CUT&Tag Applied to Zebrafish Adult Tail Fins Reveals a Return of Embryonic H3K4me3 Patterns During Regeneration

Regenerative potential is governed by a complex process of transcriptional reprogramming, involving chromatin reorganization and dynamics in transcription factor binding patterns throughout the genome. The degree to which chromatin and epigenetic changes contribute to this process remains partially understood. Here we provide a modified CUT&Tag protocol suitable for improved characterization and interrogation of epigenetic changes during adult fin regeneration in zebrafish. Our protocol generates data that recapitulates results from previously published ChIP-Seq methods, requires far fewer cells as input, and significantly improves signal to noise ratios. We deliver high-resolution enrichment maps for H3K4me3 of uninjured and regenerating fin tissues. During regeneration, we find that H3K4me3 levels increase over gene promoters which become transcriptionally active and genes which lose H3K4me3 become silenced. Interestingly, these epigenetic reprogramming events recapitulate the H3K4me3 patterns observed in developing fin folds of 24-hour old zebrafish embryos. Our results indicate that changes in genomic H3K4me3 patterns during fin regeneration occur in a manner consistent with reactivation of developmental programs, demonstrating CUT&Tag to be an effective tool for profiling chromatin landscapes in regenerating tissues.

4 on the tail of histone H3 (H3K4me3) associates with active chromatin regions and promotes RNA polymerase occupancy over genic promoter regions (2,4).Genomics patterns for these types of transcription-associated histone modi cations have been widely established for numerous tissues of Danio rerio during embryogenesis and development, but patterns during regeneration remain less well de ned (5)(6)(7).
During n regeneration in zebra sh, dramatic cellular events occur over the rst few days postamputation (dpa), including an initial phase of healing, followed by wound epidermis formation, blastema formation, cell proliferation, and redifferentiation (8).Rather than reliance on resident stem cell populations, the regeneration process involves dedifferentiation of adult n tissues in order to establish heterogenous progenitor cell populations within the blastema (9), occurring at 1-2 dpa.Prior studies have investigated how epigenetic and chromatin modi cations support the regeneration process in caudal ns, including studies which identi ed tissue regeneration-speci c enhancers (10), chromatin accessibility changes during regeneration (7), and the importance of removing tri-methylation at 27th lysine of the histone H3 tail (H3K27me3) from many genes (11).Despite these successes, knowledge of epigenetic reprogramming during caudal n regeneration is much more limited than similar reprogramming processes occurring within embryos (12)(13)(14)(15)(16), likely due to challenges associated with genome-wide characterization of epigenetic marks in adult differentiated tissues.
Chromatin immuno-precipitation combined with sequencing (ChIP-Seq) (17) is the standard methodology for pro ling histone modi cations and has proven to be a useful tool in many systems (18,19).This method enables high throughput DNA sequencing to map the genomic binding sites of target proteins and provides valuable information for pro ling the relative chromatin states of cells (18).However, ChIP-Seq methods typically require a signi cantly large number of cells (often > 1-million cells per replicate), inhibiting experimentation in many situations.Additionally, biases intrinsic to sonication and chromatin puri cations can also cause signi cant issues with ChIP-Seq, leading to decreased signal-to-noise ratios (20).Recently, a newer method called Cleavage Under Targets and Tagmentation, or CUT&Tag, (21,22) has been developed which overcomes many of these limitations, and has the potential to allow researchers to interrogate additional tissues or cell types (14,21).Like ChIP-seq, CUT&Tag is an antibodybased technology that detects protein-DNA interactions, but instead of sonication and crosslinking, CUT&Tag takes advantage of a protein A/G to Tn5 fusion, enabling users to speci cally cut and amplify DNA at precise locations where antibodies bind genomic chromatin.This difference provides a signi cant advantage, decreasing sample loss and signi cantly reducing sequencing levels over background regions.Here we have developed a modi ed CUT&Tag protocol, which has enabled us to study the active histone mark H3K4me3 in both intact and regenerating zebra sh caudal ns.
