EFB Dengue Literature Review

ATAC-seq

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ATAC-seq

Overview

ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) maps regions of open chromatin genome-wide by using a hyperactive Tn5 transposase to insert sequencing adapters into accessible DNA. In B cell immunology, ATAC-seq identifies epigenetic priming — loci that are chromatinically accessible and poised for transcription even if mRNA levels are not yet elevated — providing a complementary layer to RNA-seq transcriptomics.

Key Points from Literature

  • PRDM1 locus open in aNAV and DN2: ATAC-seq of sorted B cell subsets (rNAV, aNAV, SWM, DN2) demonstrated opening of the PRDM1 (BLIMP-1) locus in aNAV and DN2 cells. This chromatin accessibility, combined with elevated BLIMP-1 protein, indicates that these populations are epigenetically poised for PC differentiation (see Jenks2018 - DN2 B Cells and EF Pathway in SLE, ATAC-seq on 10,000–50,000 FACS-sorted cells).

  • Technical protocol: Cells resuspended in nuclei lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl₂, 0.1% IGEPAL CA-630); nuclei transposed with Tn5 for 1 hr at 37°C; low molecular weight DNA purified by SPRI-bead size selection; PCR amplified with Nextera primers; 50 bp paired-end sequencing. Reads mapped to hg19 with Bowtie; peaks called with MACS2 (see Jenks2018 - DN2 B Cells and EF Pathway in SLE, citing Scharer et al. 2016).

  • Scharer2019 — comprehensive multi-subset ATAC-seq comparison: ATAC-seq performed on 5 sorted B cell subsets (resting naive, T3 transitional, activated naive, switched memory, DN2) from 9 SLE patients and 12 healthy controls. This is the most comprehensive ATAC-seq comparison of B cell subsets in any human disease context. Key analytical outputs: (1) differentially accessible regions (DARs) between subsets and between SLE vs. HC within each subset; (2) PCA of DARs showing SLE aN and DN2 cluster more tightly than healthy counterparts; (3) HOMER motif enrichment on DARs identifying the T-BET/AP-1/EGR signature in DN2 vs. NF-κB/EBF/OCT in SM; (4) T-BET ChIP-seq integration (from ENCODE Th1 data) confirming T-BET binding at GAS7, TNFRSF1B, ITGAX, ZAP70, TBX21 loci in DN2/aN-accessible regions. Peaks called with MACS2; motifs identified with HOMER. 402 DARs distinguished SLE from HC resting naive B cells alone (see Scharer2019 - Epigenetic Programming in SLE B Cells, n=9 SLE + 12 HC).

  • AP-1/EGR disease-specific amplification detected by ATAC-seq: T-BET motif accessibility was shared between HC and SLE DN2 cells (normal DN2 programme), but AP-1 (JUN/FOSB/FOSL1/FOSL2) and EGR motif accessibility was specifically amplified in SLE aN and DN2 relative to healthy counterparts. This stratification — shared vs. disease-specific chromatin features — was only possible because ATAC-seq was performed on matched subsets from both SLE and HC donors (see Scharer2019 - Epigenetic Programming in SLE B Cells).

  • ATF3 motif accessibility highest in SLE DN2: ATAC-seq DARs in SLE DN2 cells were enriched for ATF3 binding motifs. Combined with RNA-seq (ATF3 mRNA upregulated) and intracellular flow cytometry (ATF3 protein elevated), this multi-omic validation made ATF3 the top novel TF candidate from the Scharer2019 study. 98 ATF3 target genes were identified by intersecting ATF3 motif-containing DARs with DEGs (see Scharer2019 - Epigenetic Programming in SLE B Cells).

  • PDCD1 (PD-1) locus accessibility in DN2: The PDCD1 promoter and cis-regulatory elements showed highest chromatin accessibility in DN2 cells by ATAC-seq, concordant with highest PD-1 protein expression (~60% PD-1⁺ on DN2 by flow). This multi-layered validation (chromatin → mRNA → protein) exemplifies how ATAC-seq identifies epigenetically primed loci that translate to functional protein expression (see Scharer2019 - Epigenetic Programming in SLE B Cells).

