The SWI/SNF subunit Dpf2 licenses Nrf2-dependent gene expression to enforce multilineage control of inflammation [CUT&RUN]

Purpose: Multi-omics characterization of the consequences of deleting the SWI/SNF subunit Dpf2 in the transcriptome (RNAseq), accessibility of the genome (ATACseq) and binding of SWI/SNF subunits (CUT&RUN) in hematopoietic stem/progenitor cells, resting and polarized bone-marrow derived macrophages and resting and activated CD4+ T cells obtained from Dpf2f/f and Dpf2D/D mice. Methods: Lin- cKit+ cells were sorted from bone marrow of 28-days old Dpf2f/f and Dpf2D/D mice. Bone-marrow derived macrophages were obtained from 28-days old Dpf2f/f and Dpf2D/D mice and polarized with IFNg and LPS, or IL-4 for 24hours. CD4+ splenic T helper cells were obtained from 28-days old Dpf2f/f and Dpf2D/D mice and activated by treatment with PMA and Ionomycin. Methods: For CUT&RUN experiments, Lin- cKit+ cells were obtained from bone marrow of 28-days old Dpf2f/f and Dpf2D/D mice. Cells were crosslinked (1 min, 1% formaldehyde), quenched (125mM Glycine) and washed in buffers following the CUTANA ChIC/CUT&RUN kit according to the manufacturer’s CUT&RUN cross-linking protocol (EpiCypher, 14-1048). Results: For CUT&RUN experiments, 5 million (paired-end, 75bp) reads were obtained per sample. Pair-end fastq files were processed with the ENCODE Transcription Factor and Histone ChIP-Seq processing pipeline (https://github.com/ENCODE-DCC/chip-seq-pipeline2). Reads were trimmed using cutadapt v2.5. and aligned to the mm10 genome using Bowtie2 v2.3.4.3. SAMtools v1.9 were used to convert the output file to BAM format. Duplicates were removed using Picard Tools v2.20.7. Peak calling was performed with MACS2 v2.2.4. Conclusions: The absence of Dpf2 in LK cells results in downregulation of anti-oxidative and anti-inflammatory gene expression programs in bone marrow LK cells, bone-marrow derived macrophages and CD4+ T cells. Samples were obtained from primary bone marrow or spleen cells isolated or sorted from Dpf2f/f and Dpf2D/D mice of approximately 28 days of age. For RNAseq and ATACseq datasets, each file corresponds to data from 1 independent mouse/genotype. At least 3 mice (independent biological replicates) per condition and genotype were used. CUT&RUN samples were obtained from a pool of LK cells. Antibodies used for Cut&Run are as follows: anti-Brg1 (Bethyl A300-813A, lot#4), anti-Dpf2 (Millipore SIGMA, SAB4502621, lot#3111434), anti-Nrf2 (R&D Biosystems, AF3925, lot#WID0121081), anti-IgG (Diagenode, C15410206, lot#RIG001), anti-H3K27ac (Diagenode, C15410196, lot#A1723-0041D), anti-H3K4me1 (Diagenode, C15410194, lot#A1862D).