To investigate how epigenetic changes associate with the regeneration process, we applied CUT&Tag to cells isolated from uninjured and regenerating ns.We nd that many genes which acquire H3K4me3 during regeneration are known to be involved in the establishment of embryonic morphology, including a large number of loci which possessed high levels of H3K4me3 at 24hpf (hours post fertilization) in embryonic n folds.Our results support a model in which the regeneration process relies on reactivation of dormant epigenetic programs that are utilized initially during embryogenesis (23), and demonstrate the strong utility of CUT&Tag applied during zebra sh caudal n regeneration.It is our hope that data from this study will serve as an example for future researchers investigating chromatin changes in adult zebra sh tissues, and provide a resource for subsequent investigation of regeneration.

Results
CUT&Tag detects high H3K4me3 levels over gene promoters in caudal n with strong reproducibility.
To establish baseline H3K4me3 patterns in adult ns, we performed CUT&Tag on cells harvested from 3 biological replicates of uninjured ns (Fig. 1A).For each replicate, we pooled cells dissociated from 6 uninjured ns, and each pool was divided in half for use in IgG control and H3K4me3 measurements.Similar to prior studies (5), high H3K4me3 levels were detected over gene promoter regions (Fig. 1B).After peak calling (see methods), we identi ed nearly 49-thousand sites of H3K4me3 enrichment and found there to be a high degree of correlation between replicates (Fig. 1C, S1A), demonstrating great consistency and reproducibility of this technique.Additionally, we observed a high degree of concordance in total CUT&Tag enrichment for H3K4me3 surrounding gene transcription start sites (TSS) (Figure S1B & S1C).These initial results demonstrate CUT&Tag to be reliable and consistent application for the study of epigenetic marks within the heterogeneous mixture of cells that constitute the zebra sh caudal n (24).
Measurements of H3K4me3 by CUT&Tag are consistent with prior ChIP-Seq results.
We next compared enrichment of H3K4me3 detected by CUT&Tag with published enrichment measurements acquired by ChIP-Seq.Relative to ChIP-Seq, our CUT&Tag approach detected much higher promoter enrichment scores (RPKM -see methods), demonstrating the improved enrichment signal (as measured by RPKM) (Fig. 2A & 2B).To investigate whether CUT&Tag and ChIP-Seq measurements were similar at enriched loci, we merged replicates, ranked normalized signal independently across promoters or peak regions (to overcome method-speci c enrichment differences), and then assessed overall correlations.Measurements at gene promoters were highly correlated when comparing between H3K4me3 CUT&Tag and ChIP-Seq (R = 0.72) (Fig. 2C, 2D, S2A).H3K4me3 CUT&Tag also exhibited high correlation (R = 0.83) with H3K27ac, an another histone modi cation known to be enriched at actively transcribed genes (25,26).The observed correlation at promoters was much higher than at peak regions (R = 0.48) or at randomly generated background regions (Fig. 2C), which were uncorrelated (Figure S2B).Overall, these results demonstrate a high degree of consistency across replicates for each method, especially in the context of gene promoters (Fig. 2D).
Changes in H3K4me3 localization occur during early stages of caudal n regeneration.Tissue regeneration is achieved by differential expression of a substantial number of genes.To assess regeneration-associated changes in gene promoters, we next applied our CUT&Tag approach to regenerating n tissues.We collected caudal ns at 2 dpa, a timepoint encompassing blastema formation, which is an essential event of n regeneration (8), performed CUT&Tag against H3K4me3, and then intersected peaks identi ed independently for each timepoint.Comparison of H3K4me3 enriched peaks for uninjured (0 dpa) and regenerating (2 dpa) ns identi ed 29,152 shared peaks present in both samples (Fig. 3A & 3B).Peaks de ned as "Common" had consistently elevated H3K4me3 levels across all timepoints and replicates.Peaks de ned as "Uninjured" speci c had higher H3K4me3 levels across all replicates of 0 dpa, as compared with 2 dpa samples, and peaks de ned as "Regeneration" speci c had higher H3K4me3 levels across all replicates of 2 dpa samples, as compared with 0 dpa (Fig. 3C & 3D).Interestingly, we found that common and uninjured speci c loci were largely associated with binding motifs for FOX and KLF transcription factors, which are well known to have roles in embryonic development (27,28).Loci classi ed as regeneration speci c were largely associated with motifs for FOS transcription factor, a major component of AP-1 factor which play roles broadly in regenerative context, including zebra sh ns (Figure S3A) (29,30).
