Cytoplasmic lipid droplets are highly dynamic storage organelles that are critical for cellular lipid homeostasis. While the molecular details of lipid droplet dynamics are a very active area of investigation, this work has been primarily performed in cultured cells. Taking advantage of the powerful transgenic and in vivo imaging opportunities available in zebrafish, we built a suite of tools to study lipid droplets in real time from the subcellular to the whole organism level. Fluorescently tagging the lipid droplet-associated proteins, perilipin 2 and perilipin 3, in the endogenous loci permits visualization of lipid droplets in the intestine, liver, and adipose tissue. Using these tools, we found that perilipin 3 is rapidly loaded on intestinal lipid droplets following a high-fat meal and later replaced by perilipin 2. These powerful new tools will facilitate studies on the role of lipid droplets in different tissues, under different genetic and physiological manipulations, and in a variety of human disease models. Cytoplasmic lipid droplets are cellular organelles composed of a core of neutral lipids surrounded by a monolayer of phospholipids and coated with a variety of proteins. While initially believed to be passive storage depots for lipids, it is now appreciated that lipid droplets are dynamic organelles with roles in cellular lipid homeostasis, protection from lipotoxicity and ER stress, viral and parasitic infection, and host defense .Lipid droplets are typically coated by one or more perilipins (PLINs), an evolutionarily related protein family defined by two conserved protein motifs, the N-terminal ~100 amino acid hydrophobic PAT domain followed by a repeating 11-mer helical motif of varying length . PLINs aDictyostelium to mammals, with more divergent variants in fungi and Caenorhabditis  and Fus(plin3-RFP), in which we inserted fluorescent reporters in-frame with the coding sequence at the genomic loci. These reporter lines faithfully recapitulate the endogenous expression and transcriptional regulation of plin2 and plin3 in larval zebrafish, allowing for in vivo imaging of lipid droplet dynamics in live animals at the subcellular, tissue, organ, and whole larvae level. Using these lines, we describe the ordered recruitment of Plin3 and then Plin2 to lipid droplets in intestinal enterocytes following the consumption of a high-fat meal. We also reveal a delay in hepatic expression of Plin2 and Plin3 during development and identify a population of Plin2-positive lipid droplets adjacent to neuromasts in the posterior lateral line.Here, we report the generation and validation of zebrafish PLIN reporter lines, including plin2 and plin3 mRNA expression in larval zebrafish, we performed whole-mount in situ hybridization. Our data indicate that plin2 is not expressed in any tissues of unfed larvae at 6 days post fertilization (dpf)  . However in mice  and with in mice . Plin3 mfat meal , again cfat meal . Surprisat 6 dpf  as was eat 6 dpf .To study how Plin2 and Plin3 regulate lipid droplet dynamics in vivo, we generated fluorescent Plin2 and Plin3 zebrafish reporter lines . We recoFus(EGFP-plin2) based on early data suggesting that it was necessary to tag PLIN2 on the N-terminus (plin2 (ENSDART00000175378.2) and adjacent to the termination codon in the last exon of plin3 (ENSDART00000100473.5). One-cell stage embryos were injected with TALEN mRNA and donor constructs including either EGFP for plin2 or tagRFP-t for plin3, flanked by the noted homology arms to direct homology directed repair ) or prior to feeding (Fus(plin3-RFP)). Correct integration of the fluorescent constructs was confirmed by PCR on genomic DNA of individual larvae, followed by sequencing (Fus(EGFP-plin2), a total of 40 adults were screened and three fish produced EGFP+ progeny. However, two of these clutches had abnormal tissue expression patterns and only displayed cytoplasmic EGFP fluorescence. One of these had correct integration across the left homology arm, but the right homology arm integration could not be verified; the second had no confirmed integration at the locus. Furthermore, while our founder fish produced low numbers of progeny with correct expression patterns and proper integration (8 embryos out of 853 screened), she also produced progeny with incorrect integration (22/853), suggesting mosaicism in the germ cells. To identify a Fus(plin3-RFP) founder, 26 adults were screened before a fish produced progeny expressing RFP. These embryos had correct tissue expression of RFP in the intestine and correct integration of the transgene (53 RFP+ embryos out of 613 screened). We did note a 29 bp region missing in intron 7–8; however, this does not alter the coding region, and it is unclear whether this deletion arose during integration or whether it was a naturally occurring variant in the injected embryo.From the injected F0 adult fish, we identified founders for each knock-in allele by out-crossing and screening progeny for fluorescence in the intestine at 6 dpf either following a meal /+ larvae were fed liposomes containing the fluorescent fatty acid analog BODIPY 558/568-C12. This fatty acid analog can be incorporated into both phospholipid and triglycerides for storage in lipid droplets in larval zebrafish (Fus(EGFP-plin2) and Fus(plin3-RFP), we found that lipid droplets can be labeled by both EGFP-Plin2 and Plin3-RFP proteins /+ fish at any given time-point  . While aWithin 45 min of meal onset, the green BODIPY FL-C12 is present within the intestinal lumen and throughout the cytoplasm of the enterocytes. It is also incorporated into lipids stored within nascent lipid droplets, which appear in puncta that are modestly brighter than the surrounding cytoplasm . Over thConsistent with our previous imaging , the PliTg(fabp2:EGFP-PLIN2). This promoter drives expression in the yolk syncytial layer and in the intestine (Fus(EGFP-plin2) line. Indeed, we visually detected EGFP-PLIN2 on lipid droplets at 45 min, our earliest time-point after the start of the meal  line to transgenic reporter lines for the different cell types  and Fus(plin3-RFP) zebrafish reporter lines described here provide important new tools for studying lipid droplets and the in vivo role of Plin2 and Plin3 in a vertebrate system.Studies on the role of vertebrate PLINs in modulating lipid droplet dynamics have been mostly performed in cell culture or at limited time-points in fixed tissues. While cell-based work has been, and will continue to be, crucial for detailed investigations of lipid droplet biogenesis at the ER, protein structure-function analyses, and the molecular underpinnings of how lipid droplet size and number is regulated, studies in vivo are essential for understanding how PLINs impact whole animal lipid physiology. Additionally, in systems that are difficult to model ex vivo, lipid droplet dynamics in cultured cells may not recapitulate what is observed in vivo. For instance, dietary lipid absorption in the intestinal enterocytes is profoundly influenced by bile  and micrWe chose to develop these reporters in the zebrafish system because of the ease of live imaging in the optically clear embryonic and larval tissues. Additionally, we engineered the fluorescent protein tags into the endogenous loci to minimize over-expression artifacts. Our initial validation studies indicate that these lines faithfully recapitulate the endogenous tissue expression patterns and expected subcellular localization of Plin2 and Plin3 in the larval zebrafish. Furthermore, as a demonstration of their utility to uncover new biology, we use these tools to describe the ordered recruitment of Plin3 followed by Plin2 onto lipid droplets in the intestine following a high-fat meal. Plin3-RFP, which is present in the cytoplasm of enterocytes in unfed fish, localizes rapidly to nascent lipid droplets, and remains associated for ~6–7 hr until it is once again found primarily in the cytoplasm. In contrast, EGFP-Plin2 only begins to be visible at ~2.5 hr, and it is almost exclusively associated with lipid droplets for the remainder of their lifetime.plin2 mRNA expression in zebrafish gut between 1 and 3 hr following the start of a high-fat meal (Fus(EGFP-plin2)/+ fish and wild-type siblings, suggesting that the tag did not alter the lifetime of the lipid droplets. Although an N-terminal FLAG tag was reported to decrease ubiquitination and degradation of unbound PLIN2 , small clusters of visceral adipocytes containing lipid droplets co-labeled with EGFP-Plin2 and Plin3-RFP were visible in the larval zebrafish, consistent with previous characterization of zebrafish adipose tissues using lipophilic dyes . As thesAs these knock-in reporter lines are used to further characterize lipid metabolism in different physiological and pathological conditions, we expect that Plin2- and Plin3-labeled lipid droplets will be identified in other zebrafish tissues. For example, in mammals, lipid droplets and PLINs are present in muscle tissue , and mustype2diabetesgenetics.org. 2021 July 