The human genome encodes an order of magnitude more gene expression enhancers than promoters, suggesting that most genes are regulated by the combined action of multiple enhancers. We have previously shown that neighboring estrogen-responsive enhancers exhibit complex synergistic contributions to the production of an estrogenic transcriptional response. Here we sought to determine the molecular underpinnings of this enhancer cooperativity. We generated genetic deletions of four estrogen receptor α (ER) bound enhancers that regulate two genes and found that enhancers containing full estrogen response element (ERE) motifs control ER binding at neighboring sites, while enhancers with pre-existing histone acetylation/accessibility confer a permissible chromatin environment to the neighboring enhancers. Genome engineering revealed that two enhancers with half EREs could not compensate for the lack of a full ERE site within the cluster. In contrast, two enhancers with full EREs produced a transcriptional response greater than the wild-type locus. By swapping genomic sequences, we found that the genomic location of a full ERE strongly influences enhancer activity. Our results lead to a model in which a full ERE is required for ER recruitment, but the presence of a pre-existing permissible chromatin environment can also be needed for estrogen-driven gene regulation to occur. Regulation of gene expression is a fundamental task underlying biological processes such as development and disease progression. Promoter-distal gene regulatory enhancers play a central role in metazoan gene regulation and contain binding sites for transcription factors (TFs) that recruit cofactors and influence gene expression. Most genes in the human genome are likely regulated by multiple enhancers ,2. For eet al. found that in order for PU.1 to bind at one of its upstream enhancers in myeloid cells and auto-regulate expression, a second enhancer must be active. This second enhancer likely maintains accessible chromatin at the neighboring enhancer, enabling PU.1 to bind  is a relevant model system to study combinatorial gene regulation. ER binds the genome in an estrogen-dependent manner, with the majority of binding occurring distally from promoters . The majIn this study, we explore how neighboring ER-bound enhancers impact one another. By functionally dissecting these relationships at two estrogen-responsive genes, we discovered regulatory sharing between ER-bound enhancers. Deletion of ER-bound enhancers decreased ER binding, histone acetylation, and chromatin accessibility at neighboring enhancers. Through the use of genome engineering approaches, we also investigated the role of sequence and genomic location in determining the contributions of these enhancers to estrogen-induced gene expression. We find that the presence of a full estrogen response element (ERE), ER’s preferred DNA binding sequence, in at least one enhancer is required for these genes’ transcriptional responses to estrogen. The location of the ERE containing enhancer region is also important; placement of an ERE within a region harboring histone acetylation prior to estrogen induction greatly increases the transcriptional response. Overall, we discovered that these ER-bound enhancers are cooperating at a molecular level to combine ER recruitment and permissive chromatin and drive the estrogen transcriptional response.Ishikawa cell lines containing deletions of individual ER binding sites were generated as described previously . IshikawP-value cutoff of 1e–10 and the mfold parameter bounded between 5 and 50. The input control used in peak-calling was derived from the parental Ishikawa line. The broad peak calling feature was used for H3K27ac ChIP-seq. Bedtools , 550 ng of each guide RNA , and 200CTCF, CISH, and MMP17 can be found in To harvest RNA, we used a direct on-plate lysis of cells with 300 μL of Buffer RLT Plus (Qiagen) supplemented with 1% beta-mercaptoethanol (Sigma-Aldrich). Lysates were purified using the ZR-96-well Quick-RNA kit (Zymo Research). RNA was quantified using RiboGreen (ThermoFisher Scientific) on a Wallac EnVision plate reader (PerkinElmer) or on a Qubit 2.0 (ThermoFisher Scientific). Gene expression was quantified using Power SYBR Green RNA-to-CT 1-Step Kit (ThermoFisher Scientific) and a CFX Connect light cycler (BioRad). Each reaction contained 50 ng of RNA as starting material. As per kit instructions, 40 cycles of PCR were performed following a 30-minute cDNA synthesis. Primers for To identify distances between ERBS, we used bedtools closest  on previP-values from these tests can be found in the text and in the figure legends.To quantify differences in ChIP-seq and ATAC-seq signal resulting from ERBS manipulations, we used two-way