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Cell-autonomous function of LSH ATPase in de novo DNA methylation

ABSTRACT: Purpose: We aimed to determine whether the expression of either wild-type or catalytically inactive LSH, carrying a single point mutation in its ATP binding site (K237Q), could restore the levels and patterns of DNA methylation in Lsh-/- mouse embryonic fibroblasts (MEFs). Methods: Lsh-/- MEFs were transduced with lentiviral particles carrying empty pMSCV vector, pMSCV-LSH-3xFLAG and pMSCV-LSH K/Q-3xFLAG, respectively. Clonal cell lines were generated and tested for LSH expression. Two independent cell lines expressing wild-type LSH and two expressing LSH K/Q were used for further analyses and comparison with wild-type MEFs and Lsh-/- MEFs carrying the empty vector. Genomic DNA was purified from all six cell lines and methylated DNA immunoprecipitation (MeDIP) was performed as described in Weber et al., 2007, Nat Genetics. MeDIP libraries were generated and sequenced on Illumina HiSeq 2000 instrument. Results and conclusions: Our experiments demonstrate that the expression of wild-type LSH, but not the catalytically inactive LSH K/Q, in Lsh-/- MEFs leads to reestablishment of DNA methylation at repetitive sequences and unique developmentally regulated loci in a cell-autonomous manner.

ORGANISM(S): Mus musculus

PROVIDER: GSE64384 | GEO | 2016/07/21

SECONDARY ACCESSION(S): PRJNA270864

REPOSITORIES: GEO

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Cell-autonomous function of LSH ATPase in de novo DNA methylation

Project description:Purpose: We aimed to determine whether the expression of either wild-type or catalytically inactive LSH, carrying a single point mutation in its ATP binding site (K237Q), could restore the levels and patterns of DNA methylation in Lsh-/- mouse embryonic fibroblasts (MEFs). Methods: Lsh-/- MEFs were transduced with lentiviral particles carrying empty pMSCV vector, pMSCV-LSH-3xFLAG and pMSCV-LSH K/Q-3xFLAG, respectively. Clonal cell lines were generated and tested for LSH expression. Two independent cell lines expressing wild-type LSH and two expressing LSH K/Q were used for further analyses and comparison with wild-type MEFs and Lsh-/- MEFs carrying the empty vector. Genomic DNA was purified from all six cell lines and methylated DNA immunoprecipitation (MeDIP) was performed as described in Weber et al., 2007, Nat Genetics. MeDIP libraries were generated and sequenced on Illumina HiSeq 2000 instrument. Results and conclusions: Our experiments demonstrate that the expression of wild-type LSH, but not the catalytically inactive LSH K/Q, in Lsh-/- MEFs leads to reestablishment of DNA methylation at repetitive sequences and unique developmentally regulated loci in a cell-autonomous manner. Analyses of DNA methylation upon expression of either wild-type or catalytically-inactive LSH in Lsh-/- mouse embryonic fibroblasts.

2016-07-21 | E-GEOD-64384 | biostudies-arrayexpress

Whole genome shotgun bisulfite sequencing of the Lsh-/- and Lsh+/+ murine embryonic fibroblasts and ES cells

Project description:Whole-genome single-base resolution methylcytosine map reveals profound changes that occur after Lsh deletion during embryonic development in primary WT and Lsh-/- MEFs. Lsh deletion leads to widespread decreases of CG methylation level at uniquely mapped genomic regions compared to wild type, including TSSs at protein-coding genes, and non-coding RNA genes. MethylC-Seq from Mus musculus primary MEFs.

2014-06-13 | E-GEOD-57486 | biostudies-arrayexpress

Transcription profiling of Mus musculus wild type vs mutant with knocked out Lsh to investigate mouse embryonic fibroblasts (MEFs)

Project description:Expression profiling of mouse embryonic fibroblasts (MEFs), "wild type" vs. Lsh knock-out.

2009-11-30 | E-MEXP-2385 | biostudies-arrayexpress

Genome-wide analysis of MNase digestibility in wild type or Lsh depleted murine embryonic fibroblasts

Project description:We report here differences in MNase digestibility comparing murine embryonic fibroblasts (MEFs) with and without Lsh. In the first set of samples we compare primary MEFs derived from Lsh+/+ (pMEF_WT5) or Lsh-/- (pMEF_KO6) day 13.5 gestation embryos. In a second set we compare MEFs form conditional Lsh knockout mice before (GC-OHT-NC) or after 48 hours of tamoxifen inducible cre-recombinase expression (GC-OHT-2D). In a third set we compare Lsh knockout (Lsh cko) MEFs before (IAA-0h) and after 72 hours of IAA induced proteolytical degradation of Lsh (IAA-3D). In a fourth set we compare Lsh knockout MEFs before (IAA-0h-2) and after 7 hours of IAA induced proteolytical degradation of Lsh (IAA-7h) or after 24 hours (IAA-24). Lsh depletion leads to specific changes in chromatin accessibility at potential enhancer sites.

2019-03-28 | GSE103043 | GEO

A family of unconventional deubiquitinases with modular chain specificity determinants

Project description:Interactors of human ZUFSP in HEK-293T cells. Flp-in T-REx 293 cells with stable 3xFLAG-ZUFSP integration were assayed for ZUFSP interactors under three different conditions: i) Cells expressing wild-type ZUFSP, ii) Cells expressing catalytically inactive ZUFSP, iii) Cells expressing wild-type ZUFSP with additional treatment by an unspecific nuclease (Benzonase) during purification.

