Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position

Buenrostro, Jason D.; Giresi, Paul G.; Zaba, Lisa C.; Chang, Howard Y.; Greenleaf, William J.

doi:10.1038/nmeth.2688

Cited by 5,304 publications

(6,004 citation statements)

References 38 publications

(54 reference statements)

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“…Freshly cultured ependymoma cells were prepared for ATAC-seq according to previously published methods 27 . Briefly, nuclei were prepared from ~50,000 cells by spinning at 600g for 10 minutes 4°C, followed by a PBS wash and centrifugation at 600g for 5 minutes.…”

Section: Methodsmentioning

confidence: 99%

“…The supernatant was removed and pellet re-suspended in 50ul of transposase mix (25ul of 2×TD Buffer, 2.5ul of transposase, 22.5ul of water) (FC-121-1030 Illumina; CA, USA) for 30 minutes at 37°C. Library amplification was performed using the NEBnext High Fidelity 2×PCR Master Mix (#M0541S New England Biolabs; ON, Canada) according to previously published PCR conditions 27 . PCR reactions were purified using QIAGEN miniElute kit, and a following size selection step using standard gel extraction protocol to isolate ~240-360bp.…”

Section: Methodsmentioning

confidence: 99%

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Therapeutic targeting of ependymoma as informed by oncogenic enhancer profiling

Mack¹,

Pajtler

Chávez

et al. 2017

Nature

178

214

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SUMMARY Genomic sequencing has driven precision-based oncology therapy; however, genetic drivers remain unknown or non-targetable for many malignancies, demanding alternative approaches to identify therapeutic leads. Ependymomas are chemotherapy-resistant brain tumours, which, despite genomic sequencing, lack effective molecular targets. Intracranial ependymomas are segregated based on anatomical location – supratentorial region (ST) or posterior fossa (PF) – and further divided into distinct molecular subgroups that reflect differences in age of onset, gender predominance, and response to therapy1–3. The most common and aggressive subgroup, Posterior Fossa Ependymoma Group A (PF-EPN-A), occurs in young children and appears to lack recurrent somatic mutations2. Conversely, Posterior Fossa Ependymoma Group B (PF-EPN-B) tumours display frequent large-scale copy number gains and losses yet favourable clinical outcomes1,3. Greater than 70% of supratentorial ependymomas are defined by highly recurrent gene fusions in the NFκB subunit RELA (ST-EPN-RELA), and less frequently involve fusion of the gene encoding the transcriptional activator YAP1 (ST-EPN-YAP1).1,3,4 Subependymomas, a distinct histologic variant, can also be found within the ST and PF compartments accounting for the majority of tumours in the molecular subgroups ST-EPN-SE and PF-EPN-SE, respectively1. Here, we mapped active chromatin landscapes in 42 primary ependymomas in two non-overlapping primary ependymoma cohorts with the goal of identifying essential super enhancer associated genes on which tumour cells were dependent. Enhancer regions revealed putative oncogenes, molecular targets, and pathways, which when subjected to small molecule inhibitor or shRNA treatment, diminished proliferation of patient-derived neurospheres and increased survival in mouse models of ependymomas. Through profiling of transcriptional enhancers, our study provides a framework for target and drug discovery in other cancers recalcitrant to therapeutic development because of their lack of known genetic drivers.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Therapeutic targeting of ependymoma as informed by oncogenic enhancer profiling

Mack¹,

Pajtler

Chávez

et al. 2017

Nature

178

214

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“…This aspect allows visualization of global TF binding events in a chromatin-free context but fails to capture these important tissue-specific dynamics. One powerful way to overcome this limitation is to overlay tissue-specific chromatin accessibility information from methods such as DNase-seq, ATAC-seq, and MNase-seq on DAP-seq datasets [21][22][23] . Integration of DAP-seq and DNase hypersensitivity data from multiple Arabidopsis tissue types showed that DAP-seq captures in vivo binding sites that correspond to multiple tissue-specific binding events 15 .…”

Section: Advantages and Limitationsmentioning

confidence: 99%

Mapping genome-wide transcription-factor binding sites using DAP-seq

Bartlett¹,

O’Malley²,

Huang³

et al. 2017

Nat Protoc

352

272

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In order to enable low-cost, high-throughput generation of cistrome and epicistrome maps for any organism, we developed DNA affinity purification sequencing (DAP-seq), a transcription factor (TF) binding site discovery assay that couples affinity-purified TFs with next-generation sequencing of a genomic DNA library. The method is fast, inexpensive, and more easily scaled than ChIP-seq. DNA libraries are constructed using native genomic DNA from any source of interest, preserving cell-and tissue-specific chemical modifications that are known to impact TF binding (such as DNA methylation) and providing increased specificity compared to in silico motif prediction methods such as protein binding microarrays (PBM) and systematic evolution of ligands by exponential enrichment (SELEX). The resulting DNA library is incubated with an affinity-tagged in vitro expressed TF, and TF-DNA complexes are purified using magnetic separation of the affinity tag. Bound genomic DNA is eluted from the TF and sequenced using next-generation sequencing. Sequence reads are mapped to a reference genome, identifying genome-wide binding locations for each TF assayed, from which sequence motifs can then be