ANOVA. To quantify differences in E2-induced gene expression following genetic manipulation, we used two-way ANOVA. To quantify differences in fold change in response to E2 for up-regulated genes with specific numbers of ERBS clusters within 100 kb of their TSSs, we used Wilcoxon signed-rank tests. MMP17, FHL2 and CISH each harbor pairs of ER binding sites (ERBS) that work together synergistically . These results suggest that ERBS clusters may be more effective at producing gene expression responses to estrogen compared to solitary ERBS.In order to better understand the relationship between clustered ERBS and gene expression, we examined where clusters are located in the genome relative to genes up-, down-, or not-regulated by estrogen as defined by RNA-seq . We founCISH and MMP17  regardless of treatment . As expected, deletion of a central 225 bp of CISH-1 led to a complete loss of ER binding and large reductions in accessibility and H3K27ac at CISH-1 compared to wild-type levels Figure –D. Loss MSO. As et Figure . These rCISH. Deleting 166 bp of CISH-2 resulted in loss of ER binding, H3K27ac and chromatin accessibility at CISH-2 as expected  with ER binding still detectable , histone acetylation, and, to a lesser extent, chromatin accessibility of MMP17-2, explaining why MMP17-1 is needed in order for MMP17-2 to contribute to the estrogen response.We next explored the molecular underpinnings of the hierarchical relationship between enhancers at d Figure –D. MMP17d Figure  and B. Md Figure . These rd Figure . At the MMP17 promoter in the presence of E2, indicating that loss of MMP17-2 does affect the promoter; however, chromatin accessibility is not significantly impacted by MMP17-2 loss, similar to MMP17-1 loss  The underlying DNA sequence is dictating the contribution of a site, or 2) the genomic location of a site determines the site's role in gene regulation. To investigate the importance of these features, we used Cas9 to edit the sequence of ERBS at Ligation , which rMMP17, replacing one MMP17-1 allele with the core sequence of MMP17-2 led to a loss of the estrogen response when the other MMP17-1 allele was deleted . These results indicate that MMP17-1 is an optimal location for the underlying optimal sequence of MMP17-1 and that MMP17-2’s location is suboptimal. Interestingly, both optimal locations (MMP17-1 and CISH-2) are in open chromatin surrounded by high levels of H3K27ac prior to estrogen induction, suggesting that a combination of permissive chromatin and DNA sequence (with a full ERE) combine to drive an estrogen response.When we previously deleted MMP17-2, we found that the predominant site MMP17-1 can drive approximately half of 2 Figure . While tCISH and MMP17. We performed ChIP-seq for H3K27ac in T-47D cells, an estrogen responsive breast cancer cell line. We also analyzed T-47D expression levels of CISH and MMP17 using RNA-seq   and the MMP17-1 and MMP17-2 enhancers are devoid of H3K27ac and ER binding in T-47D cells. Together, these results highlight a potential contribution of other transcription factors to an estrogen transcriptional response.Since H3K27ac is observed at MMP17-1 and CISH-2 when cells are starved for estrogens, it is likely that other transcription factors are responsible for maintaining histone acetylation at certain ERBS. To identify which factors might contribute to permissive chromatin at ERBS across the genome, we performed differential motif enrichment analysis using AME from the Meme Suite , as well RNA-seq . The gen = 2.89) . HoweverCISH and MMP17, we revisited the genome-wide analysis of ERBS clusters. Our functional dissection of ERBS clusters near MMP17 and CISH indicated that two features – a strong ERE motif and histone acetylation prior to estrogen treatment—are important for a cluster to robustly contribute to gene expression. We explored whether other clusters had similar features, and whether these features were associated with the transcriptional response to estrogen. We examined all 6211 clusters generated above, including those containing only one ERBS, and determined whether the sites within that cluster had the combination of at least one full ERE and at least one H3K27ac site in the absence of E2, which we refer to as active clusters. To investigate how these active ERBS clusters relate to gene expression, we examined the localization of active clusters relative to genes up-, down-, and not-regulated by estrogen. We found that 28% of up-regulated genes have at least one active cluster within 100 kb, compared to 9% for down-regulated genes and 9% for not-regulated genes  . When we compared transcriptional responses to E2 for up-regulated genes with active clusters nearby to up-regulated genes without active clusters nearby . These