DamID Research Articles

Single-cell detection of primary transcripts, their genomic loci and nuclear factors by 3D immuno-RNA/DNA FISH in T cells.

Over the past decades, it has become increasingly clear that higher order chromatin folding and organization within the nucleus is involved in the regulation of genome activity and serves as an additional epigenetic mechanism that modulates cellular functions and gene expression programs in diverse biological processes. In particular, dynamic allelic interactions and nuclear locations can be of functional importance during the process of lymphoid differentiation and the regulation of immune responses. Analyses of the proximity between chromatin and/or nuclear regions can be performed on populations of cells with high-throughput sequencing approaches such as chromatin conformation capture ("3C"-based) or DNA adenine methyltransferase identification (DamID) methods, or, in individual cells, by the simultaneous visualization of genomic loci, their primary transcripts and nuclear compartments within the 3-dimensional nuclear space using Fluorescence In Situ Hybridization (FISH) and immunostaining. Here, we present a detailed protocol to simultaneously detect nascent RNA transcripts (3D RNA FISH), their genomic loci (3D DNA FISH) and/or their chromosome territories (CT paint DNA FISH) combined with the antibody-based detection of various nuclear factors (immunofluorescence). We delineate the application and effectiveness of this robust and reproducible protocol in several murine T lymphocyte subtypes (from differentiating thymic T cells, to activated splenic and peripheral T cells) as well as other murine cells, including embryonic stem cells, B cells, megakaryocytes and macrophages.

An On-Station Analysis of Factors Affecting Growth Traits of Pure Red Maasai and Dorper Sheep Breeds under an Extensive Production System.

This comparative study aimed to evaluate genetic and environmental factors' effects on the growth traits of lambs in Dorper and Red Maasai (RedM) sheep breeds. The data analyzed contained the following measurements: birth weight (Bwt), weaning weight (Wwt), yearling weight (Ywt), birth type (single or twins), and details on each lamb's dam (dam ID and age) and sire. Except for the RedM, whose birth weight decreased with time, both breeds generally showed an increase in other growth weights across the study period, with yearly variations affecting both breeds. Additionally, analysis by a linear mixed model with restricted maximum likelihood (REML) showed that only breed as a factor significantly (p < 0.05) influenced birth weight; breed, sex, and birth type all significantly (p < 0.05) influenced weaning weight, whereas season, sex, and dam age significantly (p < 0.05) influenced yearling weight. The RedM breed outperformed the Dorper breed in post-weaning growth rate, demonstrating early resilience and adaptation to local environmental factors independent of maternal influence. Breed and non-genetic factors play a vital role in the growth of lambs, and the results of this study offer an opportunity for improved farm management under an extensive production system and selection for the conservation of the indigenous Red Maasai breed.

DamID-seq: A Genome-WideDNA Methylation Method that Captures Both Transient and Stable TF-DNA Interactions in Plant Cells.

Capturing the dynamic and transient interactions of a transcription factor (TF) with its genome-wide targets whose regulation leads to plants' adaptation to their changing environment is a major technical challenge. This is a widespread problem with biochemical methods such as chromatin immunoprecipitation-sequencing (ChIP-seq) which are biased towards capturing stable TF-target gene interactions. Herein, we describe how DNA adenine methyltransferase identification and sequencing (DamID-seq) can be used to capture both transient and stable TF-target interactions by DNA methylation. The DamID technique uses a TF protein fusedto a DNA adenine methyltransferase (Dam) from E. coli. When expressed in a plant cell, the Dam-TF fusion protein will methylate adenine (A) bases near the sites of TF-DNA interactions. In this way, DamID results in a permanent, stable DNA methylation mark on TF-targetgene promoters, even if the target gene is only transiently "touched" by the Dam-TF fusion protein. Herewe provide a step-by-step protocol to perform DamID-seq experiments in isolated plant cells for any Dam-TF fusion protein of interest. We also provide information that will enable researchers to analyze DamID-seq data to identify TF-binding sites in the genome. Our protocol includes instructions for vector cloning of the Dam-TF fusion proteins, plant cell protoplast transfections, DamID preps, library preparation, and sequencing data analysis. The protocol outlined in this chapter is performed in Arabidopsis thaliana, however, the DamID-seq workflow developed in this guide is broadly applicable to other plants and organisms.

