Abstract:DNA adenine methyltransferase identification (DamID) has emerged as an alternative method to profile protein-DNA interactions; however, critical issues limit its widespread applicability. Here, we present iDamIDseq, a protocol that improves specificity and sensitivity by inverting the steps DpnI-DpnII and adding steps that involve a phosphatase and exonuclease. To determine genome-wide protein-DNA interactions efficiently, we present the analysis tool iDEAR (iDamIDseq Enrichment Analysis with R). The combinati… Show more
“…We set out to test the hypothesis that Class III highly transient direct regulated targets of NLP7 are due to short-lived interactions of the TF with a set of genome-wide targets. To do this, we adapted the DNA adenine methyltransferase IDentification (DamID) method 32 to fingerprint NLP7-binding sites in the cellbased TARGET system. Our innovation of the DamID method is the incorporation of a GR-tag, enabling inducible nuclear localization of the Dam-GR-TF fusion protein.…”
Section: Resultsmentioning
confidence: 99%
“…Our innovation of the DamID method is the incorporation of a GR-tag, enabling inducible nuclear localization of the Dam-GR-TF fusion protein. The stable adenine methylation mark of targets touched by the TF in vivo, allowed us to capture even the briefest TF-DNA interaction 17,32 . Our DamID analysis identified 8625 target genes that were touched by the Dam-GR-NLP7 fusion protein in root cells.…”
Section: Resultsmentioning
confidence: 99%
“…+DEX treatment (in 10 µM in EtOH, Sigma-Aldrich) was done for 180 min at room temperature. For this analysis, we included a pDamBOB empty vector control with Dam but no TF (Dam-only) to subtract nonspecific background DNA adenine methylation, as reported in Gutierrez-Triana et al 32 . Genomic DNA was extracted from cells using the DNeasy Plant Mini kit (Qiagen).…”
Section: Methodsmentioning
confidence: 99%
“…Genomic DNA was extracted from cells using the DNeasy Plant Mini kit (Qiagen). The generation of DamID libraries was done as reported in Gutierrez-Triana et al 32 . Briefly, in a 20 µL reaction, 2 µL 10× NEB3.1 buffer, 50 ng of gDNA and 10 units of DpnII (NEB, R0543S) were mixed and incubated for 6 h at 37°C.…”
Dynamic reprogramming of gene regulatory networks (GRNs) enables organisms to rapidly respond to environmental perturbation. However, the underlying transient interactions between transcription factors (TFs) and genome-wide targets typically elude biochemical detection. Here, we capture both stable and transient TF-target interactions genome-wide within minutes after controlled TF nuclear import using time-series chromatin immunoprecipitation (ChIP-seq) and/or DNA adenine methyltransferase identification (DamID-seq). The transient TF-target interactions captured uncover the early mode-of-action of NIN-LIKE PROTEIN 7 (NLP7), a master regulator of the nitrogen signaling pathway in plants. These transient NLP7 targets captured in root cells using temporal TF perturbation account for 50% of NLP7-regulated genes not detectably bound by NLP7 in planta. Rapid and transient NLP7 binding activates early nitrogen response TFs, which we validate to amplify the NLP7-initiated transcriptional cascade. Our approaches to capture transient TF-target interactions genomewide can be applied to validate dynamic GRN models for any pathway or organism of interest.