results suggest that active clusters are more likely to drive larger estrogen transcriptional responses than inactive clusters, but that many genes are up-regulated by E2 without an ERBS cluster within 100 kb that contains both a full ERE and H3K27ac prior to E2 treatment.Based on the regulatory sharing observations at ) Figure . Active y Figure , we founCISH or the hierarchical enhancers of MMP17. To identify potential hierarchical sites (MMP17-like), we looked for clusters that had an ERBS that harbored both a strong ERE and H3K27ac in the absence of estrogens. To identify potential synergistic sites (CISH-like), we looked for clusters where one ERBS had a strong ERE and another ERBS had H3K27ac in the absence of estrogens. The vast majority of active clusters exhibited the hierarchical patterns seen at MMP17 (82%). When focused solely on ERBS clusters near up-regulated genes, 113 genes have an MMP17-like ERBS cluster, 23 genes have a CISH-like ERBS cluster, and 16 genes have both types of clusters within 100 kb. These findings suggest that a hierarchical relationship between ERBS is the more common type of collaboration between ER-bound enhancers based on associated features.We next sought to determine whether these active clusters near up-regulated genes looked more similar to the synergistic enhancers of CISH and MMP17. We discovered molecular interplay between neighboring enhancers, which provides an explanation for the collaboration that we see between ER-bound enhancers.The ER-bound enhancers that we have previously studied in detail are within 5 kb of one another and combine cooperatively to regulate gene expression in response to estrogen. In this study we find that ER-bound sites in endometrial cancer cells tend to cluster together in the genome much more than expected by chance, as previously reported in breast cancer cells ,26, suggKlf4 locus prevents other transcription factors from binding to that same enhancer and reduces chromatin accessibility at the enhancer  or the neighboring site (e.g. CISH-2). These findings are consistent with previous work showing that recruitment of p300 and subsequent histone acetylation appears to be a crucial step in estrogen-dependent gene regulation, as inhibitors of the p300 catalytic domain abrogate estrogen-dependent transcription . The facCISH and the hierarchical relationship of enhancers regulating MMP17  and GSE147140 (ATAC-seq).gkaa454_Supplemental_FileClick here for additional data file.


Accessible chromatin plays a central role in gene expression and chromatin architecture. Current accessible chromatin approaches depend on limited digestion/cutting and pasting adaptors at the accessible DNA, thus requiring additional materials and time for optimization. Universal NicE-seq (UniNicE-seq) is an improved accessible chromatin profiling method that negates the optimization step and is suited to a variety of mammalian cells and tissues. Addition of 5-methyldeoxycytidine triphosphate during accessible chromatin labeling and an on-bead library making step substantially improved the signal to noise ratio while protecting the accessible regions from repeated nicking in cell lines, mouse T cells, mouse kidney, and human frozen tissue sections. We also demonstrate one tube UniNicE-seq for the FFPE tissue section for direct NGS library preparation without sonication and DNA purification steps. These refinements allowed reliable mapping of accessible chromatin for high-resolution genomic feature studies. D = A/G/T) followed by labeling reaction with 5-methyldeoxycytidine triphosphate (5-mdCTP) and biotin-14-dCTP in the dNTPs mixture to specifically label all available deoxycytidine triphosphates at the nicking sites. This will render these sites in the newly labeled accessible chromatin DNA resistant to further nicking. Therefore, even with the excess nicking enzyme, the labeled chromatin is protected from further degradation. After the labeling reaction, biotin-labeled DNA is isolated, sonicated, and captured on streptavidin magnetic beads for library preparation and sequencing. We tested this method on a diverse set of cell lines, native, and formaldehyde-fixed tissue nuclei, fresh frozen formaldehyde-fixed 5–10 μm human tissue sections along with FFPE tissue sections and observed markedly improved accessible chromatin signals. Taken together, universal NicE-seq is a simple, cost-effective, nick translation-based method to profile accessible chromatin using NextGen sequencing. Importantly, 5mC-incorporated accessible chromatin remains resistant to further degradation eliminating time-consuming titration and would allow automation on a variety of biological samples.The eukaryotic nuclear genome is packaged into chromatin, consisting primarily of