Evaluating the effectiveness of a single application of 7% iodine tincture umbilical dip as a prevention of infection of the external umbilical structures in dairy calves

The objective of this randomized clinical trial was to evaluate the effectiveness of a single application of 7% iodine tincture-based umbilical dip for preventing infection of the external umbilical structures in dairy calves. Five dairy farms in southern Ontario were visited twice weekly from September 2020 through June 2021. Female (n = 244) and male (n = 40) Holstein calves were randomly assigned at birth to receive either a 7% iodine tincture-based umbilical dip (n = 140) or no treatment (n = 144). Calves in the 7% iodine tincture umbilical dip group had the product administered once by the producer shortly after birth. For each newborn calf, the experimental group, calving difficulty, volume and timing of colostrum administration, time of birth, calving pen cleanliness, and the dam ID were recorded. Calf body weight was recorded during the first visit after birth, and a blood sample was collected for measurement of serum IgG concentration. Calves were health scored twice weekly from enrollment until approximately 30 d of age for assessment of external umbilical infection, joint inflammation, respiratory disease, and diarrhea. The primary outcome of the study was external umbilical infection, which was defined as an enlarged umbilicus with pain, heat, or a malodorous discharge. Calves were also weighed at 30 and 60 d to determine average daily gain. Serum IgG concentration and birth weight did not differ significantly between groups. Twenty-nine calves (20%) in the umbilical dip group developed an external umbilical infections, compared with 31 calves (22%) in the control group. A mixed logistic regression model, accounting for farm as a random effect, showed no effect of treatment on the incidence of an external umbilical infection. However, for every additional hour that calves received colostrum after birth, the odds of developing an external umbilical infection increased during the first month of life (odds ratio = 1.15; 95% confidence interval: 1.04-1.26). Additionally, treatment had no effect on respiratory disease, joint inflammation, diarrhea, average daily gain, or mortality, compared with the untreated control. These findings suggest that administering a single application of 7% iodine tincture dip to the umbilicus around the time of birth may not be effective for preventing external umbilical infections. Farm-level management factors, including colostrum management, appear to have more influence on risk of this disease.

Effects of lamina associated domain on the three dimensional shapes of chromosome territories and nucleoplasmic cavities in cell nucleus

Chromatin can interact with lamin of the cellular nuclear envelope and plays important roles in 3D genome organization. Lamina Associated Domains (LAD) are regions on the chromatin where interaction with proteins in the lamin occur. Recent studies of DNA adenine methyltransferase identification (DamID) assay have mapped out LAD locations genomewide. Here we study the effects of LAD and lamin interactions on chromatin territories, chromatin shapes, and nucleopasmic cavities.

DamID identifies targets of CEH-60/PBX that are associated with neuron development and muscle structure in Caenorhabditis elegans.

Transcription factors govern many of the time- and tissue-specific gene expression events in living organisms. CEH-60, a homolog of the TALE transcription factor PBX in vertebrates, was recently characterized as a new regulator of intestinal lipid mobilization in Caenorhabditis elegans. Because CEH-60's orthologs and paralogs exhibit several other functions, notably in neuron and muscle development, and because ceh-60 expression is not limited to the C. elegans intestine, we sought to identify additional functions of CEH-60 through DNA adenine methyltransferase identification (DamID). DamID identifies protein-genome interaction sites through GATC-specific methylation. We here report 872 putative CEH-60 gene targets in young adult animals, and 587 in L2 larvae, many of which are associated with neuron development or muscle structure. In light of this, we investigate morphology and function of ceh-60 expressing AWC neurons, and contraction of pharyngeal muscles. We find no clear functional consequences of loss of ceh-60 in these assays, suggesting that in AWC neurons and pharyngeal muscle, CEH-60 function is likely more subtle or redundant with other factors.

μDamID: A Microfluidic Approach for Joint Imaging and Sequencing of Protein-DNA Interactions in Single Cells

SUMMARYDNA adenine methyltransferase identification (DamID) measures a protein’s DNA-binding history by methylating adenine bases near each protein-DNA interaction site and then selectively amplifying and sequencing these methylated regions. Additionally, these interactions can be visualized using m6A-Tracer, a fluorescent protein that binds to methyladenines. Here, we combine these imaging and sequencing technologies in an integrated microfluidic platform (μDamID) that enables single-cell isolation, imaging, and sorting, followed by DamID. We use μDamID and an improved m6A-Tracer protein to generate paired imaging and sequencing data from individual human cells. We validate interactions between Lamin-B1 protein and lamina-associated domains (LADs), observe variable 3D chromatin organization and broad gene regulation patterns, and jointly measure single-cell heterogeneity in Dam expression and background methylation. μDamID provides the unique ability to compare paired imaging and sequencing data for each cell and between cells, enabling the joint analysis of the nuclear localization, sequence identity, and variability of protein-DNA interactions. A record of this paper’s transparent peer review process is included in the Supplemental Information.