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“…To investigate a possible role of E-proteins in controlling regulatory elements at activated target genes, we mapped open chromatin regions (known as DNase I hypersensitive [DHS] sites) by assay for transposase-accessible chromatin (ATAC)-seq (Buenrostro et al, 2013) in activated WT and DKO B cells at day 2 of anti-CD40 and IL-4 stimulation. The chromatin accessibility, which was measured as read density of each DHS site, was increased at E2A peaks of activated genes in WT B cells compared with DKO B cells (Fig.…”

Section: E-proteins Induce and Maintain Open Chromatin At Activated Tmentioning

confidence: 99%

Molecular functions of the transcription factors E2A and E2-2 in controlling germinal center B cell and plasma cell development

Wöhner¹,

Tagoh²,

Bilić³

et al. 2016

J. Cell Biol.

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1201B cell immunity provides acute and long-term protection of the host against infections through the generation and secretion of high-affinity antibodies that recognize a shear unlimited number of pathogens. This enormous adaptive potential of B cells is brought about by V(D)J recombination of the immunoglobulin heavy chain (Igh) and light chain (Igk and Igl) genes in early B cell development, and by subsequent affinity maturation of the Ig heavy chain in late B cell differentiation (Victora and Nussenzweig, 2012). Somatic hypermutation alters the antigen-binding V H sequences of the Ig heavy-chain, whereas class switch recombination (CSR) exchanges the C H exons to generate Ig isotypes with distinct effector functions (Chaudhuri and Alt, 2004). Whereas the activation-induced deaminase (AID) is an essential regulator of both processes (Muramatsu et al., 2000), somatic hypermutation only takes place in germinal centers (GCs), which are formed upon antigen exposure by the interplay of T follicular helper (Tfh) cells and follicular (FO) B cells in secondary lymphoid organs (Victora and Nussenzweig, 2012). Affinity-based selection in this specialized compartment leads to clonal expansion of B cells expressing high-affinity B cell receptors, which subsequently differentiate to proliferating, antibody-secreting plasmablasts (Victora and Nussenzweig, 2012). Upon migration to specialized bone marrow niches, plasmablasts differentiate into long-lived quiescent plasma cells secreting high amounts of antibodies (Nutt et al., 2015). While many transcription factors are involved in coordinating these B cell responses, we have here studied the role of E-proteins in the regulation of these processes.Basic helix-loop-helix (bHLH) transcription factors can be subdivided into different classes based on biochemical and functional properties (Murre, 2005). Class I bHLH proteins, also known as E-proteins, consist of the three members, E2A (Tcf3), E2-2 (Tcf4), and HEB (Tcf12; Murre, 2005), which bind the E-box (CAN NTG) motif with similar sequence specificity (Fig. S1 A). E-proteins are broadly expressed and heterodimerize with class II bHLH proteins in nonlymphoid cell types. Within the lymphoid system, E-proteins function as homodimers or heterodimers with a different E-protein (Bain et al., 1993;Shen and Kadesch, 1995). E-proteins are thought to mainly function as transcriptional activators, as they interact with the co-activators p300 and CBP (Bradney et al., 2003;Bayly et al., 2004), as well as the promoter recognition factor TFI ID (Chen et al., 2013). The activity of E-proteins is controlled by the inhibitor of DNA binding proteins, which are HLH proteins lacking the basic DNA-binding domain, and are thus capable of sequestering E-proteins into DNA-bindingincompetent heterodimers (Kee, 2009).E-proteins control different aspects of B cell development (Murre, 2005). E2A is required for the commitment of lymphoid progenitors to the B cell lineage (Bain et al.,

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Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position

Cited by 5,304 publications

References 38 publications

Therapeutic targeting of ependymoma as informed by oncogenic enhancer profiling

Therapeutic targeting of ependymoma as informed by oncogenic enhancer profiling

Mapping genome-wide transcription-factor binding sites using DAP-seq

Molecular functions of the transcription factors E2A and E2-2 in controlling germinal center B cell and plasma cell development

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