26; https://t2d.hugeamp.org/region.html?chr=9&end=19199288&phenotype=LDL&start=19058373) and a PLIN2 mutation (Ser251Pro) is associated with decreased atherogenic lipids (plasma triglyceride and very low-density lipoprotein)  . While tprotein) , taken tprotein)  and estaprotein)  may provFus(EGFP-plin2) and Fus(plin3-RFP) knock-in zebrafish lines provide the opportunity to study PLINs and lipid droplet biology in vivo at the organelle, cell, tissue, organ, and whole animal level. These lines exploit the advantages of the zebrafish model and will be important tools to understand how lipid droplet dynamics are affected by different genetic and physiological manipulations.In summary, the Danio rerio) were approved by the Carnegie Institution Department of Embryology Animal Care and Use Committee (Protocol #139). Zebrafish stocks (AB line) were maintained at 27°C in a circulating aquarium facility with a 14:10 hr light:dark cycle. For propagation and stock maintenance, starting at 5.5 dpf, larvae were fed with GEMMA Micro 75 (Skretting) 3× a day until 14 dpf, GEMMA Micro 150 3× a day+ Artemia 1× daily from 15 to 42 dpf and then GEMMA Micro 500 1× daily supplemented once a week with Artemia. The nutritional content of GEMMA Micro is: 59% protein; 14% lipids; 0.2% fiber; 14% ash; 1.3% phosphorus; 1.5% calcium; 0.7% sodium; 23,000 IU/kg vitamin A; 2800 IU/kg vitamin D3; 1000 mg/kg vitamin C; 400 mg/kg vitamin E. Embryos were obtained by natural spawning and raised in embryo medium at 28.5°C in culture dishes in an incubator with a 14:10 hr light:dark cycle. Zebrafish sex is not determined until the juvenile stage, so sex is not a variable in experiments with embryos and larvae.All procedures using zebrafish  or HCS LipidTOX Red  neutral lipid stains. Fish were incubated with LipidTOX dyes at 1:5000 dilution in either embryo media  or system water  in six-well dishes  or 10 cm dishes (21 dpf) for a minimum of 2 hr prior to imaging.perilipin 2  and 900 bp of the zgc:77,486  (GRCz11) mRNA sequences were amplified from cDNA using the primers noted in Zebrafish embryos were staged according to Genomic DNA was extracted from embryos, larvae, or adult fin clips using a modified version of the HotSHOT DNA extraction protocol . Embryoslater (Thermo Fisher Scientific AM7020) at 4°C for 1 week. RNA was isolated using a Trizol-based RNA prep adapted from plin2 transcripts were previously validated (see rps18) was used as the reference gene  and stored in RNAated see  and zebrnce gene . qRT-PCRnce gene .Fus(EGFP-plin2) and Fus(plin3-RFP) lines were created with TALEN-mediated genome editing, using   flank a Fok1 restriction site just downstream of the endogenous start codon. The designed TALEN pairs for plin3  flank a Hph1 site immediately upstream of the endogenous termination codon. TALEN mRNA was in vitro transcribed using the T3 Message Machine Kit , injected into one-cell stage zebrafish embryos, and cutting efficiency of each pair was assessed by monitoring the loss of either FokI (NEB R0109) or HphI (NEB R0158) digestion due to TALEN nuclease activity. Nuclease activity was higher for plin2 TALEN pair 1 and plin3 TALEN pair 1 and these were used subsequently for genome integration. Donor plasmids used as templates for homology directed repair were assembled using the three-fragment MultiSite gateway assembly system . For plin2, the 5’ element consisted of 594 bp of genomic sequence upstream of th plin2 start codon, the middle-entry element contained the plin2 Kozak sequence followed by the EGFP coding sequence lacking a termination codon, and then a linker region coding for three glycine residues that was in-frame with the 3’ element which consisted of 900 bp of genomic sequence including and downstream of plin2 start codon. For plin3, the 5’ element consisted of the 679 bp of genomic sequence immediately upstream of the termination codon, a middle-entry element of in-frame tagRFP-t  fish with a high-fat meal in order to detect EGFP-Plin2 fluorescence when integrated correctly. Correct in-frame integration of the fluorescent reporters was confirmed by PCR and sequencing. Following verification, Fus(EGFP-plin2) fish were genotyped using primers for EGFP. For primer information, see g, using  as a gui8.2) see  and discign tool  and clonign tool . The desPLIN2 (ENSG00000147872 GRCh38.p13) or PLIN3 (ENSG00000105355) under the control of various promoters were generated with the Tol2-Gateway molecular cloning system  coding sequence was obtained from Flexgene clones collection  and re-cloned into pCR8. The intestine-specific intestinal fatty acid binding protein  promoter was provided by Michel Bagnat (fabp10a [–2.8 kb]), originally described in hsp70l) promoter (p5E-hsp70l), p3E-EGFPpA, pME-EGFP no stop, and p3E-polyA plasmids were originally provided by Chi-bin Chien (Tg(fabp2:EGFP-PLIN2), Tg(fabp10a:EGFP-PLIN2), Tg(hsp70l:EGFP-PLIN2), Tg(fabp2:PLIN3-EGFP), and Tg(hsp70l:PLIN3-EGFP) transgene constructs. Plasmids were injected (25–50 pg) along with 40 pg tol2 transposase mRNA into one-cell stage AB embryos. Zebrafish were raised to adulthood, out-crossed to wild-type fish, and resulting embryos were screened for progeny stably expressing the fluorescent constructs. Transgenic larvae expressing a heat shock-inducible construct were incubated at 37°C for 45 min in 15 ml of embryo media and screened a few hours later. Embryos expressing a fabp2-driven construct were screened at 2–4 dpf for EGFP expression in the yolk syncytial layer and embryos expressing the fabp10a-driven construct were screened at 5 or 6 dpf following liver development. At least two stable lines per construct were initially generated, the pattern of expression was verified to be the same in each line and subsequently, a single line for each construct was used for experiments and propagated by out-crossing to wild-type AB fish.Additional transgenic zebrafish expressing human g system . The codLauchlan  and re-cl Bagnat ; the livin Chien . GatewayFor whole-mount imaging, zebrafish larvae were anesthetized with tricaine (Sigma-Aldrich A5040) and mounted in 3% methylcellulose in embryo media on glass slides and imaged live with a Zeiss Axio Zoom V16 microscope equipped with a Zeiss PlanNeoFluar Z 1×/0.25 FWD 56 mm objective, AxioCam MRm camera, EGFP, Cy3, and mCherry filters and Zen 2.5 Blue edition software. The EGFP filter was used for both EGFP and LipidTOX Green imaging, the Cy3 filter was used for Plin3-RFP imaging, and the mCherry filter was used for LipidTOX Red imaging. For confocal imaging of lipid droplets in the tissues of live larvae, fish were anesthetized with tricaine and mounted in 3% methylcellulose on glass slides with bridged coverslips. Images were obtained with a Leica DMI6000 inverted microscope and Leica 63×/1.4 HCX PL Apo oil-immersion objective with a Leica TCS-SP5 II confocal scanner with photomultiplier detectors using Leica Application Suite Advanced Fluorescence 2.7.3.9723 image acquisition software. Images were obtained using four-line averaging and recorded with 12-bit dynamic range. EGFP and BODIPY FL-C12 were excited with an argon laser (488 nm) and had a collection window of 498–530 nm. BODIPY (558/568)-C12 was imaged with 561 laser and collection window of 571–610 nm and mTagRFP-t and LipidTOX Red were imaged with 561 laser and collection window of 575–650 nm. When using green BODIPY FL-C12 or LipidTOX dyes, imaging was performed sequentially for red and green channels.Fus(EGFP-plin2) genotyping at the 0–5 and 24–30 hr time-points when the EGFP fluorescence is absent or very dim. Fluorescence intensity (raw integrated density) of all images was quantified in Fiji /+ fish at 10 hr was also quantified to obtain an estimate of the amount of lipid consumed.To investigate changes in Plin3-RFP and EGFP-Plin2 fluorescence in the whole intestine following a high-fat meal, larvae were fed with a 5% egg yolk solution for 90 min as described above. Whole-mount images of the intestine were obtained every hour for the first 8 hr and subsequently every 2 hr thereafter for a total of 30 hr from the onset of the feed. Following the meal, all larvae were maintained at room temperature for the duration of the experiment. Fish were collected for Fus(plin3-RFP)/+; Fus(EGFP-plin2)/+ larvae were fed for 60 min with 5% egg yolk in embryo media as described above. Confocal images of the anterior region of the intestine were obtained every hour starting at 30 min and continuing until ~8 hr . Costes autothresholding and Manders’ colocalization analysis of EGFP and RFP fluorescence was performed on a subset of the 2× zoom, 8-bit images in Fiji. A region-of-interest was used for each pair of images to ensure the analysis was only being performed in the intestine.To study changes in Plin3-RFP and EGFP-Plin2 localization in the enterocytes at the tissue, cell, and subcellular level, Fus(plin3-RFP)/+ fish were pre-sorted for RFP prior to feeding and imaging, larvae from the Fus(EGFP-plin2)/+ × AB crosses and Tg(fabp2:EGFP-PLIN2)/+ × AB crosses were collected for genotyping