DamID to Map Genome-Protein Interactions in Preimplantation Mouse Embryos.

Investigating the chromatin landscape of the early mammalian embryo is essential to understand how epigenetic mechanisms may direct reprogramming and cell fate allocation. Genome-wide analyses of the epigenome in preimplantation mouse embryos have recently become available, thanks to the development of low-input protocols. DNA adenine methyltransferase identification (DamID) enables the investigation of genome-wide protein-DNA interactions without the requirement of specific antibodies. Most importantly, DamID can be robustly applied to single cells. Here we describe the protocol for performing DamID in single oocytes and mouse preimplantation embryos, as well as single blastomeres, using a Dam-LaminB1 fusion to generate high-resolution lamina-associated domain (LAD) maps. This low-input method can be adapted for other proteins of interest to faithfully profile their genomic interaction, allowing us to interrogate the chromatin dynamics and nuclear organization during the early mammalian development.

Simultaneous quantification of protein-DNA interactions and transcriptomes in single cells with scDam&T-seq.

Protein-DNA interactions are essential for establishing cell type-specific chromatin architecture and gene expression. We recently developed scDam&T-seq, a multi-omics method that can simultaneously quantify protein-DNA interactions and the transcriptome in single cells. The method effectively combines two existing methods: DNA adenine methyltransferase identification (DamID) and CEL-Seq2. DamID works through the tethering of a protein of interest (POI) to the Escherichia coli DNA adenine methyltransferase (Dam). Upon expression of this fusion protein, DNA in proximity to the POI is methylated by Dam and can be selectively digested and amplified. CEL-Seq2, in contrast, makes use of poly-dT primers to reverse transcribe mRNA, followed by linear amplification through in vitro transcription. scDam&T-seq is the first technique capable of providing a combined readout of protein-DNA contact and transcription from single-cell samples. Once suitable cell lines have been established, the protocol can be completed in 5 d, with a throughput of hundreds to thousands of cells. The processing of raw sequencing data takes an additional 1-2 d. Our method can be used to understand the transcriptional changes a cell undergoes upon the DNA binding of a POI. It can be performed in any laboratory with access to FACS, robotic and high-throughput-sequencing facilities.

Transient genome-wide interactions of the master transcription factor NLP7 initiate a rapid nitrogen-response cascade

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 genome-wide can be applied to validate dynamic GRN models for any pathway or organism of interest.

Methyl Adenine Identification (MadID): High-Resolution Detection of Protein-DNA Interactions.

Mapping the binding sites of DNA- or chromatin-interacting proteins is essential to understand many essential biological processes. Methyl Adenine Identification (MadID) is a proximity methylation-based assay that allows the visualization, quantification, and identification of binding sites from DNA-interacting proteins in eukaryotic cells. Chromatin-binding proteins of interest are fused to the newly described bacterial methyltransferase M.EcoGII. This enzyme catalyzes the methylation of adenine residues with no sequence specificity. Consequently, adenines within and in the vicinity of the protein binding sites will be decorated with a methyl group (m6A), a modification that can be further detected using different methods. M.EcoGII-dependent DNA methylation can be monitored in situ using immunostaining, at the genome-wide level using a combination of m6A-specific immunoprecipitation and whole-genome sequencing, or locally at DNA regions of interest purified by chromatin immunoprecipitation or probe-based capture techniques. MadID is conceptually similar to DNA adenine methyltransferase identification (DamID) that relies on the methylation of GATC motifs. However, MadID provides a higher resolution, deeper coverage, and opens ways for identification of binding sites in genomic regions that were largely inaccessible such as telomeres, centromeres, and repeated elements.

The transcriptional regulator CBX2 and ovarian function: A whole genome and whole transcriptome approach

The chromobox homolog 2 (CBX2) was found to be important for human testis development, but its role in the human ovary remains elusive. We conducted a genome-wide analysis based on DNA adenine methyltransferase identification (DamID) and RNA sequencing strategies to investigate CBX2 in the human granulosa cells. Functional analysis revealed that CBX2 was upstream of genes contributing to ovarian function like folliculogenesis and steroidogenesis (i.e. ESR1, NRG1, AKR1C1, PTGER2, BMP15, BMP2, FSHR and NTRK1/2). We identified CBX2 regulated genes associated with polycystic ovary syndrome (PCOS) such as TGFβ, MAP3K15 and DKK1, as well as genes implicated in premature ovarian failure (POF) (i.e. POF1B, BMP15 and HOXA13) and the pituitary deficiency (i.e. LHX4 and KISS1). Our study provided an excellent opportunity to identify genes surrounding CBX2 in the ovary and might contribute to the understanding of ovarian physiopathology causing infertility in women.