2018-04-06 | PXD008731 | Pride

Gene expression signatures in IRF7 overexpressed 3T3-L1 adipocytes

Project description:To identify the genes that are regulated by IRF7, we have performed DNA microarray in 3T3-L1 adipocytes differentiated from precursor cells infected with retrovirus empty or carrying IRF7. Plasmids (Empty- and IRF7-pMSCV) were kindly provided from Dr. Eguchi at the Okayama University Graduate School of Medicine, Okayama, Japan [Cell Metab. 2008;7: 86-94.]. Infected 3T3-L1 preadipocytes were selected by puromycin treatment and differentiated into adipocytes. 7 days after the induction of adipogenesis, total RNA was isolated.

2020-04-01 | GSE147857 | GEO

Comprehensive genomic map of cytosine methylation for wild-type (WT) and Lsh-/- mouse embryonic fibroblasts

Project description:DNA methylation is critical for normal development and plays important roles in genome organization and transcriptional regulation. Although DNA methyltransferases have been identified, the factors that establish and contribute to genome-wide methylation patterns remain elusive. Here, we report a high-resolution cytosine methylation map of the murine genome modulated by Lsh, a chromatin remodeling family member that has previously been shown to regulate CpG methylation at repetitive sequences. We provide evidence that Lsh also controls genome-wide cytosine methylation at nonrepeat sequences and relate those changes to alterations in H4K4me3 modification and gene expression. Deletion of Lsh alters the allocation of cytosine methylation in chromosomal regions of 50 kb to 2 Mb and, in addition, leads to changes in the methylation profile at the 5′ end of genes. Furthermore, we demonstrate that loss of Lsh promotes—as well as prevents—cytosine methylation. Our data indicate that Lsh is an epigenetic modulator that is critical for normal distribution of cytosine methylation throughout the murine genome. We used microarrays to detail a high-resolution cytosine methylation map of the murine genome modulated by Lsh, a chromatin remodeling family member that has previously been shown to regulate CpG methylation at repetitive sequences To investigate how genome-wide DNA methylation patterns are established in mice and how they may specifically depend on Lsh, we generated a comprehensive genomic map of cytosine methylation for wild-type (WT) and Lsh−/− mouse embryonic fibroblasts (MEFs) using methylated DNA immunoprecipitation (MeDIP) combined with whole-genome tiling microarray. In addition, we generated a histone 3 lysine 4 trimethylation (H3K4me3) chromatin map using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) and also evaluated genome-wide gene expression using cDNA microarrays

2011-05-19 | E-GEOD-29394 | biostudies-arrayexpress

The chromatin remodeler Lsh modulates genome-wide cytosine hydroxymethylation

Project description:TET proteins convert 5-methylcytosine to 5-hydroxymethylcytosine, an emerging dynamic epigenetic state of DNA that can influence transcription. While Tet proteins have been associated to epigenetic repression or activation complexes, our understanding of the molecular mechanisms involved in Tet-mediated regulation of gene transcription remains limited. Here, we showed that Tet directly interact with lymphoid-specific helicase (Lsh), a chromatin remodelling factor belonging to the ISWI family. This specific interaction seems to regulate Tet enzymatic activity since Lsh knock-out leads to a substantial reduction of 5-hydroxymethylation global level in mouse embryonic fibroblasts (MEFs) and in embryonic stem cells (ES). Whole genome sequencing of 5hmC in wild-type versus Lsh knock-out MEFs and ESCs showed that in absence of Lsh some regions of the genome gain while others loose 5hmC marks, with a weak correlation to gene expression changes . We further demonstrated that 5hmC modifications upon Lsh loss are not a direct consequence of 5mC decrease as DhMR (differentially hydroxymethylated regions) did not overlap with DMR (differentially methylated regions) underlying that these modifications occurred at different genomic loci. Altogether, our results suggest that DNA 5-hydroxymethylation and nucleosome folding are linked phenomena, highlighting novel means by which Tet proteins may influence gene regulation.

2021-01-31 | GSE110129 | GEO

Genome-wide analysis of H3K4me1 in Lsh-/- mouse embryonic fibroblasts

Project description:We report here differentiated enrichment of H3K4me1 at Lsh WT and KO mouse embryonic fibroblasts (MEFs). We found a subset of differentially enriched H3K4me1 regions in Lsh KO MEFs, and they clustered at neuronal lineage genes and overlapping with known cis-regulatory elements present in brain tissue. Reprogramming of Lsh?/? MEFs into induced pluripotent stem (iPS) cells leads to increased neuronal lineage gene expression of premarked genes and enhanced differentiation potential of Lsh?/? iPS cells toward the neuronal lineage pathway compared with WT iPS cells in vitro and in vivo. The state of H3K4me1 enrichment is partially maintained in Lsh?/? iPS cells, suggesting the regions are preserved as potential enhancers. Genome-wide maps of H3K4me1 in Lsh WT and KO primary MEFs.

2014-03-25 | E-GEOD-56151 | biostudies-arrayexpress

Transcription profiling of Mus musculus MEFs wide type and knocked out gene Lsh to investigate DNA methylation status

Project description:MBD-affinity purification (MAP) was employed to investigate the DNA methylation status at promoters of mouse embryonic fibroblasts (MEFs), both wild type and Lsh knock-out cells.<br>MAP is conceptually identical to ChIP, using an affinity column rather than an antibody.

2009-11-30 | E-MEXP-2383 | biostudies-arrayexpress

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