after imaging to identify wild-type and transgenic fish at the early time-points when EGFP-Plin2 is absent. To quantify the RFP-Plin2 and EGFP-Plin2 fluorescence present in the cytoplasm vs. associated with the BODIPY C12-labeled lipid droplets, we first segmented the BODIPY C12 lipid droplets automatically using Ilastik V1.3.3post3 (https://ilastik.org). The pixel classifier was trained on example images from different time-points using color/intensity features ranging from σ 1.6 to 5.0 pixels and edge and texture features ranging from σ 0.07 to 5.0 pixels. This classification resulted in a probability map of the brighter BODIPY puncta that represent lipid droplets, vs. the BODIPY C12 signal in the intestinal lumen and cytoplasm of enterocytes. Training and segmentation were performed separately on green BODIPY FL-C12 images and red BODIPY 558/568-C12 images. Following training, images were batch-analyzed and probability maps were exported as ‘simple segmentation’ files in .tiff format. These files were opened in Fiji and the threshold was adjusted so the image contained only the lipid droplet segmentation as a binary image. Because the lipid droplets are often tightly clustered, it is difficult to segment them into single objects. Therefore, all the lipid droplets were unified into one object mask and this mask was expanded by three pixels  using the Process> Binary > Dilate function in Fiji. This expansion was necessary to ensure that the mask would encompass the rim of the lipid droplets where the majority of the Plin fluorescent signal is located, especially on larger lipid droplets. This expanded lipid droplet mask was applied to the corresponding fluorescent Plin3-RFP or EGFP-Plin2 image in Fiji and the mean fluorescence intensity in the mask was quantified in addition to the total mean fluorescence intensity without the mask. Because some images included regions of liver or other organs, when necessary, an additional region of interest delineating the intestine was combined with the lipid droplet mask to ensure only the intestine was analyzed. The mean fluorescence in the cytoplasm was obtained by subtracting the fluorescence associated with the lipid droplets from the total fluorescence. In addition, individual data points were also transformed by the mean of all data points (per genotype) to better compare the relative changes in Plin3-RFP, zebrafish EGFP-Plin2, and human EGFP-PLIN2 fluorescence over time. Data from 0.75 to 8.5 or 16–30 hr for each data set was fit using linear regression with the least squares method to determine the coefficient of correlation, R.To assess subcellular localization of Plin3-RFP and EGFP-Plin2 proteins in relation to lipid droplets in the intestinal enterocytes over time following a high-fat meal, larvae were fed for 60 min with a 5% egg yolk solution containing either green BODIPY FL-C12 or red BODIPY (558/568)-C12 at 4 µg/ml as described above. Confocal images of the anterior region of the intestine were obtained approximately every 30 min for the first 3.5 hr, every hour from 4.5 to 8.5 hr, and every 2 hr from 16 to 30 hr (63× + 2× zoom). Following the meal, all larvae were maintained at room temperature for the duration of the experiment. While 3.3post3  . Data are presented as mean ± standard deviation (SD). Statistical tests used are noted in figure legends or text. All experiments were performed with two to three independent replicates (N). The sample size (n) for each experiment is noted in the figure legends.Graphing was performed with GraphPad Prism (GraphPad Software). DNA, mRNA, and protein sequence alignments were performed with MacVector V15.5 . Micrographs were adjusted and cropped as needed in Fiji (NIH) and figures were assembled in Adobe Illustrator CS5 (Adobe Systems). Microsoft Word and Excel were used for manuscript preparation and data analysis, and references were compiled with EndNote x8.Fus(EGFP-plin2) and Fus(plin3-RFP) zebrafish lines are available at the Zebrafish International Resource Center (ZIRC): plin2c847Tg  and plin3875Tg .All transgenic zebrafish lines are available upon request. The  public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Acceptance summary:Your revised manuscript addresses most of the requests of the reviewers; your rationale for not performing detailed fasting studies is acceptable, in light of the heat shock issue and the ubiquitous transgenic paper you cited. The quantification of data Figures 1 and 4; and the inclusion of standard length makes your resource of broader utility to the field in that it allows comparison with other studies. Likewise, the additional details on the knock-in strategy provide greater transparency and validation of your tool. You rightly note some errors in selecting citations for revision-thank you for this description of your choices. Your suite of knock-in and transgenic tools will serve as a resource of broad utility.Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Amnon Schlegel as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Didier Stainier at Senior Editor.Thank you for submitting your article "Imaging cytoplasmic lipid droplets in vivo with fluorescent perilipin 2 and perilipin 3 knockin zebrafish" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this single summary and list of essential revisions to help you prepare a revised submission.Summary:The authors sought to develop fluorescent protein reports for lipid droplets for use in live imaging of an intact vertebrate organism. These subcellular structures are under dynamic regulation by assorted intrinsic and extrinsic factors; their study would be enhanced by having a convenient, in vivo detection system. The authors present knock-in lines of two lipid droplet coat proteins  toward this end. These tools could be valuable to the cell biological and metabolic research communities. The authors find that plin2 transcript is induced in liver and intestine of 6 dpf zebrafish larvae following a single feeding, while plin3 transcript is expressed in the fasted and fed states in the liver. They use TALENS to knock-in EGFP and TagRFP1 into the plin2 and plin3 loci, with the encoded gene products being the fusion proteins Plin2-EGFP and Plin3-TagRFP1. The Plin2-EGFP protein shows greater induction of fluorescence following a meal. The overall aim of these initial expression characterizations and development of lipid droplet reporter knock-ins is to be able to monitor the life cycle of these organelles in a living whole organism.Higher resolution photomicrographs of lipid droplets with these knock-in lines concurrently stained with the fluorescent lipid dyes BODIPY C12 and BODIPY FL-C12 are presented with a time series following feeding in intestine; additional cell types beyond enterocytes  are presented.The authors are in a position to provide a technical advance to the field of lipid droplet biology and, potentially, assorted diseases marked by lipid derangements. With the tractable revisions set out below, their tools set the stage for chemical and genetic screens for factors and compounds that modulate the normal life cycle of these dynamic organelles.Essential revisions:1) Please present data with greater quantitative rigor throughout the manuscript.a. Figure 1E and 1F, for instance, require quantification of the effects of induction. More generally, the number of animals studied (5 to 10) per experimental time point was felt to be low given the large number of embryos obtained in a cross, and the potential to reveal whether lipid droplet  is gaussian in distribution or has a threshold; previous work by the Rawls and Sadler labs have pointed to standard length as the driver (not age) of detecting lipid droplets with fluorescent (and other) dyes.b. Relatedly, statistical rigor in these analyses is requested; clear statements throughout of what tests are used is required.2) Please improve the description and validation of the knock-in and transgenic lines.C. elegans literature) whether this choice matters. Does this merely reflect the cDNAs obtained from others? Was there a deliberate strategy?a. Why was the 5'-end used to insert one fluorescent reporter, but the 3'-end was used for the other? Please describe  please move Figure Supplement 1 relating to the assorted transgenic lines to the main figures and include co-labeling with a fluorescent lipid dye, and (ii) please provide whole-mount, low magnification views of adults subjected to a fast along the lines of the studies conducted by Minchin and Rawls. This experiment will address similar issues to that raised in points 2c and 2e.e. For the transgenic lines, please state why human orthologs were selected? For instance, if you can detect these with commercially available antibodies, that could be a major advantage to their use. While PLIN2 and 3 are generally considered permissive to the lipolytic cascade  without hormonal input, it is important to assess whether the over-expressed proteins impact  lipid droplet biology. A fasting series of up to 7 days in larvae is