EXPERIMENT_TITLE: GSM6239266: human_MDM_HiC_TET2ASO_HiC3; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Total RNA was isolated using Direct-zol RNA MicroPrep kit (Zymoresearch, #11-33MB) and treated with DNase I on column. 500 ng-1 μg of RNA solution was enriched in poly-A tailed RNA by incubation with Oligo d(T) Magnetic Beads (NEB, #S1419S). Single cell RNA-Seq:For normal mDM, mDM from three healthy donors were washed twice with PBS, pelleted, and resuspended in PBS containing 0.04% BSA. Single cells were linked to beads using the 10X Chromium system (10X Genomics). Single-cell sequencing libraries were prepared following manufacturer ́s instructions using v3 chemistry. The demultiplexed fastq files produced by cellranger mkfastq were processed by cellranger count (10X genomics) using the human genome reference 'refdata-cellranger-GRCh38-3.0.0'. For each sample, the filtered_feature_bc_matrix output from cellranger count was analyzed by the Seurat R package (Stuart et al, 2019), by adapting the code from the Guided Clustering Tutorial (https://satijalab.org/seurat/archive/v3.1/pbmc3k_tutorial.html). Single cell RNA-Seq data from atherosclerotic macrophages was provided by KHMP and is available in (Depuydt et al., 2020). The single cell expression data were filtered to include only the genes of interest: DNMT3A, TET2 and genes identified in the REACTOME Interferon alpha and beta signalling gene set. As surrogate of reduced DNMT3A or TET2, we compared cells in the top and bottom quartile of DNMT3A or TET2 levels for mDM or top two quartiles versus bottom two quartiles for macrophages isolated from the atherosclerotic plaque, for the expression of genes in the Reactome Interferon alpha and beta signalling gene set. RNA-Seq library preparation: It was performed as described elsewhere (Gosselin et al., 2017); (Seidman et al., 2020)]. Briefly, mDM were lysed in TRIzol (ThermoFisher, #15596026). RNA and DNase treatment was carried out using Direct-zol RNA MicroPrep kit (Zymoresearch, #11-33MB). 500ng-1 μg total RNA was enriched in poly-A tailed RNA transcripts by double incubation with Oligo d(T) Magnetic Beads (NEB, S1419S) and fragmented for 9 min at 94oC in 2X Superscript III first- strand buffer containing 10mM DTT (Invitrogen, #P2325). The 10 μl of fragmented RNA was added to 0.5 μl of Random primers (Invitrogen, #48190011), 0.5 μl of Oligo d(T) primer (Invitrogen, #18418020), 0.5 μl of SUPERase inhibitor (Ambion, #AM2696), 1 μl of 10mM dNTPs and incubated at 50oC for three minutes. Then, 5.8 μl of water, 1 μl of 10mM DTT, 0.1 μl of 2 μg/μl Actinomycin D (Sigma, #A1410), 0.2 μl of 1% Tween-20 (Sigma) and 0.5 μl of SuperScript III (Invitrogen, #ThermoFisher 18080044) was added to the mix. Reverse- transcription (RT) reaction was performed at 25oC for 10 min followed by 50oC for 50 min. RT product was purified with RNAClean XP (Beckman Coulter, #A63987) and eluted in 10 μl in 0.01% Tween-20. The RNA-cDNA complex was then added to 1.5 μl of 10X Blue Buffer (Enzymatics, #B0110-L), 1.1 μl of dUTP mix (10 mM dATP, dCTP, dGTP and 20 mM dUTP), 0.2 μl of 5U/μl RNAseH (Enzymatics, #Y9220L), 1 μl of 10U/μl DNA polymerase I (Enzymatics, #P7050L), 0.15 μl of 1% Tween-20 and 1.05 μl of nuclease free water; and incubated at 16oC for 2.5h or overnight. The resulting dsDNA product was purified using 28 μl of SpeedbBead Magnetic Carboxylate (GE Healthcare, #651521050 50250) diluted in 20%PEG8000:2.5M NaCl to a final 13% PEG concentration, washed twice with 80% etOH, air dry and eluted in 40 μl of 0.05% Tween-20. The purified 40 μl of dsDNA