Single-fiber nucleosome density shapes the regulatory output of a mammalian chromatin remodeling enzyme

Abdulhay, Nour J.; Hsieh, Laura J; McNally, Christopher A.; Ketavarapu, Mythili; Kasinathan, Sivakanthan; Nanda, Arjun Scott; Ostrowski, Megan S; Wu, Ke; Moore, Camille M.; Goodarzi, Hani; Narlikar, Geeta J.; Ramani, Vijay

doi:10.1101/2021.12.10.472156

Cited by 5 publications

(15 citation statements)

References 76 publications

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“…= 1.24; p ~ 0). Both results are broadly consistent with our previous in vivo SAMOSA observations of active promoters and heterochromatin in human K562 cells 4 and murine embryonic stem cells (mESCs) 31 , pointing to a conserved pattern of single fiber chromosome structure within these domains. Together, these analyses demonstrate that SAMOSA-Tag can easily generate genome-wide, multi-omic single-molecule chromatin fiber accessibility data from tens of thousands of cells.…”

Section: Integrative Measurement Of Cpg Methylation and Single-molecu...supporting

confidence: 92%

“…In prior work, we demonstrated that single-fiber accessibility data could be used to cluster the genome based on nucleosome regularity and average distance between regular nucleosomes (nucleosome-repeat length, or NRL) 4, 31 . These studies relied entirely on complementary epigenomic datasets to assess how the distribution of so-called ‘fiber-types’ ( i.e.…”

Section: Resultsmentioning

confidence: 99%

“…In addition to filtering out all clusters that together comprised less than 10% of all fibers, we also manually filtered out unmethylated / lowly methylated fibers, which fell out of the leiden clustering analysis and together accounted for 317,768 fibers (12.5% of all clustered fibers). Fiber type enrichment Fisher's exact tests to determine fiber type enrichment across were performed as in 31 . ThermoT4DNAPolymerase(5U/uL)_10U_Ampligase(5U/uL)_20U_ThermoT4DNAPolRxnBuffer_1mM-dNTPs_30min@37ºC_add-(2.5mMNAD+) ThermoT4DNAPolymerase(5U/uL)_12.5U_Ampligase(5U/uL)_25U_ThermoT4DNAPolRxnBuffer_1mM-dNTPs_30min@37ºC_add-(0.5mMNAD+) ThermoT4DNAPolymerase(5U/uL)_5U_NEBTaqDNALigase(40U/uL)_80U_TaqDNALigaseRxnBuffer_1mM-dNTPs_30min@37ºC ThermoT4DNAPolymerase(5U/uL)_5U_NEBT7DNALigase(3000U/uL)_3000U_StickTogetherLigaseBuffer_1mM-dNTPs_30min@37ºC ThermoT4DNAPolymerase(5U/uL)_5U_NEBHiFiTaqLigase_1U_HiFiTaqDNALigaseBuffer_1mM-dNTPs_30min@37ºC ThermoT4DNAPolymerase(5U/uL)_5U_NEB9ºNLigase_80U_9ºNLigaseBuffer_1mM-dNTPs_30min@37ºC NEBPhusion(2U/uL)_0.8U_Ampligase(5U/uL)_2U_AmpligaseBuffer_0.05mM-dNTPs_30min@37ºC_add-(50mMKCl,20%DMF) NEBPhusion(2U/uL)_0.8U_Ampligase(5U/uL)_2U_AmpligaseBuffer_0.05mM-dNTPs_30min@37ºC_add-(50mMKCl,10%DMF) NEBPhusion(2U/uL)_0.8U_Ampligase(5U/uL)_2U_AmpligaseBuffer_0.05mM-dNTPs_30min@37ºC, 15min@45ºC_add-(50mMKCl,10%DMF) NEBPhusion(2U/uL)_0.8U_Ampligase(5U/uL)_2U_AmpligaseBuffer_0.8mM-dNTPs_60min@37ºC_add-(25mMKCl,10%DMF) NEBPhusion(2U/uL)_4U_Ampligase(5U/uL)_10U_AmpligaseBuffer_0.05mM-dNTPs_30min@37ºC_add-(50mMKCl,20%DMF) NEBPhusion(2U/uL)_4U_Ampligase(5U/uL)_10U_AmpligaseBuffer_0.05mM-dNTPs_30min@37ºC_add-(50mMKCl,10%DMF) NEBPhusion(2U/uL)_4U_Ampligase(5U/uL)_10U_AmpligaseBuffer_0.05mM-dNTPs_30min@37ºC, 15min@45ºC_add-(50mMKCl,10%DMF) NEBPhusion(2U/uL)_4U_Ampligase(5U/uL)_10U_AmpligaseBuffer_0.8mM-dNTPs_60min@37ºC_add-(25mMKCl,10%DMF) NEBPhusion(2U/uL)_4U_Ampligase(5U/uL)_10U_AmpligaseBuffer_0.8mM-dNTPs_60min@37ºC_add-(25mMKCl) NEBPhusion(2U/uL)_0.32U_NEBTaqDNALigase(40U/uL)_80U_TaqDNALigaseRxnBuffer_0.8mM-dNTPs_30min@37ºC NEBPhusion(2U/uL)_0.32U_NEBTaqDNALigase(40U/uL)_80U_TaqDNALigaseRxnBuffer_0.8mM-dNTPs_30min@37ºC_add-(10%DMF) NEBPhusion(2U/uL)_0.8U_NEBTaqDNALigase(40U/uL)_80U_AmpligaseBuffer_0.05mM-dNTPs_30min@37ºC_add-(50mMKCl,10%DMF) NEBPhusion(2U/uL)_2U_NEBTaqDNALigase(40U/uL)_80U_TaqDNALigaseRxnBuffer_0.8mM-dNTPs_30min@37ºC NEBPhusion(2U/uL)_2U_NEBTaqDNALigase(40U/uL)_80U_TaqDNALigaseRxnBuffer_0.8mM-dNTPs_60min@37ºC NEBPhusion(2U/uL)_4U_NEBTaqDNALigase(40U/uL)_80U_TaqDNALigaseRxnBuffer_0.8mM-dNTPs_60min@37ºC NEBPreCRMix(1U/uL)_1U_None_0U_ThermoPolRxnBuffer_0.1mM-dNTPs_30min@37ºC_add-(0.5mMNAD+) NEBBstFullLengthPolymerase(5U/uL)_0.8U_NEBTaqDNALigase(40U/uL)_60U_ThermoPolRxnBuffer_1mM-dNTPs_30min@37ºC_add-(0.5mMNAD+) NEBPhusion(2U/uL)_2U_NEB9ºNLigase_80U_9ºNLigaseBuffer_0.8mM-dNTPs_30min@37ºC…”