feasible and might reveal neomorphic aspects to these transgenics.3) Please provide broader context regarding the value of these new tools in a dedicated Discussion section.The summary paragraph (lines 331-340) should be expanded to describe the potential uses of these new reagents for studying obesity and lipodystrophy, along with fatty liver disease , diabetes mellitus (types 1 and 2), specific cardiovascular diseases and atherosclerosis ? More narrowly, please describe how these disease processes would be explored in zebrafish: are there established dietary models of fatty liver disease, type1 or type 2 diabetes, or atherosclerosis in which these tools could be used? Are there genetic or chemical screens you foresee would benefit from these lines? Could they be crossed to established mutants for live imaging purposes?Paralleling this disease modeling discussion, please discuss the limitations of using a perilipins on lipid droplet biogenesis investigations: early lipid droplet biogenesis proceeds without perilipins, discussion of what other tagged proteins might be required for visualizing those early steps in a whole vertebrate also merits presentation.4. Improve citations to primary literature or key review articles of direct relevance to the Tools and Resources format. Original citations can be added to address point 3.Please remove the following citations, unless you feel a particular review article raises a unique hypothetical or synthetic point:1. Bickel et al.,2. Chien et al., ;3. Kimmel and Sztalryd.4. Mather et al.,5. Miura et al.,6. Olzmann and Carvalho .7. Roberts and Olzmann .8. Sztalryd and Brasaemle9. Thomas et al., 201510. Zeituni and Farber Essential revisions:1) Please present data with greater quantitative rigor throughout the manuscript.a. Figure 1E and 1F, for instance, require quantification of the effects of induction. More generally, the number of animals studied (5 to 10) per experimental time point was felt to be low given the large number of embryos obtained in a cross, and the potential to reveal whether lipid droplet  is gaussian in distribution or has a threshold; previous work by the Rawls and Sadler labs have pointed to standard length as the driver (not age) of detecting lipid droplets with fluorescent (and other) dyes.b. Relatedly, statistical rigor in these analyses is requested; clear statements throughout of what tests are used is required.Based on reviewer requests, we have provided more quantitation throughout the manuscript. In particular, we quantified the effects of induction associated with Figure 1E at both the whole mount and sub-cellular level and in doing so increased the numbers of animals studied. These new analyses can be found in Figure 2  and Figure 4  in the revised manuscript. With regard to the number of animals studied, during a confocal time-course, the number of animals used in the analysis is dependent on how many fish we can mount and image at a given time-point. In the first hours after feeding, our time-points are closer together, so fewer fish are imaged. Furthermore, we can sort for Plin3-RFP before feeding, but it is not possible to do so for the EGFP-Plin2 line. Typically, we have imaged at least 2x as many fish as noted in the figure legend and it is only after collecting and genotyping the imaged larvae that we know how many EGFP-Plin2 fish were present. At later time-points when the EGFP signal is visible, it is easier to ensure that we have higher numbers of fish. While we could repeat these time-courses additional times to increase the numbers of larvae, these are very time-intensive experiments and we feel that the data we have collected in Figure 4 is more than sufficient to show the clear transition from Plin3 to Plin2 on the lipid droplets after a meal. As requested, we have also performed and reported statistical analyses where appropriate.With regard to the use of standard length and not age, for any imaging performed in the liver, adipose tissue and lateral line, we have now provided standard lengths of the fish in addition to the day post fertilization at which the imaging was performed. For images provided in the initial submission for which no standard length measurements were available, as a proxy, we have provided standard length data for fish that were treated similarly with regard to food and rearing.For Example, see Figure 5 legend:“Standard length of the imaged fish is noted on the upper left corner of each set of images, see source data for additional standard length data.”2) Please improve the description and validation of the knock-in and transgenic lines.C. elegans literature) whether this choice matters. Does this merely reflect the cDNAs obtained from others? Was there a deliberate strategy?a. Why was the 5'-end used to insert one fluorescent reporter, but the 3'-end was used for the other? Please describe . Since we began the synthesis of these lines, C-terminal fusions have now also been shown to be functional. The tags were placed on the C-terminal end of Plin3 because our human transgenic lines with C-terminal fusions were known to be functional before we made the knock-in line.Results section now reads:“We designed our engineering strategy for Fus(EGFP-plin2) based on early data suggesting that it was necessary to tag PLIN2 on the N-terminus , although subsequent work has now shown that C-terminal tags are also functional . Additionally, we had evidence from our generation of Tol2-based transgenic reporter lines that over-expression of both human EGFP-PLIN2 and PLIN3-EGFP resulted in labeling of lipid droplets .”b. Were insertions or deletions introduced beyond the sites of knock-in? Are there off-targets?We have now included additional details about the numbers of fish screened to find our knock-in lines. We noted the incorrect insertions discovered and the one intronic deletion that we found in the plin3 line.Results section now reads:“For Fus(EGFP-plin2), a total of 40 adults were screened, and 3 fish produced EGFP+ progeny. However, two of these clutches had abnormal tissue expression patterns and only displayed cytoplasmic EGFP fluorescence. One of these had correct integration across the left homology arm, but the right homology arm integration could not be verified; the second had no confirmed integration at the locus. Furthermore, while our founder fish produced low numbers of progeny with correct expression patterns and proper integration (8 embryos out of 853 screened), she also produced progeny with incorrect integration (22/853), suggesting mosaicism in the germ cells. To identify a Fus(plin3-RFP) founder, 26 adults were screened before a fish produced progeny expressing RFP. These embryos had correct tissue expression of RFP in the intestine and correct integration of the transgene (53 RFP+ embryos out of 613 screened). We did note a 29 bp region missing in intron 7-8, however, this does not alter the coding region, and it is unclear whether this deletion arose during integration or whether it was a naturally occurring variant in the injected embryo.”c. Since GFP and TagRFP-T are not fast folding proteins and are very stable, a kinetic analysis directly comparing lipid dye signals to fluorescent proteins in both growing and shrinking lipid droplets is required. For instance, please provide fluorescent dye labeling of cell types beyond enterocytes in Figure 2. Any cases where this is not feasible can be informative. For example, if the live liver is not well-labeled, then the transgenic reporters will clearly provide an advantage for imaging.We have now provided extensive time-course imaging and kinetic analysis of Plin3-RFP and EGFP-Plin2 in the intestine in larvae fed with fluorescent BODIPY fatty acids . We imaged both the induction (0 – 8.5 h) and the degradation phase (16 – 30 h) of the lipid droplets. We find that at all times, the BODIPY-labeled lipid droplets in the intestine are labeled with either Plin3 or Plin2.Unfortunately, this analysis does not allow us to directly determine whether the EGFP or TagRFP-t tags affect the folding or stability of the Plin proteins compared to untagged versions (and thus possibly influence lipid droplet growth and lifetime). Since we do not have antibodies that recognize the zebrafish plin proteins, this direct comparison is not possible at this time.The text of the Discussion now reads:“Therefore, we have little reason to believe the fluorescent tags used in this study are interfering with lipid droplet dynamics, however we cannot test these hypotheses directly due to the current lack of antibodies that recognize the zebrafish perilipin 2 and 3 orthologs. Despite these caveats, these reporters indicate a clear progression from Plin3 to Plin2 on intestinal lipid droplets following a high-fat meal in zebrafish.”As requested, we have now provided additional images of fluorescent LipidTOX labeling of the lipid droplets in fish expressing the fluorescent Plin proteins .d. Relatedly, (i) please move Figure Supplement 1 relating to the assorted transgenic lines to