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RNAseq to determine gene expression changes following depletion of SETDB1 in THP-1 AML Cells

ABSTRACT: Purpose: To determine how loss of SETDB1 effects gene expression in THP-1 AML cells. Methods: THP-1 cells were treated with two different SETDB1 sgRNAs (6, and 9) or Non-targeting control sgRNA (NTC) for 4 and 7 days. RNA was isolated and prepared for RNAseq with Qiagen RNeasy kits. Results: Using an optimized data analysis workflow, we mapped 23 million or more sequence reads per sample to the human genome (GRCh38) with GSNAP for obtaining standard gene expression measurements. Since SETDB1 is also know to regulate repetitive elements we also mapped sequences to a pseudogenome containing tranposable elements from hg19 repeatmasker annotations using RepEnrich and following the pipeline published on GitHub (https://github.com/nskvir/RepEnrich). Conclusions: Our data determined that immediately following the disruption of SETDB1, a strong type I Interferon response can be observed at day 4. In addition, many repetitive elements are also significanly induced, including L1 LINEs, Endogenous Retroviruses, and Satellite repeats.

ORGANISM(S): Homo sapiens

PROVIDER: GSE103409 | GEO | 2017/09/06

SECONDARY ACCESSION(S): PRJNA401801

REPOSITORIES: GEO

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RNAseq to determine whether bidirectional transcription occurs over transposable elements following depletion of SETDB1 in THP-1 AML Cells

Project description:SETDB1 disruption leads to an increase in the expression of transposable elements as determined by our standard RNAseq, where bidirectional transcription has been reported. We repeated RNA seq on our samples with a stranded prep to determine whether bidirectional transcription occurs of transposable elements following depletion of SETDB1 in THP-1 AML Cells Methods: THP-1 cells were treated with two different SETDB1 sgRNAs (6, and 9) or Non-targeting control sgRNA (NTC) for 4 and 7 days. RNA was isolated and prepared for RNAseq with Qiagen RNeasy kits. Results: Consensus sequences for elements for upregulated TEs were downloaded from the Dfam database (Hubley et al., 2016). Stranded RNA-seq data was then aligned to consensus sequences using BWA-MEM and quantified using HTseq-count(Anders et al., 2015; Li, 2013). Reads were visualized using the IGV genome browser, using a custom pseudogenome that was generated from the Dfam consensus sequences (Robinson et al., 2011; Thorvaldsdottir et al., 2013). Conclusions: Our data determined that following the disruption of SETDB1, there is increased bidirectional transcription over a subset of transposable elements.

2017-09-06 | GSE103410 | GEO

DNA methylation profiling of promoter DNA associated to the T2D.

Project description:The Mouse Promoter Array is designed for chromatin immunoprecipitation (ChIP) experiments. Sequences used in the design of the Mouse Promoter array were selected from NCBI human genome assembly (Build 34). Repetitive elements were removed by RepeatMasker.

2018-06-30 | GSE100677 | GEO

DNA methylation profiling of promoter DNA associated to the T2D patients in Taiwan

Project description:The GeneChip® Human Promoter 1.0R Array is designed for chromatin immunoprecipitation (ChIP) experiments. Sequences used in the design of the Human Promoter array were selected from NCBI human genome assembly (Build 34). Repetitive elements were removed by RepeatMasker.

2016-05-26 | GSE81868 | GEO

DNA methylation and SETDB1/H3K9me3 regulate predominantly distinct sets of genes, retroelements and chimaeric transcripts in mouse ES cells

Project description:DNA methylation and histone H3 lysine 9 trimethylation (H3K9me3) play important roles in silencing of genes and retroelements. However, a comprehensive comparison of genes and repetitive elements repressed by these pathways has not been reported. Here we show that in mouse embryonic stem cells (mESCs), the genes up-regulated following deletion of the H3K9 methyltransferase Setdb1 are distinct from those de-repressed in mESC deficient in the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b, with the exception of a small number of primarily germline-specific genes. Numerous endogenous retroviruses (ERVs) lose H3K9me3 and are concomitantly de-repressed exclusively in SETDB1 knockout mESCs. Strikingly, ~15% of up-regulated genes are induced in association with de-repression of promoter proximal ERVs, half in the context of "chimaeric" transcripts that initiate within these retroelements and splice to genic exons. Thus, SETDB1 plays a previously unappreciated yet critical role in inhibiting aberrant gene transcription by suppressing the expression of proximal ERVs. NChIP-seq and mRNA-seq of WT, SETDB1 KO and DMNT1 TKO mESCs