Assembling the jigsaw puzzle: CBX2 isoform 2 and its targets in disorders/differences of sex development

Background One of the defining moments of human life occurs early during embryonic development, when individuals sexually differentiate into either male or female. Perturbation of this process can lead to disorders/differences of sex development (DSD). Chromobox protein homolog 2 (CBX2) has two distinct isoforms, CBX2.1 and CBX2.2: the role of CBX2.1 in DSD has been previously established, yet to date the function of the smaller isoform CBX2.2 remains unknown. Methods The genomic DNA of two 46,XY DSD patients was analysed using whole exome sequencing. Furthermore, protein/DNA interaction studies were performed using DNA adenine methyltransferase identification (DamID) to identify putative binding partners of CBX2. Finally, in vitro functional studies were used to elucidate the effect of wild-type and variant CBX2.2 on selected downstream targets. Results Here, we describe two patients with features of DSD i.e. atypical external genitalia, perineal hypospadias and no palpable gonads, each patient carrying a distinct CBX2.2 variant, p.Cys132Arg (c.394T>C) and p.Cys154fs (c.460delT). We show that both CBX2.2 variants fail to regulate the expression of genes essential for sexual development, leading to a severe 46,XY DSD defect, likely because of a defective expression of EMX2 in the developing gonad. Conclusion Our study indicates a distinct function of the shorter form of CBX2 and by identifying several of its unique targets, can advance our understanding of DSD pathogenesis and ultimately DSD diagnosis and management.

Simultaneous quantification of protein-DNA contacts and transcriptomes in single cells.

Protein-DNA interactions are critical to the regulation of gene expression, but it remains challenging to define how cell-to-cell heterogeneity in protein-DNA binding influences gene expression variability. Here we report a method for the simultaneous quantification of protein-DNA contacts by combining single-cell DNA adenine methyltransferase identification (DamID) with mRNA sequencing of the same cell (scDam&T-seq). We apply scDam&T-seq to reveal how genome-lamina contacts or chromatin accessibility correlate with gene expression in individual cells. Furthermore, we provide single-cell genome-wide interaction data on a Polycomb-group protein, RING1B, and the associated transcriptome. Our results show that scDam&T-seq is sensitive enough to distinguish mouse embryonic stem cells cultured under different conditions and their different chromatin landscapes. Our method will enable analysis of protein-mediated mechanisms that regulate cell type-specific transcriptional programs in heterogeneous tissues.

DamC reveals principles of chromatin folding in vivo without crosslinking and ligation.

Current understanding of chromosome folding largely relies on chromosome conformation capture (3C)-based experiments, where chromosomal interactions are detected as ligation products after chromatin crosslinking. To measure chromosome structure in vivo, quantitatively and without crosslinking and ligation, we implemented a modified version of DamID named DamC, which combines DNA-methylation based detection of chromosomal interactions with next-generation sequencing and biophysical modelling of methylation kinetics. DamC performed in mouse embryonic stem cells provides the first in vivo validation of the existence of topologically associating domains (TADs), CTCF loops and confirms 3C-based measurements of the scaling of contact probabilities. Combining DamC with transposon-mediated genomic engineering shows that new loops can be formed between ectopic and endogenous CTCF sites, which redistributes physical interactions within TADs. DamC provides the first crosslinking- and ligation-free demonstration of the existence of key structural features of chromosomes and provides novel insights into how chromosome structure within TADs can be manipulated.

Expressional Profiling of Carpet Glia in the Developing Drosophila Eye Reveals Its Molecular Signature of Morphology Regulators.