the main figures and include co-labeling with a fluorescent lipid dye, and (ii) please provide whole-mount, low magnification views of adults subjected to a fast along the lines of the studies conducted by Minchin and Rawls. This experiment will address similar issues to that raised in points 2c and 2e.i) We have moved the figure supplement to the main text  and we have now included co-labeling with a fluorescent lipid dye for each transgenic line.ii) We did not generate our fusion lines in a pigment mutant background, so whole-mount imaging of the perilipins in the adult pigment mutant fish would require many months of breeding. However, in Figure 6, we have now provided whole-mount images of EGFP-Plin2 and Plin3-RFP in the adipose tissue in juvenile larvae .It was unclear whether the reviewers were requesting the fasting experiment in the knock-in lines or in the over-expression transgenic lines. We did not perform a fasting experiment with our fusion lines because in the recent Lumaquin et al., eLife 2021 study, stable over-expression Plin2-tdtomato did not show differences in adipose area, standard length or adipose area/standard length following a 7 day fast. Given that our knock-in lines are expressing the fusion proteins at endogenous levels, it is therefore unlikely that the fluorescent tags would cause alterations in lipolysis. The Tol2 transgenic lines we have generated over-express human PLINs either in the intestine, liver or ubiquitously following heat-shock. Therefore, to have overexpression of PLIN2 or PLIN3 in the adipocytes, a daily heat-shock would likely be required and would add additional stress to the fish being starved. While it is feasible that over-expression of PLIN2 in the liver may alter the effects of a fast on adipocytes or other tissues in some way, it is unlikely that over-expression in the intestine would have any effect if the fish is not consuming any dietary lipids and have no lipid droplets in the intestine. While these are interesting questions and could be pursued in the future, we feel that they are not essential to the description/validation of the new tools that we are highlighting in the current manuscript.e. For the transgenic lines, please state why human orthologs were selected? For instance, if you can detect these with commercially available antibodies, that could be a major advantage to their use. While PLIN2 and 3 are generally considered permissive to the lipolytic cascade  without hormonal input, it is important to assess whether the over-expressed proteins impact  lipid droplet biology. A fasting series of up to 7 days in larvae is feasible and might reveal neomorphic aspects to these transgenics.We have included information in the text about why we chose to over-express the human orthologs and why they may provide an advantage over the zebrafish orthologs.The Results section now reads:“The decision to over-express the human orthologs was mostly out of convenience of already having the clones present in our laboratory and evidence from cell culture studies that fusions of human PLIN proteins with fluorescent proteins were functional . However, we also appreciated that human proteins are more amenable to detection with commercially available antibodies in downstream applications, and might provide an opportunity to maintain tissue-specific or temporal control of perilipin expression when targeting the endogenous loci for gene editing.”To begin to address whether the over-expressed proteins impact lipid droplet dynamics, we performed the kinetic BODIPY C12 intestinal time-course imaging in the Tg(fabp2:EGFP-PLIN2) transgenic line alongside the fusions lines . This allowed us to ask whether the lipid droplets are amenable to PLIN2 binding if the protein is present earlier in the enterocyte and to ask whether the lifetime of the lipid droplets was increased due to the presence of the over-expressed human EGFP-PLIN2 when compared with endogenously-expressed zebrafish EGFP-Plin2. These experiments indicate that when expressed under the control of the fabp2 promoter, the human PLIN2 protein is present earlier in the enterocyte following onset of a high-fat meal and localizes primarily to the lipid droplets. Thus, the nascent lipid droplets are amenable to PLIN2 binding. However, we did not find a significant difference in the % area occupied by lipid droplets at 30 h. At this time, fish of each genotype had a range of lipid droplets remaining, some had none and whereas others had droplets occupying upwards of 15-20% of the intestine area, the mean for all genotypes was 6.2%. Furthermore, at 6 months of age, we did not find differences between the Tg(fabp2:EGFP-PLIN2)/+ fish and their WT siblings in either standard length or mass, suggesting that the subtle differences noted in the intestine did not have lasting impacts on their physiology.The following paragraph has been added to the results:“Based on this analysis, we wondered whether the timing of the transition to Plin2 simply reflects the time needed to both transcribe and translate Plin2 prior to its association with the droplets, and whether Plin2 could bind to nascent lipid droplets sooner if the protein was already present. To investigate this question, we repeated the intestinal time-course imaging in transgenic fish stably over-expressing human PLIN2 under the control of the zebrafish fatty acid binding protein 2 promoter, Tg(fabp2:EGFP-PLIN2). This promoter drives expression in the yolk syncytial layer and in the intestine . In unfed fish, EGFP-PLIN2 signal cannot be detected above the autofluorescence visible in the intestine at 6 dpf , likely because any protein produced is rapidly degraded in the absence of lipid droplets . However, we hypothesized that the mRNA would be available immediately for translation and PLIN2 protein would be produced more quickly than in the Fus(EGFP-plin2) line. Indeed, we visually detected EGFP-PLIN2 on lipid droplets at 45 min, our earliest time-point after the start of the meal  and the difference between fluorescence on lipid droplets vs. in the cytoplasm reached statistical significance by 3.5 h , suggesting that the droplets are not prohibitive to plin2 binding at the early times. Although we also hypothesized that over-expression of EGFP-PLIN2 might delay the degradation of lipid droplets in the enterocytes, the fraction of intestine area covered by remaining lipid droplets at 30 h was not different between the three transgenic lines or wild-type fish . Furthermore, over-expression of EGFP-PLIN2 in the intestine does not affect the standard length or mass of the fish at 6 months of age .”As discussed above, the tissue in which our human transgenic lines would be most appropriate to be used in a fasting series would be the liver. While we did perform short periods of fasting (48 – 72 h) in the Tg(fabp10a:EGFP-PLIN2) line, we could not draw sufficient conclusions from these experiments to warrant addition in this manuscript. Although we expected to find that these fish had many more lipid droplets in the liver than wild-type siblings or the fusion lines subjected to the same period of fasting, the variability between fish in lipid droplet number and size, irrespective of the genotype of the fish, made it very difficult to interpret the effects of fasting and/or over-expression. We feel strongly that many further experiments are necessary to even begin to understand lipid metabolism and perilipins in the developing liver.3) Please provide broader context regarding the value of these new tools in a dedicated Discussion section.The summary paragraph (lines 331-340) should be expanded to describe the potential uses of these new reagents for studying obesity and lipodystrophy, along with fatty liver disease , diabetes mellitus (types 1 and 2), specific cardiovascular diseases and atherosclerosis ? More narrowly, please describe how these disease processes would be explored in zebrafish: are there established dietary models of fatty liver disease, type1 or type 2 diabetes, or atherosclerosis in which these tools could be used? Are there genetic or chemical screens you foresee would benefit from these lines? Could they be crossed to established mutants for live imaging purposes?Paralleling this disease modeling discussion, please discuss the limitations of using a perilipins on lipid droplet biogenesis investigations: early lipid droplet biogenesis proceeds without perilipins, discussion of what other tagged proteins might be required for visualizing those early steps in a whole vertebrate also merits presentation.As suggested by the Reviewers, we have now extensively expanded the Discussion section to provide broader context as to the value of these new tools and to further discuss our findings regarding the ordered recruitment of plin3 and plin2 in the intestine following a high-fat meal. We specifically discuss the need to study lipid droplet biology in the context of whole animal studies.For example, here is a section from the penultimate paragraph of the Discussion:“Using