To assess biological pathways associated with H3K4me3 enrichment, we performed the gene ontology (GO) analysis (Fig. 3E) (31).Common peaks tended to reside in close proximity to promoters of genes involved in cell metabolism (Fig. 3F).While uninjured speci c peaks generally lacked associations, regeneration speci c peaks were associated with embryonic development, morphogenesis, and differentiation (Fig. 3F).For instance, promoters for igfbp6b and lepb were enriched for H3K4me3 in 2 dpa samples.Interestingly, lepb is highly regulated upon n amputation in zebra sh, and homologs to igfbp6 are known to be important for regeneration in other systems (10,32).Additional examples include several genes previously described to have putative roles in n regeneration (33-36) (Figure S3C).Overall, these analyses provide initial insight into the H3K4me3 changes that occur during zebra sh n regeneration and highlight locations in the genome where epigenetic alterations occur.
Changes H3K4me3 levels correspond with moderate changes in chromatin accessibility.
Active gene promoters are often characterized by high levels of H3K4me3 and elevated chromatin accessibility (37,38), leading us to explore whether changes in chromatin accessibility during the n regeneration may accompany the observed H3K4me3 changes.To investigate this, we compared enrichment for H3K4me3 at 0 dpa and 2 dpa with previously published chromatin accessibility measurements at 0 dpa and 1 dpa obtained from ATAC-Seq analysis (7,39).Initial comparisons of H3K4me3 enrichment at gene promoters (Fig. 4B & S4A) indicated a considerable amount of correlation between CUT&Tag and ATAC-Seq signal (Fig. 4A, 4B, S4A), analogous to associations observed in other biological systems (37,38).We next utilized the previously classi ed H3K4me3 peaks regions to investigate similar changes in chromatin accessibility, relying on the aforementioned "common" peaks, as well as uninjured speci c and regeneration speci c loci.As anticipated, regions which gained H3K4me3 between 0 dpa and 2 dpa (classi ed as regeneration speci c peaks) also become signi cantly more accessible between 0 dpa and 1 dpa (Fig. 4C).Accordingly, regions with lost H3K4me3 during regeneration (classi ed as uninjured speci c) tended to become less accessible (p = 0.072).These results indicate that the majority of already accessible loci (including promoters) remain accessible during n regeneration, and regions which gain H3K4me3 experienced a moderate but statistically signi cant increase in chromatin accessibility during regeneration.
H3K4me3 accumulates during n regeneration over regions which possessed H3K4me3 in embryos.
Development-related GO terms are enriched in regeneration status samples (Fig. 3E), leading us to hypothesize that changes in H3K4me3 localization during n regeneration might embody a "return" to embryonic chromatin patterns.To compare regeneration and development samples, we sought embryonic timepoint matching those of 2 dpa regenerating ns.Key transcription factors for appendage development and regeneration include the Msx family of homeodomain-containing transcription factors (40,41).Upon n amputation, msx1b (msxB) is strongly induced in blastema at 2 dpa (40,41).A previous study reported that msx1b is transiently expressed in embryonic n folds as msx1b transcript is uniformly detectable in caudal n folds at 24 hours post-fertilization (hpf) but restricted to the distal cells at 36 hpf (40,41).Given the strong and uniform expression pattern of msx1b at 24 hpf in caudal n folds, we chose 24 hpf caudal n fold as representative n samples for development.