Section: Fiber Type Clusteringmentioning

confidence: 99%

“…Per-CpG methylation estimates were made using tools available at https://github.com/PacificBiosciences/pb-CpG-tools. Predicting nucleosome footprints in SAMOSA-Tag data SAMOSA-Tag data was preprocessed as above, and subsequently analyzed using a computational pipeline previously developed for detecting m 6 dA methylation in HiFi reads 31 . In brief, the kinetics of polymerase base addition were extracted per read, and a series of neural networks trained on methylated and unmethylated controls were used to predict the probability of m 6 dA methylation at all adenines on both the forward and reverse strands.…”

Section: Predicting Cpg Methylation In Single Molecule Readsmentioning

confidence: 99%

“…FRITSS / FRICBS was calculated as the fraction of read-ends falling within the 5 kilobase window. CTCF CpG and accessibility analyses m 6 dA accessibility signal surrounding predicted CTCF sites was extracted from accessibility pickle files and leiden clustered as in 31 . In addition to filtering out clusters that together accounted for less than 10% of all data, we also manually filtered out 1 cluster that corresponded to completely unmethylated fibers.…”

Section: Insertion Bias Analyses At Tss and Ctcf Sitesmentioning

confidence: 99%

See 4 more Smart Citations

Sensitive multimodal profiling of native DNA by transposase-mediated single-molecule sequencing