these zebrafish Plin reporter lines in the context of diets  and established zebrafish mutations and disease models  may provide mechanistic insights that connect perilipin cell biology to metabolic and cardiovascular diseases.”4. Improve citations to primary literature or key review articles of direct relevance to the Tools and Resources format. Original citations can be added to address point 3.Please remove the following citations, unless you feel a particular review article raises a unique hypothetical or synthetic point:1. Bickel et al.,2. Chien et al., ;3. Kimmel and Sztalryd.4. Mather et al.,5. Miura et al.,6. Olzmann and Carvalho .7. Roberts and Olzmann .8. Sztalryd and Brasaemle9. Thomas et al., 201510. Zeituni and FarberWe have augmented many of the reviews with primary literature, however, there were a number of primary references in the above list, so we have addressed each reference request individually below:1. The Bickel et al., 2009 review provides important comparisons amongst the plin family proteins that are not readily appreciated when reading all of the original primary research articles and additionally provides relevant historical context. However, we have removed this reference to satisfy the reviewers’ request.Drosophila model system. While the reference noted by the reviewers describes the use of confocal Raman spectroscopic imaging in zebrafish and briefly describes lipid droplets, we feel that this suggested reference does not adequately make the point that CARS and SRS microscopy can be used to image lipid droplets and should not replace our original reference. Furthermore, the sentence in which our reference occurs is not strictly describing zebrafish model systems.2. The Chien et al., 2012 reference is a primary article using CARS microscopy to elucidate lipid droplets within the 3. Kimmel and Sztalryd 2016 – We have added a number of primary references to support our statements, however, given the complexity of the PLIN protein family  we feel that this review article is still an important addition to this manuscript for readers who may not be as familiar with the lipid droplet/perilipin field .4. We have replaced the Mather et al., 2019 symposium review article reference with the Masedunskas et al., 2017 article, which is the source of the data provided in the Mather et al., 2019 symposium paper.5. Miura et al., 2002. We do not understand why the reviewers have requested this reference removed. This is a primary article which beautifully shows fluorescently-tagged perilipin proteins from different species associating with lipid droplets in cultured cells. It is also the first study to show that proteins identified as perilipins in other species based on sequence identity, associate with lipid droplets.6 and 7. Olzmann and Carvalho, 2019 and Roberts and Olzmann, 2020. These reviews are some of the most recently written on lipid droplets and provide a solid background for readers less familiar with the lipid droplet field. Together, they provide a thorough review of lipid droplet functions and dynamics, including budding and growth, organelle contacts, and roles in lipotoxicity and ER stress, as well as the role of the lipid droplet proteome in these dynamics and functions. If we were to try to replace these reviews, it would require hundreds of primary references. Given that the role of these reviews was to introduce readers to the fact that lipid droplets are not just bags of fat, but that they are important organelles with many roles in physiology, we feel that they accomplish this purpose more completely than a handful of randomly chosen primary references. To reduce the reviews cited in this section of the introduction, we have removed the Coleman 2020 reference because this is not as comprehensive a review as the Olzmann and Carvalho, 2019 and Roberts and Olzmann, 2020 references.8. Sztalryd and Brasaemle, 2017. We have added a number of additional primary references to this section of the introduction, but we feel strongly that this review concisely summarizes our understanding of the regulation of lipolysis by the perilipins and think that it should remain in the paper.9. Thomas et al., 2015. We have replaced this reference with more appropriate primary references.10. Zeituni and Farber 2016 is a methods paper that provides step-by-step protocols for a reader interested in repeating the feeding and imaging assays described in this manuscript. Given eLife’s commitment to enhancing data reproducibility, we think that this reference should remain in our Methods and Materials section.


While these data implicate the ERα as a Pol III transcriptional regulator, how widespread this regulation is across the 631 tRNA genes has yet to be revealed. Through analyses of ERα ChIP-seq datasets, we show that ERα interacts with hundreds of tRNA genes, not only in MCF-7 cells, but also in primary human breast tumours and distant metastases. The extent of ERα association with tRNA genes varies between breast cancer cell lines and does not correlate with levels of ERα binding to its canonical target gene GREB1. Amongst other Pol III-transcribed genes, ERα is consistently enriched at the long non-coding RNA gene RMRP, a positive regulator of cell cycle progression that is subject to focal amplification in tumours. Another Pol III template targeted by ERα is the RN7SL1 gene, which is strongly implicated in breast cancer pathology by inducing inflammatory responses in tumours. Our data indicate that Pol III-transcribed non-coding genes should be added to the list of ERα targets in breast cancer.tRNA gene transcription by RNA polymerase III (Pol III) is a tightly regulated process, but dysregulated Pol III transcription is widely observed in cancers. Approximately 75% of all breast cancers are positive for expression of Estrogen Receptor alpha (ERα), which acts as a key driver of disease. MCF-7 cells rapidly upregulate tRNA gene transcription in response to estrogen and ChIP-PCR demonstrated ERα enrichment at  RNA polDysregulated transcription by Pol III has been widely observed in many types of tumour, including ovarian and breast cancers . FurtherERα is a nuclear receptor that specifically coordinates transcriptional changes for many target genes in response to estradiol. Its paradigm mechanism of gene induction involves an estrogen-responsive element (ERE), which is a palindromic consensus sequence of two half site motifs separated by a 3 bp spacer (5′-GGTCAnnnTGACC-3′); they are found at enhancer and promoter regions of many ERα-regulated genes that enable the hormone receptor to directly bind to the gene via its DNA-binding domain and bring about transcription by recruitment of additional transcription factors, such as histone acetyltransferases. An alternative, less-characterized mechanism involves ERα tethering to other transcription factors that are themselves DNA-bound and act to either stabilize transcription complexes, or recruit additional cofactors; in this instance, the ERα itself is not directly bound to the DNA.Stimulation of MCF-7 breast cancer cells with estrogen induces rapid and profound transcriptional changes, in which many tRNA genes are affected . FurtherRMRP gene, which has been implicated by recurrent mutation as an oncogenic driver  were analysed. This revealed widespread interaction of ERα with many tRNA genes in MCF-7 cells and in ERα + ve patient breast cancer samples, as well as in distant breast cancer metastases. However, minimal interaction with tRNA genes was detected in ERα-negative MDA-MB-231 cells with a stably-integrated ERα expression vector, despite robust binding to established targets with consensus ERE sequence motifs. These data suggest that recruitment of ERα to tRNA genes may depend on unknown factors present in MCF-7 and ERα + human breast cancer cells, but absent from the MDA-MB-231 cell line. Possibilities include specific pioneer factors and/or proteins involved in chromatin regulation or ERα tethering. Two other pol III-transcribed loci that attract ERα in breast cancer cells are the c driver , and thec driver .22.1insertion, using CRISPR to generate an MCF-7 cell line stably expressing a C-terminal LAP-tag containing eGFP, fused to ESR1) were downloaded in Binary Alignment Map (BAM) file format from ENCODE  “Genomic File Manipulation” tools.ERα ChIP-seq was performed in patient samples and in the MCF-7 and ZR-75–1 cell lines . Dataset2.3ChIP-seq was performed in the ERα -ve MDA-MB-231 cell line that was stably expressing a wild type ERα construct  and the 2.4https://genome.ucsc.edu) and imported as EaSeq “regionsets” to be used as regions of interest.BAM or BED files containing ERα ChIP-Seq replicates were uploaded into EaSeq “DataSets”. Complete tables of tracked GRCh38 (hg38) and NCBI36 (hg18) tRNA genes were downloaded from the UCSC Table Browser, . The top 50 ERα-bound tRNA genes  confirmed in the ENCODE ERα MCF-7 ChIP-seq data analysis described above were independently compared against these tRNA gene categories.2.6Motif investigations were