We amputated n folds of ~ 200 embryos at 24 hpf and performed CUT&Tag with IgG and H3K4me3 antibodies.Despite performing measurements on drastically different staged samples, we observed remarkably similar H3K4me3 enrichment patterns at gene promoters in the 24hpf embryonic n folds compared with regenerating caudal ns (Fig. 5A & S5C).Furthermore, correlation values resulting from comparisons of development and uninjured or regenerating caudal n samples were only slightly lower (R = 0.82 and R = 0.86, respectively) than values obtained from comparisons between n timepoints (Fig. 3A, R = 0.92), indicating that H3K4me3 patterns at gene promoters were not drastically different among sample types.This was not the case when we compared H3K4me3 patterns across peaks, which included many intergenic regions.Correlation between development and uninjured or regenerating n samples was quite modest (R = 0.38 and 0.41, respectively) (Fig. 5A -right), indicating more substantial differences between tissues.
To explore these differences further, we partitioned peak regions with respect to enrichment for each sample type, enabling us to classify peaks as "shared", when enrichment occurred across all sample types, or "speci c", when enrichment occurred speci cally in development, uninjured, or regeneration samples (Fig. 5B & 5C).Remarkably, 35% of regions which acquired H3K4me3 during n regeneration (5,055 peaks out of 14,369) also possessed H3K4me3 in development (24 hpf embryo samples), as compared with only 24% of regions that lost H3K4me3 (1,793 peaks out of 7,573).In further support of maintained H3K4me3 enrichment over genic loci (as in Figs.3A & 5A), a relatively large portion of "shared" peaks occurred within gene promoters (21% of peaks).Whereas uninjured-and regenerationspeci c peaks tended to occur more frequently over intergenic regions (Fig. 5D).GO analysis revealed that shared peaks were associated with "housekeeping" genes, loci possessing H3K4me3 in both regenerative ns (2 dpa) and in 24 hpf embryos were associated with developmental genes, and no signi cant ontology terms were identi ed for H3K4me3 peaks that were lost during n regeneration (possessing H3K4me3 at 0 dpa but not at 2 dpa) (Fig. 5E).These results support a mechanism in which accumulation of H3K4me3 occurs during caudal n regeneration over regions which previously possessed H3K4me3 at the earlier developmental timepoints (24hpf), including many developmentally regulated gene promoters.
Changes H3K4me3 levels at gene promoters are accompanied by gene expression changes.
As noted, high H3K4me3 levels are indicative of gene activation, and loss of H3K4me3 leads to gene expression reduction (38).We therefore investigated whether the observed CUT&Tag H3K4me3 changes during n regeneration associated with altered gene expression patterns.For this analysis, we rst categorized gene promoters based on changes in H3K4me3 levels between 0 dpa and 2 dpa (see methods).Promoters were categorized in a manner similar to our parsing of peak regions, classifying loci as common, uninjured-speci c, and regeneration-speci c (Figure S6A).In agreement with our prior measurements, chromatin accessibility levels remained mostly stable over promoters during regeneration, and we observed modest but statistically signi cant increases at 1 dpa for promoters which gained H3K4me3 (regeneration-speci c) (Fig. 6A -red pro les & S6B).Changes in RNA transcript levels also followed a pattern highly similar to the observed changes in H3K4me3.Promoters which gained H3K4me3 had higher levels of RNA at 1 dpa compared with 0 dpa, and promoters which lost H3K4me3 experienced a decrease in RNA transcript levels over this same period (Fig. 6A -grey pro les & S6B).Additionally, promoters which acquired H3K4me3 during regeneration also exhibited higher levels of H3K4me3 and a greater abundance of RNA transcripts within 24hpf embryonic n folds, as compared with promoters that lost H3K4me3 (Fig. 6A -brown and green pro les, respectively & S6C).