was end-repaired by blunting followed by A-tailing and adapter ligation as described elsewhere (Heinz et al., 2010) using BIOO Barcodes (BIOO Scientific, #514104), IDT TruSeq Unique Dual Indexes or Kapa Unique Dual-Indexed Adapters using 15 μl Rapid Ligation Buffer (Enzymatics, #L603-LC-L), 0.33 μl 1% Tween-20 and 0.5 μl T4 DNA ligase HC (Enzymatics, #L6030-HC- L). Libraries were amplified by PCR for 11-15 cycles using Solexa IGA and Solexa IGB primers (AATGATACGGCGACCACCGA and CAAGCAGAAGACGGCATACGA, respectively), purified using 1 μl of SpeedbBead Magnetic Carboxylate in 15.2 μl of 20%PEG8000:2.5M NaCl, washed with 80% etOH and eluted in 0.05% Tween-20. Eluted libraries were quantified using a Qubit dsDNA HS Assay Kit and sequenced on a NextSeq 500 or Hi-Seq 4000 (Illumina, San Diego, California). ChIP-Seq library preparation: For DNMT3A and TET2 ChIP was performed as described previously (Eichenfield et al., 2016; Troutman et al., 2020). Briefly, mDM were fixed with 3mM Disuccinimidyl-glutarate, DSG, (Proteochem, #C1104) in PBS for 30 min at room temperature followed by 10 min incubation with 1% formaldehyde (ThermoFisher, #28906) at room temperature. Next, 2.625 M Glycine was added to a final concentration of 125 mM to quench fixation. Cells were washed with 0.01%Triton-X-100:PBS, scraped and centrifuged for 10 min at 3,000 rpm at 4oC. Cells were washed once again with 0.01%Triton-X-100 in PBS, pelleted, snap frozen and stored at -80oC. For ChIP experiments, cells were thawed and permeabilised in 1 ml of ice-cold buffer containing 10mM HEPES/KOH pH7.9, 85mM KCl, 1mM EDTA, 0.2% IGEPAL CA-630 (Sigma Aldrich, #I8896), 1X protease inhibitor cocktail (Sigma, #11836145001) and 1 mM PMSF for 10 min on ice. Cells were then spun down and lysed in 130 μl of lysis buffer containing 20 mM Tris/HCl pH7.5, 1 mM EDTA, 0.5 mM EGTA, 0.1% SDS, 0.4% Sodium Deoxycholate, 1% NP-40, 0.5 mM DTT, 1x protease inhibitor cocktail and 1 mM PMSF and chromatin was sheared by sonication. In all buffers, DTT, protease inhibitor and PMSF were added freshly. Cell lysates were sonicated in a 96 microTUBE Rack (Covaris, #500282) using a Covaris E220 for 25 cycles with the following settings: time, 60 s; duty, 5.0; PIP, 140; cycles, 200; amplitude, 0.0; velocity, 0.0. Sonicated lysates were recovered and spun at 10,000 rpm for 10 min at 4oC to remove cell debris. One percent of sonicated lysate was kept as ChIP input for analysis. Immunoprecipitation mix consisting of Protein G Dynabeads (Invitrogen, #10003D) and DNMT3A antibody cocktail (2 μg of DNMT3A, #ab2850 plus 2 μg of DNMT3A, #C15410085), 5 μl of TET2 antibody (kindly provided by Ali Shilatifard, (Wang et al., 2018), 1.5 μl of ZNF143 (H00007702-M01) or 2 μl of RBPJ (#ab25949) was added to sonicated chromatin solution and incubated overnight on rotator at 4oC. Next day, immunocomplexes were placed on a magnet and bead complexes were washed for one minute with 150 μl of cold buffers as indicated: 3 times lysis buffer, 6 times with wash buffer containing 10 mM Tris/HCl pH7.5, 250mM LiCl, 1 mM EDTA, 0.7% Na-Deoxycholate and 1% NP-40 alternative; 3 times with TET buffer containing 10 mM Tris/HCl pH 8.0, 1 mM EDTA, 0.2% Tween-20; and 1 time with IDTE buffer containing 10 mM Tris/HCl pH 8.0 and 0.1 mM EDTA. Bead complexes were resuspended in 25 μl of TT buffer containing 10 mM Tris/HCl pH 8.0, 0.05% Tween-20. All wash buffer contained 1X Protease Inhibitor cocktail. ChIP libraries for sequencing were prepared while remained on beads using NEBNext Ultra II Library kit (NEB, #E7645L) by reducing 50% the reaction