2011-05-21 | E-GEOD-29413 | biostudies-arrayexpress

Epigenetic Silencing by SETDB1 Suppresses Tumor-cell Intrinsic Immunogenicity

Project description:Griffin GK, Wu J, Iracheta-Vellve A, Patti JC, Hsu J, Davis T, Dele-Oni D, Du PP, Halawi A, Ishizuka JJ, Kim S, Klaeger S, Knudsen NH, Miller BC, Nguyen T, Olander K, Papanastasiou M, Rachimi S, Robitschek EJ, Schneider EM, Yeary M, Zimmer M, Jaffe JD, Carr SA, Doench JG, Haining WN, Yates KB, Manguso RT, Bernstein BE. 2020. Epigenetic dysregulation is a defining feature of tumorigenesis and has been implicated in immune escape, yet mechanisms that drive immune evasion are poorly understood. To systematically identify epigenetic factors that modulate the immune sensitivity of tumor cells, we performed in vivo CRISPR-Cas9 screens targeting 936 chromatin regulators in mouse tumor models treated with immune checkpoint blockade. We identified the H3K9-methyltransferase SETDB1 and other members of the HUSH and KAP1 complexes as cell-intrinsic mediators of immune escape in tumor cells. We also found that amplification of SETDB1 (1q21) in human tumors is associated with reduced cytotoxic T-cell infiltration and resistance to immune checkpoint blockade. Mechanistically, we demonstrate that SETDB1 represses broad domains, hundreds of kilobases in size, many of which reside within the open genome compartment. These SETDB1 domains are enriched for transposable elements (TEs) and immune gene clusters associated with segmental duplication events, a central mechanism of mammalian genome evolution. SETDB1 loss derepresses latent TE-encoded regulatory elements and proximal immune genes within these repetitive regions, including canonical co-stimulatory ligands, and induces hundreds of putative TE-encoded viral antigens. Our study establishes SETDB1 as an epigenetic checkpoint that suppresses intrinsic immunogenicity in cancer cells, and thus represents a candidate target for immunotherapy.

2020-12-11 | MSV000086580 | MassIVE

RNA-seq identifies a distinct response in macrophages infected with Mycobacterium tuberculosis for a serial time points.

Project description:Purpose: The goals of this study are to obtain the NGS-derived transcriptome profiling (RNA-seq) for THP-1 macrophages response to Mycobacterium tuberculosis (H37Rv and H37Ra) Methods: mRNA and long noncoding RNA profiles of THP-1 macrophages infected with H37Rv and H37Ra for 1, 4, 12, 24, 48 hours were generated by deep sequencing, using Illumina Hiseq3000. The sequence reads that passed quality filters were first mapped to the latest UCSC transcript set using Bowtie2 (version 2.1.0). Then the gene expression level was estimated using RSEM (RNA-Seq by Expectation Maximization, v1.2.15), for lncRNA analysis, reads were mapped to lncRNA transcript set from LNCipedia.org. The sequence reads were normalized with TMM (trimmed mean of M-values) to identify differentially expressed genes (DEGs) using the edgeR package edgeR. qRT–PCR validation was performed using SYBR Green assays. Results: Using an optimized data analysis workflow, we mapped about 20 million sequence reads per sample to the human genome (GRCh38/hg38) and identified 25,343 mRNA and 47877 long non-coding RNA transcripts. Conclusions: Our study represents the detailed analysis of transcriptomes for THP-1 macrophages response to H37Rv and H37Ra, generated by RNA-seq technology.

2021-01-04 | GSE108731 | GEO

RNA-seq identifies a distinct response in macrophages stimulated with early secreted antigenic target 6-KDa from Mycobacterium tuberculosis

Project description:Purpose: The goals of this study are to obtain the NGS-derived transcriptome profiling (RNA-seq) for THP-1 macrophages response to early secreted antigenic target 6-KDa (ESAT6) from Mycobacterium tuberculosis Methods: mRNA profiles of THP-1 macrophages treated with ESAT6 were generated by deep sequencing, using Illumina Hiseq3000. The sequence reads that passed quality filters were first mapped to the latest UCSC transcript set using Bowtie2 (version 2.1.0). Then the gene expression level was estimated using RSEM (RNA-Seq by Expectation Maximization, v1.2.15), and normalized with TMM (trimmed mean of M-values) to identify differentially expressed genes (DEGs) using the edgeR package edgeR. qRT–PCR validation was performed using SYBR Green assays. Results: Using an optimized data analysis workflow, we mapped about 23 million sequence reads per sample to the human genome (GRCh38/hg38) and identified 25,343 transcripts. Conclusions: Our study represents the first detailed analysis of transcriptomes for macrophages response to ESAT6, generated by RNA-seq technology.