conducted in tDNA genes which had the largest ERα Q-values following ChIP-seq analysis in the MCF-7 dataset (n = 19 for ENCODE data). The tRNA genomic coordinates were obtained  using the NCBI Gene database and supplied to the Multiple EM for Motif Elicitation ChIP (MEME) Suite (v 5.3.0) to search for known sequences, using default settings. Sequence motifs of the conserved A and B box and of the ERE and half EREs were supplied to the program.2.7https://www.ncbi.nlm.nih.gov/genome/tools/remap) was used to retrospectively remap the hg38 tRNA gene coordinates to the NCBI36 hg18 gene coordinates. Of the 631 hg38 tRNA gene coordinates provided, 80% of the tRNA genes were successfully remapped.To determine the overlap between the tRNA genes in which ERα strongly associates in the hg38 MCF-7 cells and the hg18 breast cancer samples, the NCBI genome remapping service  were grown in DMEM  with 10% FBS  and 1% Penicillin Streptomycin . Cells were crosslinked with 1% formaldehyde for 8 min at room temperature. Crosslinking was inhibited by adding equal volume of ice cold quenching buffer (250 mM Glycine/ 2 mM EDTA / TBS) to the cells, followed by centrifugation . This step was repeated for a total of two washes in the quenching buffer. MCF-7 cells were then lysed in Lysis Buffer 1  for 10 min at 4℃. Cell lysate was collected by centrifugation  and resuspended in Lysis Buffer 2  for 10 min at 4℃. MCF-7 nuclei were harvested by centrifugation  and resuspended in Lysis Buffer 3 , before sonication . 0.1% of Triton X-100 was added to nuclear extract and debris was pelleted by centrifugation .2O and heating for 5 min at 65℃, for a total of two elution steps. Eluates were then pooled together.Antiserum 1901 was raised by immunizing rabbits with synthetic peptide PKRPLTFDTNEFHIPLVT (residues 1374–1391 of the RPC155 subunit of human pol III), coupled to KLH. For immunoprecipitation, protein A Dynabeads were washed in blocking solution (0.5% BSA in PBS). Dynabeads were next incubated in blocking solution containing either 15 μl pre-immune serum (PI), 15 μl anti-pol III serum (1901) or 6 μg of anti-ERα antibody  for 2 h at 4℃. Protein A:antibody-containing beads were then added to nuclear lysate and mixed overnight at 4℃. Next, the Dynabeads were collected on a magnetic rack and washed in RIPA buffer  for a total of 6 washes . The sample-containing beads were then washed with 50 mM NaCl Tris EDTA buffer for a total of 2 washes  before elution with elution buffer (100 mM NaHCO3 + 1% SDS) at 50℃ for 10 min. Crosslinking was then reversed by heating at 55℃, overnight. To collect chromatin, 1.8x SPRY beads were added to the samples and collected on a magnetic stand after 10 min. Beads were washed twice with 80% ethanol. DNA was then eluted from the beads by resuspending in dHReal time quantitative PCR (qPCR) was performed using 2 ng of chromatin extraction (input) and 1 μl of ChIP sample per reaction on a QuantStudio™ 3 qPCR system (Thermo Fisher). Triplicate reactions of each sample were performed using LUNA Universal qPCR Master Mix  on 96 well plates. The cycling parameters are shown in 33.1tRNA-Pro-TGG-1–1, tRNA-Arg-CCG-2–1 and tRNA-Met-CAT-1–1 genes . To investigate how extensively ERα targets the 631 tRNA genes annotated in the human genome, we interrogated ChIP-seq datasets from two replicate experiments. This revealed widespread interaction of ERα with>300 tRNA genes in MCF-7 cells . A heatm–1 genes D. Exampl control E. This f3.2Previous analysis of tRNA expression identified transcripts that are induced preferentially during cell differentiation and a distinct group that is expressed preferentially in proliferating cell types, including bladder, colon and prostate cancers . Although breast cancers were not included in those data, we analysed whether the tRNA targets of ERα identified by ChIP-seq in MCF-7 cells are enriched in either the differentiation or proliferation categories. We found that the top fifty ERα-bound tRNA genes (Supplementary Table ST1) are slightly depleted in the differentiation category relative to the proliferation group, although the difference is not statistically significant A. Simila3.3At least two distinct mechanisms have been shown to recruit ERα to enhancer and promoter regions that regulate protein-coding genes: i) direct binding to a DNA sequence motif (ERE) via its DNA-binding domain; ii) protein/protein interactions with other DNA-binding transcription factors, such that ERα is not bound to the DNA directly. To investigate the predominant mechanism directing the ERα to tRNA genes, motif analysis was conducted to search for the full ERE or half ERE consensus elements around the tRNA genes that have the strongest ERα binding events in MCF-7 cells. The search included 20 kb up- and downstream of the target genes themselves. Motif analysis conducted in MEME-ChIP failed to identify either a full or half ERE in these regions; this was not due to technical failure, as the conserved A and B box promoter sequences were strongly enriched in all tRNA genes, as expected . This do3.4GREB1 gene was at least as strong in transfected MDA-MB-231 cells as in MCF-7 cells (If recruitment of ERα to tRNA genes requires interactions with other protein(s) rather than direct recognition of DNA, it might vary between cell types according to availability of the necessary factor(s) and/or chromatin accessibility. To begin to explore this possibility, we investigated binding by exogenous ERα stably transfected into the MDA-MB-231 breast cancer cell line that lacks endogenous ERα . Binding-7 cells A. Howeve-7 cells  and aver-7 cells C. This s3.5ChIP-seq data are publicly available that analyse genome-wide binding of ERα in primary breast tumour samples from patients with invasive ductal carcinomas . HeatmapBiopsy samples of distant metastases were also analysed from three patients. These again showed clear accumulations of ERα at many tRNA genes, relative to the 10 kb upstream and downstream flanking regions, as revealed in heatmaps  and plot3.6BCYRN1) is a long non-coding RNA that is synthesized by pol III. Predominantly expressed in the brain, it has been implicated as a regulator of protein synthesis (BC200 (ynthesis . BC200 lynthesis ; these iynthesis . Manipulynthesis . Analysiynthesis . In contynthesis A. The reynthesis . AnalysiRN7SL1 gene in MCF-7 cells (RN7SL1 includes sequences upstream of the transcription start site, as well as A- and B-box motifs within the transcribed region; ERα reads were evident in both these areas in MCF-7 cells, whereas ERα is localized to the upstream region in Met 3 and, much more weakly, in the primary tumour biopsies; the significance of this difference is unclear. No enrichment of ERα was observed at RN7SL1 in Met 1 and MDA-MB-231 cells expressing the exogenous receptor. These data suggest significant heterogeneity in the interaction of ERα with the RN7SL1 gene in breast cancer cells.7SL RNA has been shown to be overexpressed in breast tumours . In addi-7 cells D The proRMRP is a 267 nucleotide ncRNA involved in rRNA processing, which also associates with the telomerase catalytic subunit to produce an RNA-dependent RNA polymerase (RMRP promoter lead to elevated expression in breast cancers and the locus also undergoes focal amplification in several tumour types, including breast (RMRP locus, relative to a pre-immune control (RMRP promoter was also detected in MDA-MB-231. Thus, the RMRP promoter is targeted by ERα, to a greater or lesser extent, in every case we have examined.lymerase . It has lymerase . Germlinlymerase . Somaticg breast . The RMRg breast C. ChIP-q control D. In con4Estradiol has been shown to rapidly increase transcription of rRNA and most tRNA genes in MCF-7 cells . WhetherMotif analysis of the top ERα-bound tRNA genes did not detect canonical ERE or even half ERE sequences within 40 kb windows centred on the tDNAs. The possibility remains of degenerate or non-canonical motifs that are recognised directly by ERα. Despite some well-characterized exceptions, in the majority of cases of estrogen-responsive protein-coding genes ERα acts at a distance after binding to remote enhancer sites, rather than targeting proximal promoters . Our dattRNA-Arg-CCG-2–1 and tRNA-Met-CAT-1–1 genes (RN7SL1 and RMRP ((tRNA-Pro-TGG-1–1 and tRNA-Leu-AAG-2–4 genes is downstream of where TFIIIB is expected (tRNA-Pro-TGG-1–1 and tRNA-Leu-AAG-2–4.ERα can also be brought to tDNAs through interactions with other proteins, rather than direct recognition of DNA sequences, and this has been estimated to account for ∼ 10% of its binding events . At pol –1 genes D, as welnd RMRP (B and 8C.expected D. One ortRNA-Leu-AAG-2–4 (As TFIIIB is required for expression of all pol III-transcribed genes, it is predicted to be available for exogenous ERα to access at active tDNAs in MDA-MB-231 cells and yet no interaction was detected at these loci B and 5C.