To con rm these results, we next parse promoters based on changes in RNA transcript levels, or changes in chromatin accessibility, and then assessed H3K4me3 patterns.For these measurements we again classi ed promoters using a strategy similar to the one we previously described for H3K4me3 (see methods).Interestingly, H3K4me3 levels increased at promoters which become more accessible, and decreased at loci which lost accessibility (Fig. 6B).In the context of gene expression, we observed a signi cant increase in H3K4me3 levels at genes which became more transcriptionally active during regeneration (from 0 dpa to 1 dpa) and H3K4me3 signi cantly decreased at gene promoters which underwent silencing (Fig. 6B).As in our comparisons with 24hpf embryonic n folds, GO analysis revealed that promoters which maintained or experienced a decrease in H3K4me3 levels were associated with metabolism and housekeeping processes, whereas gene promoters which gained H3K4me3 associated with the developmental processes and establishment of embryonic morphology (Fig. 6C), such as kat7a and hoxc11a (43,44).Examples of genes which acquire H3K4me3 during early n regeneration post amputation and embryonic n development included shha (45,46) and foxm1 (47), and examples of genes associated with n fold-speci c H3K4me3 included tal1 (48) and sgk2a (49) (Fig. 6D) (24,(50)(51)(52)(53).

Discussion
Our study demonstrates CUT&Tag to be an effective tool for investigating epigenetic changes during zebra sh caudal n regeneration.We nd there to be a high degree of reproducibility between biological replicates, a strong concordance between CUT&Tag and ChIP-Seq datasets, and a robust agreement with results acquired from RNA-Seq.Furthermore, the relatively few number of cells required for CUT&Tag, the higher signal-to-noise ratio (21), and the feasibility of this technique, as compared with ChIP-Seq, make CUT&Tag particularly amenable to investigations of the adult zebra sh ns.The high degree of sensitivity this technique offers is likely to enable future researchers to assess chromatin changes within discrete cell types, perhaps including puri ed populations within regenerating tissues (52).Additionally, the feasibility and robustness of CUT&Tag will allow researchers to gain access to more re ned timepoints during regeneration, potentially attaining higher resolution of molecular mechanisms underlying the reprogramming process.
Recent technological advances have enabled researchers to characterize numerous tissues at single-cell resolution through measurements of RNA (54) or chromatin accessibility (55).In the very recent past, CUT&Tag methods have been similarly applied (56), and it is therefore conceivable that studies of caudal n will soon include single-cell epigenetic characterization.It is also likely that improvements in CUT&Tag methods or the closely related CUT&RUN method (57) will allow researchers to investigate changes in transcription factor binding using single-cell approaches (21,22).Such advances can drastically improve our molecular understanding of the regeneration process, in which numerous epigenetic modi cations and transcription factors are known to play critical roles (8, 10, 58).
Our ndings revealed a substantial overlap of H3K4me3 localization in 24 hpf embryonic n folds and 2 dpa regenerating adult n tissues, providing evidence that genetic and epigenetic programs that are important for embryonic development are repurposed during adult n regeneration.The regenerative blastema, which forms during 1-2 dpa, is comprised of dedifferentiated cells that arise from a mixture of adult n tissues, including osteoblasts and broblast/mesenchymal cells (9).The mechanisms permitting blastema formation remain poorly understood, but our study raises the interesting possibility that chromatin and epigenetic factors which facilitate development in embryos play important roles in regeneration-based reprogramming processes.So called "bivalent" chromatin modi cations reside at developmental genes within embryonic stem cells in a wide range of organisms (59).Bivalent chromatin is characterized by the dual presence of H3K4me3 and H3K27me3 (a silencing histone modi cation) at gene promoters.This combination of epigenetic marks enables developmental genes to remain silently poised in undifferentiated stem cells, so that they can become rapidly activated during later developmental stages (59).Here we nd that one component of bivalent chromatin, H3K4me3, accumulates at developmental genes during the precise timepoint when mature n cells dedifferentiate to progenitor-like state.Whether H3K4me3 and/or H3K27me3 function as 'bivalent' epigenetic factors within regenerative progenitor cells remains unknown and is a compelling topic for future investigation.
