H2A and H2A.Z mainly occurred at less condensed chromatin (open), suggesting that condensed chromatin (closed) is a barrier for histone incorporation. To overcome this barrier, H2A, but not H2A.Z, uses a replication-coupled deposition mechanism. Our study revealed that the combination of chromatin structure and DNA replication dictates the differential histone deposition to maintain the epigenetic chromatin states.

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Section: Resultssupporting

confidence: 93%

Chromatin structure-dependent histone incorporation revealed by a genome-wide deposition assay

Tachiwana

Dacher

Maehara

et al. 2021

eLife

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“…Further, as we find here (fig. S3), H3.3 is enriched at inflammatory genes, though its function in this context is unknown 6,30 . To study the function of H3.3 in inflammatory gene induction we generated H3f3a/H3f3b double knockout (DKO) RAW264.7 (macrophage-like) mouse cell lines through CRISPR targeting of both H3f3a and H3f3b .…”

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confidence: 99%

Phosphorylation of the ancestral histone variant H3.3 amplifies stimulation-induced transcription

Armache

Yang

Robbins

et al. 2019

Preprint

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115In vitro kinase assay and dot blot: Recombinant CHK1 kinase (Sigma) was incubated with kinase buffer (40mM 116 HEPES pH7.4, 20mM MgCl2), Magnesium/ATP cocktail (EMD) and histone tail peptides for overnight at 37C (Total 117 reaction 15ul, 2ug Peptide, Mg(4.5mM)/ATP(30uM) cocktail and 4ng Enzyme). The samples were then added with 118 5ul of 0.5%SDS followed by boiling for 5min at 95C. The samples were dropped on a dry nitrocellulose membrane 119 and probed with a-H3S31ph antibody. 121CRISPR targeting of H3.3: CRISPR targeting H3f3b and H3f3a was performed in RAW264.7 cells using methods 122 described in Ran et al. 2013 18 . Targeting was done consecutively first targeting H3f3b, then using H3f3b mutants 123 to target H3f3a. 124The gRNAs (Primers caccTAGAAATACCTGTAACGATG forward aaacCATCGTTACAGGTATTTCTA reverse for 125 H3f3a and caccGAAAGCCCCCCGCAAACAGC forward aaacGCTGTTTGCGGGGGGCTTTC reverse for H3f3b)

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“…MEFs were also isolated from these mice and immortalized as described previously (24). The expression levels of ISGs, Viperin , bone marrow stromal cell antigen-2 ( Bst-2 ), Isg15 , Isg54 , myxovirus resistance dynamin like GTPase 2 ( Mx2 ), guanylate binding protein 2 ( Gbp2 ), and Isg56 that are known to be highly increased in cells upon IFN stimulation (3638394041), were measured in WT and viperin KO BMDMs or MEFs (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

Viperin Differentially Induces Interferon-Stimulated Genes in Distinct Cell Types

Kim

Eom

et al. 2019

Immune Netw

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Viperin is an IFN-stimulated gene (ISG)-encoded protein that was identified in human primary macrophages treated with IFN-γ and in human primary fibroblasts infected with cytomegalovirus (CMV). This protein plays multiple roles in various cell types. It inhibits viral replication, mediates signaling pathways, and regulates cellular metabolism. Recent studies have shown that viperin inhibits IFN expression in macrophages, while it enhances TLR7 and TLR9-mediated IFN production in plasmacytoid dendritic cells, suggesting that viperin can play different roles in activation of the same pathway in different cell types. Viperin also controls induction of ISGs in macrophages. However, the effect of viperin on induction of ISGs in cell types other than macrophages is unknown. Here, we show that viperin differentially induces ISGs in 2 distinct cell types, macrophages and fibroblasts isolated from wild type and viperin knockout mice. Unlike in bone marrow-derived macrophages (BMDMs), viperin downregulates the expression levels of ISGs such as bone marrow stromal cell antigen-2, Isg15, Isg54, myxovirus resistance dynamin like GTPase 2, and guanylate binding protein 2 in murine embryonic fibroblasts (MEFs) treated with type I or II IFN. However, viperin upregulates expression of these ISGs in both BMDMs and MEFs stimulated with polyinosinic-polycytidylic acid or CpG DNA and infected with murine CMV. The efficiency of viral entry is inversely proportional to the expression levels of ISGs in both cell types. The data indicate that viperin differentially regulates induction of ISGs in a cell type-dependent manner, which might provide different innate immune responses in distinct cell types against infections.

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A versatile mouse model of epitope-tagged histone H3.3 to study epigenome dynamics

Cited by 12 publications

References 36 publications

Chromatin structure-dependent histone incorporation revealed by a genome-wide deposition assay

Chromatin structure-dependent histone incorporation revealed by a genome-wide deposition assay

Phosphorylation of the ancestral histone variant H3.3 amplifies stimulation-induced transcription

Viperin Differentially Induces Interferon-Stimulated Genes in Distinct Cell Types

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