volume as previously described (Heinz et al., 2018; Troutman et al., 2020). Crosslinks were revered by adding 33.5 μl of mix containing 18.4 μl nuclease free water, 4 μl 10% SDS, 3 μl 0.5 M EDTA, 1.6 μl 0.2M EGTA, 1 μl 10mg/ml Proteinase K (Biolabs, #P8107S), 1 μl 10mg/ml RNase A, 4.5 μl 5M NaCl by incubating at 55oC for 1h followed by 75oC for 30 min. Dynabeads were removed and libraries were cleaned by adding 2 μl of SpeadBeads in 124 μl of 20% PEG 8000/1.5M NaCl, washed by adding 150 μl 80% etOH, air dry and eluted in 12.5 μl of buffer containing 10 mM Tris/HCl pH 8.0 and 0.05% Tween-20. DNA was PCR- amplified for 14 cycles using NEBNext Ultra II PCR master mix using Solexa 1GA and Solexa 1GB primers. Libraries were size selected 200-500 bp by running in 10% TBE acrylamide gels (ThermoFisher, #EC62752BOX) and sequenced using using either a HiSeq 4000 or a NextSeq 500.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Deleterious somatic mutations in DNMT3A or TET2 are associated with clonal expansion of haematopoietic cells and higher risk of cardiovascular disease (CVD). In mice, most studies have indicated an over-activation of the inflammatory program in myeloid cells with reduced DNMT3A or TET2 expression. However, the effect of loss of function of DNMT3A and TET2 in human myeloid cells remains poorly understood. Here, we investigated the roles of DNMT3A and TET2 in normal human monocyte- derived macrophages (mDM), in mDM isolated from individuals with DNMT3A or TET2 mutations and in macrophages isolated from human atherosclerotic plaques. Using a combination of transcriptomic, epigenetic, and imaging assays, we provide evidence that, in addition to distinct roles in regulation of macrophage gene expression, DNMT3A and TET2 share a common role in maintaining mitochondrial DNA integrity by regulating the expression of Transcription Factor A, Mitochondria ( TFAM ). This shared function is dependent on interactions of DNMT3A and TET2 with RBPJ and ZNF143 at regulatory elements in the TFAM gene. Experimental reduction of DNMT3A or TET2 expression in normal human mDMs results in downregulation of TFAM, release of mtDNA into the cytoplasm and consequent activation of a type I interferon response mediated by cGAS. Single-cell RNA-seq analysis of mDMs from individuals with heterozygous loss-of-function mutations in DNMT3A and TET2 revealed a similar transcriptional signature. In addition, single-cell RNA-seq analysis of normal mDM and macrophages residing in human atherosclerotic plaques revealed higher expression of type I IFN signature genes specifically in cells with lower DNMT3A or TET2 expression. The production of IFN as a component of the type I IFN response induces activation of interferon-stimulated genes in neighbouring cells, representing a mechanism by which partial loss of function of DNMT3A or TET2 in a small fraction of cells could exert pathogenic effects in the artery wall. These findings suggest that targeting the type I IFN pathway may have therapeutic value in reducing risk of cardiovascular disease in patients with DNMT3A or TET2 mutations. Overall design: RNA-Seq, scRNA-Seq, ChIP-Seq and Hi-C
SAMPLE_TITLE: human_MDM_HiC_TET2ASO_HiC3
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "Monocyte-derived macrophages"}, {"TAG": "cell type", "VALUE": "macrophages"}, {"TAG": "sample type", "VALUE": "Pool of three donors"}]
Pool_Member_@attributes_sample_title: human_MDM_HiC_TET2ASO_HiC3
RUN_SET_RUN_Pool_Member_@attributes_sample_title: human_MDM_HiC_TET2ASO_HiC3
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