-AAG-2–4 D, where Even in MCF-7 cells, the ChIP-seq signal intensity for ERα at tDNAs is generally much lower than that seen for well-established targets such as GREB1. Several possibilities might account for this difference. For example, tethering through other proteins might lower the efficiency of ERα crosslinking to tDNAs. Additional components of transcription factor complexes could restrict antibody accessibility and thereby reduce ChIP efficiency. In principle, such confounding factors may contribute to the low signal intensities on most tDNAs and explain why they have not been identified previously as ERα targets. It may also be that ERα is attracted to tDNAs primarily by their accessibility in chromatin and is often rapidly released unless captured by other proteins. Binding that is weak and/or transient may nevertheless have functional impact, as demonstrated by the robust transcriptional induction of these genes by oestradiol .The Brf1 gene encodes an essential subunit of TFIIIB and is itself bound and induced by ERα in MCF-7 cells . As alcoAbnormally elevated tRNA levels have been documented in breast cancers by several studies . Direct in vitro and metastasis in mouse models through codon-dependent effects on stability and translation of specific pro-metastatic mRNAs (RN7SL1, RMRP and hundreds of tDNAs are targeted by ERα in breast cancer tumours and metastases.Abnormal expression of tRNAs is likely to have functional impact. Elevated levels of 14 tRNAs are significantly associated with poor prognosis in breast cancer  and specic mRNAs ; it is tic mRNAs , which hic mRNAs . 7SL RNAic mRNAs . These oJodie R. Malcolm: Methodology, Investigation, Writing – original draft, Visualization. Natasha K. Leese: Methodology, Investigation, Visualization. Philippa I. Lamond-Warner: Methodology, Investigation, Visualization. William J. Brackenbury: Supervision. Robert J. White: Supervision, Conceptualization, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


DPF1–3 and PHF10 genes from unicellular to vertebrate organisms. The data reviewed suggest that the DPF domain of PHF10 plays an important role in SWI/SNF-dependent chromatin remodeling during transcription activation.Transcription activation factors and multisubunit coactivator complexes get recruited at specific chromatin sites via protein domains that recognize histone modifications. Single PHDs (plant homeodomains) interact with differentially modified H3 histone tails. Double PHD finger (DPF) domains possess a unique structure different from PHD and are found in six proteins: histone acetyltransferases MOZ and MORF; chromatin remodeling complex BAF (DPF1–3); and chromatin remodeling complex PBAF (PHF10). Among them, PHF10 stands out due to the DPF sequence, structure, and functions. PHF10 is ubiquitously expressed in developing and adult organisms as four isoforms differing in structure (the presence or absence of DPF) and transcription regulation functions. Despite the importance of the DPF domain of PHF10 for transcription activation, its structure remains undetermined. We performed homology modeling of the human PHF10 DPF domain and determined common and distinct features in structure and histone modifications recognition capabilities, which can affect PBAF complex chromatin recruitment. We also traced the evolution of  Gene expression is regulated by a variety of protein complexes. The chromatin epigenetic landscape, essential to the manifestation of genetic information, is shaped by histone modifications. Both individual proteins and protein complex subunits capable of interacting with chromatin possess domains that recognize different modifications of N-terminal histone tails. PHD (plant homeodomain fingers) are zinc-finger-containing domains that recognize modified N-terminal tails of H3 histones ,2. PHDs Two of these proteins, MOZ and MORF, are histone acetyltransferases, either of which can be part of the MYST complexes family acetylating H3 histone tails at lysines 9, 14, and 23  . Four other proteins, DPF1–3 and PHF10, are subunits of the SWI/SNF chromatin remodeling complex, which participates in replication, reparation, and transcription, changing chromatin topology and restructuring nucleosomes in the course of ATP hydrolysis. SWI/SNF complexes activate gene expression by shifting nucleosomes along the DNA strand, transferring them to another strand, removing H2A and H2B dimers, and, as a result, decreasing nucleosome density at gene promoter regions and regulatory elements, thereby activating transcription . SWI/SNFThe SWI/SNF family includes PBAF, BAF, and ncBAF complexes consisting of many proteins. These complexes possess identical cores and the ATPase subunit BRG1/BRM; however, their specific chromatin-binding modules differ. PHF10 and DPF1–3 proteins are such specific subunits of the PBAF and BAF complexes, respectively . DPF domIn mammals, the expression of PHF10 begins at the earliest stages of embryonic development: during gastrulation, PHF10 isoforms can be detected in all future organs and tissues  20]. Th. Th20]. PHF10 N[D/A]D[N/Q] in the others A. At theH3K14ac, H4K16ac, and H3K4me3 histone modifications are characteristic of actively transcribed genes . By bindThe H3K14ac modification is found at promoters and genes coding sequences (CDS), as well as at active enhancer regions of actively transcribed genes . H3KH3K4me3 is a well-studied modification, localized at promoters of actively transcribed genes and sometimes at the 3′ ends; it is absent at enhancer or CDS regions . SimilarWe therefore hypothesize that DPF domains carrying PHF10-P isoforms in PBAF complex can selectively bind CDS, active enhancers, and some specific metabolic and digestion genes enriched in H3K14ac, and also participate in transcription activation. However, in the case of stably transcribed genes, the H3K4me3 mark prevents the DPF from interacting with nucleosomes, the implication being that PHF-P is not required to maintain transcription at this stage. Meanwhile, PBAF complexes containing PHF10-S isoforms (lacking DPF domains) interact with H3K4me3-enriched chromatin via other subunits than PHF10 and are active during constant gene transcription, but not during transcription activation. In line with this suggestion, we observed sequential replacement of PHF10-P by PHF10-S isoforms in PBAF complexes during activation of specific myeloid genes in the HL60 cells, which corroborates our hypothesis about the specialization of PBAF complexes containing PHF10-P and PHF10-S isoforms .PHF10 and DPF1–3 genes group was unicellular or a simple multicellular. This conclusion can be drawn from the fact that unicellular Naegleria species, according to the NCBI Protein database, only has one homologous gene, while all other multicellular species carry at least two. The following scenario might be suggested: initially, a simple organism carried just one gene, the ancestor of all genes encoding DPF-containing proteins . It would be worthwhile to understand whether they have a common ancestor and how they evolved. Apparently, the organism which possessed the ancestral gene of proteins . Gene duF groups . Such a tebrates  53]. Th. ThPHF10hylogeny A; howevehylogeny . PHF10 gene-ancestor took place for the first time, how gene functioning was organized in that organism, or which domain of the two, PHD-1 or PHD-2, it had initially. It might be that duplication of PHD-1 resulted in the formation of the PHD-2 domain, which is shorter in length and does not possess the long α-helix (Naegleria species already have two PHDs (PHF10 gene was a commonplace event in eukaryotes in general and was triggered by different molecular mechanisms or genomics features [Amino acid sequences of PHD-1 and PHD-2 of PHF10 are quite similar, and their tandem location most probably indicates that they are a result of domain duplication. It is difficult to say in what species domain duplication in the  α-helix A, possibtwo PHDs . In thisfeatures ,56,57. IPHF10 is a unique protein expressed as two isoforms that have the DPF domain and two isoforms without it. Isoforms are subunits of the PBAF chromatin remodeling complex and determine its nucleosome binding specificity. We performed homology modeling and determined that the PHF10 DPF domain has unique conserved structural features that enable PHF10-P isoforms to bind the H3K14ac, H3R2, and H4K16ac markers of actively transcribed chromatin, but not the functionally similar marker H3K4me3. The differential distribution of histone modifications across the genome determine the specificity of recruitment of PBAF complexes that contain either the PHF10-P or PHF10-S isoforms. Via the DPF domain, PHF10-P isoforms can bind CDS gene regions or enhancers, enriched in H3K14ac but depleted of H3K4me. H3K14ac appears at chromatin during gene transcription activation and attracts PHF10-P isoforms to specifically participate in the activation. H3K4me3 markers are located on genes that are already actively transcribed and are not required for the activation process. Therefore, PBAF complexes containing PHF10-S isoforms could potentially bind H3K4me3-enriched chromatin via subunits of the PBAF complex other than PHF10.