“…We set out to test the hypothesis that Class III highly transient direct regulated targets of NLP7 are due to short-lived interactions of the TF with a set of genome-wide targets. To do this, we adapted the DNA adenine methyltransferase IDentification (DamID) method 32 to fingerprint NLP7-binding sites in the cellbased TARGET system. Our innovation of the DamID method is the incorporation of a GR-tag, enabling inducible nuclear localization of the Dam-GR-TF fusion protein.…”
Section: Resultsmentioning
confidence: 99%
“…Our innovation of the DamID method is the incorporation of a GR-tag, enabling inducible nuclear localization of the Dam-GR-TF fusion protein. The stable adenine methylation mark of targets touched by the TF in vivo, allowed us to capture even the briefest TF-DNA interaction 17,32 . Our DamID analysis identified 8625 target genes that were touched by the Dam-GR-NLP7 fusion protein in root cells.…”
Section: Resultsmentioning
confidence: 99%
“…+DEX treatment (in 10 µM in EtOH, Sigma-Aldrich) was done for 180 min at room temperature. For this analysis, we included a pDamBOB empty vector control with Dam but no TF (Dam-only) to subtract nonspecific background DNA adenine methylation, as reported in Gutierrez-Triana et al 32 . Genomic DNA was extracted from cells using the DNeasy Plant Mini kit (Qiagen).…”
Section: Methodsmentioning
confidence: 99%
“…Genomic DNA was extracted from cells using the DNeasy Plant Mini kit (Qiagen). The generation of DamID libraries was done as reported in Gutierrez-Triana et al 32 . Briefly, in a 20 µL reaction, 2 µL 10× NEB3.1 buffer, 50 ng of gDNA and 10 units of DpnII (NEB, R0543S) were mixed and incubated for 6 h at 37°C.…”
Dynamic reprogramming of gene regulatory networks (GRNs) enables organisms to rapidly respond to environmental perturbation. However, the underlying transient interactions between transcription factors (TFs) and genome-wide targets typically elude biochemical detection. Here, we capture both stable and transient TF-target interactions genome-wide within minutes after controlled TF nuclear import using time-series chromatin immunoprecipitation (ChIP-seq) and/or DNA adenine methyltransferase identification (DamID-seq). The transient TF-target interactions captured uncover the early mode-of-action of NIN-LIKE PROTEIN 7 (NLP7), a master regulator of the nitrogen signaling pathway in plants. These transient NLP7 targets captured in root cells using temporal TF perturbation account for 50% of NLP7-regulated genes not detectably bound by NLP7 in planta. Rapid and transient NLP7 binding activates early nitrogen response TFs, which we validate to amplify the NLP7-initiated transcriptional cascade. Our approaches to capture transient TF-target interactions genomewide can be applied to validate dynamic GRN models for any pathway or organism of interest.
“…The basic DamID experimental pipeline is outlined below, and is largely similar in many of the newly developed variants.Generation of animals or cells with Dam-fusion transgene and Dam-only control.Extraction of genomic DNA from tissue or organism of interest.Digestion of DNA with DpnI (digests methylated GATC motifs only).Ligation of PCR adapters to digested DNA.Digestion of remaining unmethylated DNA with DpnII (digests unmethylated GATC motifs only).PCR amplification of adapter ligated GATC fragments.Sequencing library preparation using standard protocols.Detailed protocols have been published for DamID (Vogel et al, 2007) and targeted DamID (Marshall et al, 2016; Tosti et al, 2018). Variations on this basic protocol have also been described (Gutierrez-Triana et al, 2016) and bioinformatic tools for the analysis of DamID data are readily available (Li et al, 2015; Marshall and Brand, 2015). …”
The interaction of proteins and RNA with chromatin underlies the regulation of gene expression. The ability to profile easily these interactions is fundamental for understanding chromatin biology
in vivo
. DNA adenine methyltransferase identification (DamID) profiles genome-wide protein-DNA interactions without antibodies, fixation or protein pull-downs. Recently, DamID has been adapted for applications beyond simple assaying of protein-DNA interactions, such as for studying RNA-chromatin interactions, chromatin accessibility and long-range chromosome interactions. Here, we provide an overview of DamID and introduce improvements to the technology, discuss their applications and compare alternative methodologies.
Highlights d Depletion of ttk69 in fly ISCs causes a genome-wide program switch to NSC-like d The ectopically expressed NSC factor Dpn drives NET tumorigenesis d The ectopically expressed pan-neural factor Seq generates a neuroendocrine phenotype d Seq also converts the differentiation-promoting role of Notch into a self-renewal role
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