2019-03-10 | GSE107387 | GEO

Epigenetic Silencing by SETDB1 Suppresses Tumor-cell Intrinsic Immunogenicity

Project description:Epigenetic dysregulation is a defining feature of tumorigenesis and has been implicated in immune escape. However, the epigenetic mechanisms that drive immune evasion in cancer are poorly understood. To systematically identify epigenetic factors that modulate the immune sensitivity of tumor cells, we performed in vivo loss of function screens targeting 936 chromatin regulators in mouse tumor models treated with immune checkpoint blockade. We identified the H3K9-methyltransferase SETDB1 and other members of the HUSH and KAP1 complexes as cell-intrinsic mediators of immune escape in tumor cells. We also found that amplification of SETDB1 (1q21) in human tumors is associated with reduced cytotoxic T-cell infiltration and resistance to immune checkpoint blockade. Mechanistically, we demonstrate that SETDB1 targets broad domains, hundreds of kilobases in size, many of which reside within the open genome compartment. These SETDB1 domains are enriched for transposable elements (TEs) and immune gene clusters associated with segmental duplication events, a central mechanism of mammalian genome evolution. SETDB1 loss derepresses latent TE-encoded regulatory elements and proximal immune genes within these repetitive regions, including canonical NKG2D ligands, and induces hundreds of putative TE-encoded viral antigens. Our study establishes SETDB1 as an epigenetic checkpoint that suppresses intrinsic immunogenicity in cancer cells, and thus represents a candidate target for immunotherapy.

2021-03-18 | GSE155972 | GEO

Cell-type differential targeting of Setdb1 prevents aberrant CTCF binding, chromatin looping, and cis-regulatory interactions

Project description:SETDB1 is a histone methyltransferase with essential roles in mouse development. It mediates transcriptional repression of genes and repetitive elements in different cell-types by depositing histone H3 lysine 9 trimethylation (H3K9me3). However, the function of differential H3K9me3 enrichment between cell- and tissue-types remains unclear. In this study, we demonstrate a global mutual exclusivity of H3K9me3 and CTCF across mouse tissues from different developmental time points. To investigate the mechanistic relationship, we analyzed SETDB1 depleted mouse embryonic stem cells and discovered that H3K9me3 prevents aberrant CTCF binding independently of DNA methylation and H3K9me2. Such sites are enriched with retrotransposons such as SINE B2 elements, which are not previously defined as targets of SETDB1. While the epigenetic modifier is known to silence transcriptional enhancers, its effects on higher-order chromatin structures have not been clearly defined. Overall, large chromatin structures including topologically associated domains and subnuclear compartments, remain intact. However, chromatin loops and local interactions are disrupted. Such perturbations lead to transcriptional changes by altering pre-existing chromatin landscapes, where specific genes have differential interactions with dysregulated cis-regulatory elements. This mode of transcriptional regulation is distinct from the direct repression of genic promoters or enhancers. Collectively, we find that cell-type specific targets of SETDB1 contributes to maintaining cellular identities through shaping genome architecture and transcriptomic networks by modulating CTCF binding, representing a novel function for SETDB1 and H3K9me3.

2023-11-01 | GSE184471 | GEO

RNA-seq identifies a distinct response in macrophages stimulated from phagosome with early secreted antigenic target 6-KDa from Mycobacterium tuberculosis

Project description:Purpose: The goals of this study are to obtain the NGS-derived transcriptome profiling (RNA-seq) for THP-1 macrophages, which were stimulated from phagosome with early secreted antigenic target 6-KDa (ESAT6) Methods: First we fabricated a nanocapsule enclosing ESAT6 (nESAT6) and a nanocapsule enclosing BSA (nBSA) as control. mRNA profiles of THP-1 macrophages treated with nESAT6 were generated by deep sequencing, using Illumina Hiseq3000. The sequence reads that passed quality filters were first mapped to the latest UCSC transcript set using Bowtie2 (version 2.1.0). Then the gene expression level was estimated using RSEM (RNA-Seq by Expectation Maximization, v1.2.15), and normalized with TMM (trimmed mean of M-values) to identify differentially expressed genes (DEGs) using the edgeR package edgeR. qRT–PCR validation was performed using SYBR Green assays. Results: Using an optimized data analysis workflow, we mapped about 23 million sequence reads per sample to the human genome (GRCh38/hg38) and identified 25,343 transcripts. Conclusions: Our study represents the first detailed analysis of transcriptomes for macrophages response to ESAT6 stimulation in phagosome, generated by RNA-seq technology.

2020-12-21 | GSE108393 | GEO

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