  • ★ Chromatin accessibility as a record of antigenic history — the proposed readout for memory B cell fate. Accumulated stimulation progressively increases accessibility at plasma-cell-associated gene loci, notably Prdm1, thereby raising baseline BLIMP-1 expression and biasing memory B cells toward PC differentiation over germinal-center reentry; DN and DP memory subsets carry distinct epigenetic profiles that may predetermine their responsiveness to secondary antigen encounter, acting in part through BACH2/BLIMP1 regulation (see Glaros2025 - Multilayered Identity of B Cell Memory, review, no original data; flagged by the authors as remaining to be tested; primary is Shao 2024 Nat Immunol — audit entry #64, not yet ingested). Because the corresponding transcriptomes differ only subtly, accessibility profiling is the assay positioned to detect what expression profiling misses.

  • Application this suggests for dengue (wiki-generated, untested). ATAC-seq at the PRDM1 locus on sorted DENV-specific memory B cells from primary vs secondary donors would test whether repeat DENV exposure epigenetically pre-commits memory to a plasmablast recall — the candidate mechanism for the secondary-dengue plasmablast-burst / low-SHM / OAS triad. See Original Antigenic Sin and Glaros2025 - Multilayered Identity of B Cell Memory Questions Raised.

  • ★ Motif-proximal accessibility as a transcription-factor-activity readout — and how to control it. Rather than reporting differentially accessible regions alone, Stone quantified chromatin accessibility within 100 bp surrounding the consensus binding motifs of named transcription factors, giving a per-TF activity estimate (T-bet n=963 motif-containing DARs, Blimp-1 n=871, PAX5 n=193, SpiB n=389, Bcl6 n=605, XBP1 n=105, IRF4 n=538, OCT2 n=175). The design’s power comes from holding the motif set fixed across two different perturbations: the same n=871 Blimp-1 motif set was strongly opened by Th1 priming (p=3.8 × 10⁻⁹⁰) yet unchanged by T-bet deletion (ns), while T-bet’s own motifs moved in the same panel (see Stone2019 - T-bet Promotes ASC Differentiation by Limiting IFN-gamma Inflammation, mouse, 3 independent samples/group; GEO GSE118984). This is a template worth reusing: a negative result at one motif is interpretable only when a positive control motif in the same assay moves.

  • A 10,000-cell ATAC-seq protocol with the full downstream motif pipeline. Tagmentation of 10,000 sorted cells in 25 µl (2.5 µl Tn5, 1× Tagment DNA Buffer, 0.2% digitonin, 1 hr at 37°C), lysis with SDS/Proteinase-K, SPRI size selection, Nextera PCR; Bowtie to hg19, Picard MarkDuplicates, MACS2 peak calling, edgeR v3.18.1 with a generalized linear model for differentially accessible regions at >2-fold and FDR<0.05, then HOMER v4.8.2 findMotifsGenome.pl/annotatePeaks.pl for motif enrichment and motif footprinting (read depth at the motif and surrounding sequence via GenomicRanges) (see Zumaquero2019 - IFN-gamma Programs T-bet-hi B Cells for ASC Differentiation, human, 2 independent samples/group; GEO GSE119726). Yielded 15,917 DARs across the four priming conditions. The footprinting step is what turns “a region is open” into “this TF’s motif is open”, and is the same readout Stone2019 - T-bet Promotes ASC Differentiation by Limiting IFN-gamma Inflammation uses — the two papers are directly comparable on that axis.

Contradictions & Debates

None documented in current wiki sources.

RNA Sequencing, RRBS, BLIMP-1, DN2 B Cell, Activated Naive B Cell, FACS Sorting, ATF3, EGR, PD-1, T-bet, BACH2, BLIMP-1, Memory B Cell, Original Antigenic Sin

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