Homeostasis in the nervous system requires intricate regulation and is largely accomplished by the blood–brain barrier (BBB). The major gate keeper of the vertebrate BBB is vascular endothelial cells, which form tight junctions (TJs). To gain insight into the development of the BBB, we studied the carpet glia, a subperineurial glial cell type with vertebrate TJ-equivalent septate junctions, in the developing Drosophila eye. The large and flat, sheet-like carpet glia, which extends along the developing eye following neuronal differentiation, serves as an easily accessible experimental system to understand the cell types that exhibit barrier function. We profiled transcribed genes in the carpet glia using targeted DNA adenine methyl-transferase identification, followed by next-generation sequencing (targeted DamID-seq) and found that the majority of genes expressed in the carpet glia function in cellular activities were related to its dynamic morphological changes in the developing eye. To unravel the morphology regulators, we silenced genes selected from the carpet glia transcriptome using RNA interference. The Rho1 gene encoding a GTPase was previously reported as a key regulator of the actin cytoskeleton. The expression of the pathetic (path) gene, encoding a solute carrier transporter in the developing eye, is specific to the carpet glia. The reduced expression of Rho1 severely disrupted the formation of intact carpet glia, and the silencing path impaired the connection between the two carpet glial cells, indicating the pan-cellular and local effects of Rho1 and Path on carpet glial cell morphology, respectively. Our study molecularly characterized a particular subperineurial cell type providing a resource for a further understanding of the cell types comprising the BBB.

DamID as a versatile tool for understanding gene regulation.

ABSTRACTThe 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.

Genome-wide analysis of ETV1 targets: Insights into the role of ETV1 in tumor progression.

ETS variant 1 (ETV1) is a key player in metastatic progression in several types of human cancers, yet the direct target genes of ETV1 and the mechanisms by which ETV1 exerts its deleterious function remain largely elusive. Here, we performed large-scale mapping and analysis of target loci of ETV1 in the prostate cancer cells LNCaP using the DNA adenine methyltransferase identification technique, we identified close to 800 direct targets for ETV1. Expression analysis using quantitative reverse transcriptionpolymerase chain reaction confirmed a positive regulation by ETV1 in most of the genes examined. Furthermore, gene and pathway analysis unraveled new signaling pathways and biological networks that interact with ETV1. Our findings cast light on genes and networks regulated by ETV1, it also opens new fronts for studying the role of ETV1 and its target genes in tumorigenesis.

MadID, a Versatile Approach to Map Protein-DNA Interactions, Highlights Telomere-Nuclear Envelope Contact Sites in Human Cells

SummaryMapping the binding sites of DNA- or chromatin-interacting proteins is essential to understanding biological processes. DNA adenine methyltransferase identification (DamID) has emerged as a comprehensive method to map genome-wide occupancy of proteins of interest. A caveat of DamID is the specificity of Dam methyltransferase for GATC motifs that are not homogenously distributed in the genome. Here, we developed an optimized method named MadID, using proximity labeling of DNA by the methyltransferase M.EcoGII. M.EcoGII mediates N6-adenosine methylation in any DNA sequence context, resulting in deeper and unbiased coverage of the genome. We demonstrate, using m6A-specific immunoprecipitation and deep sequencing, that MadID is a robust method to identify protein-DNA interactions at the whole-genome level. Using MadID, we revealed contact sites between human telomeres, repetitive sequences devoid of GATC sites, and the nuclear envelope. Overall, MadID opens the way to identification of binding sites in genomic regions that were largely inaccessible.

Assembling the jigsaw puzzle: CBX2 isoform 2 and its targets in disorders/differences of sex development

BackgroundOne of the defining moments of human life occurs early during embryonic development, when individuals sexually differentiate into either male or female. Perturbation of this process can lead to disorders/differences of sex development (DSD). Chromobox protein homolog 2 (CBX2) has two distinct isoforms, CBX2.1 and CBX2.2: the role of CBX2.1 in DSD has been previously established, yet to date the function of the smaller isoform CBX2.2 remains unknown.MethodsThe genomic DNA of two 46,XY DSD patients was analysed using whole exome sequencing. Furthermore, protein/DNA interaction studies were performed using DNA adenine methyltransferase identification (DamID) to identify putative binding partners of CBX2. Finally, in vitro functional studies were used to elucidate the effect of wild‐type and variant CBX2.2 on selected downstream targets.ResultsHere, we describe two patients with features of DSD i.e. atypical external genitalia, perineal hypospadias and no palpable gonads, each patient carrying a distinct CBX2.2 variant, p.Cys132Arg (c.394T>C) and p.Cys154fs (c.460delT). We show that both CBX2.2 variants fail to regulate the expression of genes essential for sexual development, leading to a severe 46,XY DSD defect, likely because of a defective expression of EMX2 in the developing gonad.ConclusionOur study indicates a distinct function of the shorter form of CBX2 and by identifying several of its unique targets, can advance our understanding of DSD pathogenesis and ultimately DSD diagnosis and management.