Nanda¹,

Wu²,

Kasinathan³

et al. 2022

Preprint

Self Cite

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We present SMRT-Tag: a multiplexable, PCR-free approach for constructing low-input, single-molecule Pacific Biosciences (PacBio) sequencing libraries through Tn5 transposition. As proof-of-concept, we apply SMRT-Tag to resolve human genetic and epigenetic variation in gold-standard human reference samples. SMRT-Tag requires 1-5% as much input material as existing protocols (15,000 - 50,000 human cell equivalents) and enables highly-sensitive and simultaneous detection of single nucleotide variants, small insertions / deletions, and CpG methylation comparable to the current state-of-the-art. We further combine SMRT-Tag with in situ adenine methyltransferase footprinting of nuclei (SAMOSA-Tag) to facilitate joint analysis of nucleosome repeat length, CTCF occupancy, and CpG methylation on individual chromatin fibers in osteosarcoma cells. SMRT-Tag promises to enable basic and clinical research by offering scalable, sensitive, and multimodal single-molecule genomic and epigenomic analyses in rare cell populations.

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Section: Integrative Measurement Of Cpg Methylation and Single-molecu...supporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Fiber Type Clusteringmentioning

confidence: 99%

Section: Predicting Cpg Methylation In Single Molecule Readsmentioning

confidence: 99%

Section: Insertion Bias Analyses At Tss and Ctcf Sitesmentioning

confidence: 99%

See 3 more Smart Citations

Sensitive multimodal profiling of native DNA by transposase-mediated single-molecule sequencing

Nanda¹,

Wu²,

Kasinathan³

et al. 2022

Preprint

Self Cite

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show abstract

DNA-m6A calling and integrated long-read epigenetic and genetic analysis with fibertools

Jha

Bohaczuk

Mao

et al. 2023

Preprint

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Single-molecule chromatin fiber sequencing is based on the single-nucleotide resolution identification of DNA N6-methyladenine (m6A) along individual sequencing reads. We present fibertools, a semi-supervised convolutional neural network that permits the fast and accurate identification of both endogenous and exogenous m6A-marked bases using single-molecule long-read sequencing. Fibertools enables highly accurate (>90% precision and recall) m6A identification along multi-kilobase DNA molecules with a ~1,000-fold improvement in speed and the capacity to generalize to new sequencing chemistries.

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The single-molecule accessibility landscape of newly replicated mammalian chromatin

Ostrowski,

Yang,

McNally

et al. 2023

Preprint

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The higher-order structure of newly replicated (i.e.nascent) chromatin fibers remains poorly-resolved, limiting our understanding of how epigenomes are maintained across cell divisions. To address this, we presentReplication-AwareSingle-moleculeAccessibilityMapping (RASAM), a long-read sequencing method that nondestructively measures genome-wide replication-status and protein-DNA interactions simultaneously on intact chromatin templates. We report that individual human and mouse nascent chromatin fibers are hyperaccessible compared to steady-state chromatin. This hyperaccessibility occurs at two, coupled length-scales: first, individual nucleosome core particles on nascent DNA exist as a mixture of partially-unwrapped nucleosomes and other subnucleosomal species; second, newly-replicated chromatin fibers are significantly enriched for irregularly-spaced nucleosomes on individual DNA molecules. Focusing on specific cis-regulatory elements (e.g.transcription factor binding sites; active transcription start sites [TSSs]), we discover unique modes by which nascent chromatin hyperaccessibility is resolved at the single-molecule level: at CCCTC-binding factor (CTCF) binding sites, CTCF and nascent nucleosomes compete for motifs on nascent chromatin fibers, resulting in quantitatively-reduced CTCF occupancy and motif accessibility post-replication; at active TSSs, high levels of steady-state chromatin accessibility are preserved, implying that nucleosome free regions (NFRs) are rapidly re-established behind the fork. Our study introduces a new paradigm for studying higher-order chromatin fiber organization behind the replication fork. More broadly, we uncover a unique organization of newly replicated chromatin that must be reset by active processes, providing a substrate for epigenetic reprogramming.

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Single-fiber nucleosome density shapes the regulatory output of a mammalian chromatin remodeling enzyme

Cited by 5 publications

References 76 publications

Sensitive multimodal profiling of native DNA by transposase-mediated single-molecule sequencing

Sensitive multimodal profiling of native DNA by transposase-mediated single-molecule sequencing

DNA-m6A calling and integrated long-read epigenetic and genetic analysis with fibertools

The single-molecule accessibility landscape of newly replicated mammalian chromatin

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