It is also interesting to note that cells within the blastema are able to re-use developmental programs/pathways to regenerate ns rather than applying regeneration-speci c mechanisms -if such processes exist at all.Markedly, these same developmental pathways are highly conserved in mammals, yet mammals lack the ability to regenerate limbs.It is plausible that an ancestor of mammals maintained these pathways for use in development but lost the ability to reactive them following injury in adults.Like mammals, certain teleost species of cartilaginous and ray shes like Cottus gobio cannot regenerate limbs (60) despite a much closer common ancestor with zebra sh.While it is unknown how divergence among vertebrates occurred, our results indicate that the genes necessary for regeneration are likely present in mammals, but these genes can no longer be activated at the precise time and place for limbs to regrow.It is also worth noting that many mammals are highly regenerative as infants or neonates, but lose the ability to regenerate tissues in adulthood (58, 61, 62).Thus, it is quite conceivable that temporal regulation of chromatin and epigenetic features (as opposed to gene speci c mutation or adaptation) are involved in these species-speci c limb regeneration mechanisms.
Although the data presented in this study are robust, and we offer an optimistic perspective for the regeneration community, we expect that CUT&Tag technologies will continue to be re ned and optimized, and newer adaptations are likely to emerge (22).We anticipate that our data will serve as a useful resource for continued investigation of regeneration-speci c chromatin or transcription control mechanisms.With the publication of our study, and the accompanying detailed protocol, it is our hope that CUT&Tag methods will be widely adopted, and the regeneration community will continue to advance as a result.

Method
Zebra sh Husbandry and Care Care and maintenance of zebra sh were conducted in strict compliance with guidelines for animal care and use, securing ethical clearance from the University Committee on Animal Resources at both the University of Rochester Medical Center and the University of Wisconsin School of Medicine and Public Health.The zebra sh were housed and nurtured under conditions that conformed to relevant protocols and ethical standards.

Harvesting of Fin and Embryonic Tissues
To anesthetize animals for amputation, shes were submerged in a diluted tricaine solution as per IACUC approved methods.Once immobilized, zebra sh placed one by one on a cutting mat, and their n tissues were transversally cut at 50% location and carefully transferred to 190ul PBS solution in an Eppendorf tube.For uninjured tissues, ns were cut again at the length expected to be regrown at 2 dpa.Two days after amputation, the regenerated ns were cut for 2 dpa samples.3 ns per antibody were combined as one sample.After n amputation, the zebra sh were transferred to a recovery tank for several mi before being returned to their original tanks.For development samples, embryos were cultured in egg water and maintained at 28°C for 24 hours.At 24 hpf, dead embryos were removed, and live embryo were dechorionated using Pronase (Roche,165921) diluted at 2mg/ml nal concentration in egg water.Dechorionated embryos were vigorously rinsed multiple times and then moved to a dish containing HBSS (no phenol, no magnesium, no calcium).Embryos were anesthetized with tricaine, and any remaining chorions were removed manually with forceps.Using a curved blade, the n folds were cut transversally to include a portion of the notochord (see more detail in supplementary protocol).A total of 100 n folds per antibody were collected into HBSS (no phenol, no magnesium, no calcium) (14,21).

Cell Processing and CUT&Tag
The detailed protocol is attached as Supplementary Protocol.The protocol was adopted and modi ed from previously described methods (14,21).Uninjured or 2dpa ns were collected in 250µL per 6 ns of cold HBSS (no calcium, no magnesium) in a low-bind microcentrifuge tube.A total of 2-3 ns per antibody were used for each condition.Fins were brie y centrifuged and HBSS was replaced with freshly made digestion buffer (HBSS no calcium, no magnesium, 12.5µM CaCl2, 5mg/mL collagenase type IV (Gibco), and 0.26U/mL Liberase DH (Roche)).A microcentrifuge stir bar (1.5 x 8mm) was placed in each tube, and the tubes were incubated on a stir plate set to 120 rpm in a 35°C incubator.The tubes were either icked or gently pipetted every 15 min for 45 min -1 hour.

Sequencing data
The CUT&Tag libraries from zebra sh ns were pooled and sequenced using services from UW-Biotechnology center on the Illumina NovaSeq 6000 platform.Raw sequencing data generated in this study can be found at NCBI GEO with the accession number (GSE261540).The publicly available RNA data used in this study can be found at NCBI GEO Datasets with accession number GSE146960.The publicly available H3K4me3 & H3K27ac ChIP data used in this study can be found at NCBI BioProject with accession number PRJNA559885.The publicly available ATAC data used in this study can be found at NCBI GEO with accession number GSE146960.

ChIP and ATAC data analysis
The ChIP and ATAC sequencing data were aligned to the zebra sh genome assembly (GRCz.11,Ensembl release 103) utilizing Bowtie2, followed by conversion to bam format using SAMtools.Unmapped reads were ltered out using samtools, and PCR duplicates were eliminated with picard MarkDuplicates.The H3K4me3 replicate data were merged using UCSC bigwigMerge, and genome browser tracks were generated with deepTools bamCoverage, employing the --normalizeUsing RPKM option for normalization.
Peak calling for ChIP data was performed using macs2 bdgpeakcall with the parameters -c 10 -l 100 -g 50.The comparison of peak locations between samples was conducted using Bedtools intersect.For the visualization of ChIP read distribution, deepTools bamCoverage was used to compute normalized read counts in each 100 bp genomic window, with the results visualized in the Integrated Genome Viewer (version 73).The matrix of read counts of all samples was generated and converted by deeptools Multibigwigsummary to the CSV format to be processed in R, enabling us to generate scatterplots and rank-normalized correlation plots.
RNA data analysis 40-50 n folds amputated from 24 hpf embryos were pooled for RNA-seq analysis.24 hpf n fold RNAseq analysis was done by Novogene with 40 Million of 150bp paired-end using Novaseq6000.Initial processing steps for RNA-Seq data involved mapping reads to the latest zebra sh genome assembly (GRCz.11,Ensembl release 103) employing STAR-aligner, generating the sorted BAM les.To further identify the relationship between genomic features and gene expression, the matrix of read counts of all samples was generated and converted to the CSV format using deeptool Multibigwigsummary.For visualization of RNA read distribution, deepTools bamCoverage was used to compute normalized read counts in each 100 bp genomic window, with the results visualized in the Integrated Genome Viewer.

CUT&Tag data analysis
The processing of H3K4me3 CUT&Tag paired-end sequencing reads were aligned to the zebra sh genome assembly (GRCz.11,Ensembl release 103) using Bowtie2.Samtools was employed to lter out unmapped reads, and Picard MarkDuplicates was applied to eliminate PCR duplicates.The H3K4me3 replicate data were then merged using UCSC bigwigMerge, leading to the creation of bigwigs (used for genome browser tracks) through deepTools bamCoverage with the setting --normalizeUsing RPKM.Peak calling was executed with macs2 bdgpeakcall, adopting parameters of -c 30 -l 100 -g 50.The matrix of read counts of all samples was generated using deeptools Multibigwigsummary to generate a CSV format, which was further analyzed using standard tools in R for generation of pro le plots, ranknormalized correlation plots, and boxplots.Promoters with increased or decreased H3K4me3 were those with log2FC scores greater than 1 or less than − 1, respectively, as calculated in R from CSV table outputs.For the visualization of the data, deepTools plotHeatmap and plotPro le were utilized.Overlapping peak analysis was conducted using bedtools intersect.Motif identi cation and genomic element percentage piecharts were carried out using the Hypergeometric Optimization of Motif EnRichment (HOMER) software package.Lastly, Gene Ontology Analysis was performed using the ChIP-Seeker R package,

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