DNA, proteins, and RNA, which is further condensed into larger folded chromosome structures during cell division. During cellular events, chromatin undergoes remodeling providing accessibility to DNA-binding proteins including transcription factors –3. Gene During the previously published NicE-seq labeling reaction, the nucleotide mixture contained dNTPs supplemented with biotin-14-dATP and biotin-14-dCTP . This reTo determine the effect of 5mC incorporation in accessible region for library preparation, we performed UniNicE-seq in the presence of ×10 and ×20 (25 and 50 units) excess Nt. CviPII nicking enzyme. Indeed, neither of the two reaction conditions showed loss of accessible chromatin in HCT116 cells  0.19–0.14), demonstrating the robustness of 5mdC-labeled accessible chromatin protection against degradation  and control (off beads) either with 5-mdCTP or dCTP in the dNTP mix  compared to UniNicE-seq with ATAC-seq (Pearsons correlation = 0.60) or ATAC-seq with DNase-seq (Pearsons correlation = 0.60), demonstrating most DHS (DNase Hypersensitive Sites) are indeed captured in this modified protocol Fig. . Since nr = 1; 5 K vs. 25 K, r = 0.97; 500 vs. 25 K, r = 0.95). Since accessible DNA quantity varied between different cell numbers, we further tested if the read density between the common peaks are similar. For validation, average log2  in 6062 peaks that were detected in all 6 samples were plotted on a 3-dimensional scatter plot. The observation that all data points line up on a straight line in the 3-dimensional space shows that signal in 25000, 5000, and 500 T cells are highly correlated and that there was no loss of signal with decreased numbers of cells , suggesting both samples are compatible with the UniNicE-seq protocol, and is robust without the enzyme titration requirement for low cell numbers  demonstrating the high quality of common accessible regions  cells were cultured in McCoy’s 5A media supplemented with 10% fetal bovine serum. MCF7 and HeLa cells  were cultured in DMEM plus 10% FBS.2, 60 mM KCl, 0.5 mM DTT, 15 mM NaCl, 300 mM sucrose, and 1% NP40) for 10 min on ice with occasional agitation. Nuclei were precipitated by spinning at 1000 × g, 4 °C for 5 min and supernatant were discarded. The nuclei pellet was resuspended in 100 μl of ×1 PBS. Open chromatin DNA was labeled with biotin by incubating the nuclei in the presence of 2.5 U of Nt.CviPII , 50 U of DNA polymerase I  and 30 μM of each dGTP and dTTP, 24 μM of 5-mdCTP  and dATP 24 μM plus 6 μM of biotin-14-dATP  and 6 μM of biotin-14-dCTP  at 37 °C for 2 h with occasional mixing. The labeling reaction was stopped with 20 μL of 0.5 M EDTA, and 2 μg of RNase A was added to the labeling reaction and incubated at 37 °C for 0.5 h to digest RNA. The DNA was isolated using phenol-chloroform extraction and ethanol precipitation method or Qiagen spin column . For NicE-seq libraries using 25 or 50 units of enzymes, the reaction conditions were identical except that the incubation was at 800 RPM at 37 °C for maximal NicE-seq labeling.Twenty-five thousand HCT116 cells were used for routine library construction. Cells were cross-linked using 1% formaldehyde for 10 min at room temperature and quenched by using 125 mM glycine. Nuclei were isolated by incubating the cross-linked cells in cytosolic buffer  for 10 min at 4 °C. 200 μl of the labeling buffer was placed on the tissue section, and the slide was transferred to a humidified chamber at 37 °C for 2 h. Please refer to supplementary text Frozen tissue sections of lungs on slides were procured from US Biomax, Inc.  HuFTS251). For frozen 5–10-μm tissue section fixing and labeling, the slides were treated with 1% formaldehyde for 10 min at room temperature and quenched by using 125 mM glycine. After ×1 PBS wash, the slide was treated with cytosolic buffer , followed by gradual ethanol wash  in jars at room temp for 5 min each step. The final wash was in milli-Q water for 2 min. Then, the slides were incubated in PBST buffer (×1 PBS in 0.1% Tween 20) at 65 °C for 1 h, and incubated again in ×1 PBST buffer containing protease inhibitor  at room temperature for 10 min, washed by ×1 PBS buffer, and dried at the room temperature. The slides were rehydrated with ×1 PBS buffer and treated with the cytosolic buffer for 10 min at 4 °C. Cytosolic buffer was washed away, and the NicE-seq labeling reaction was identical to the frozen tissue section procedure.For FFPE samples, after the labeling reactions, the labelled nuclei sample is collected in 200 μl of ATL buffer (Qiagen), 20 µl (200 units) of proteinase K  was added, and the reaction was incubated at 65 °C for o/n for decrosslionking. Proteinase K was inactivated by incubating the reaction tube at 95 °C for 2 min. We next added 2.5 units of Nt.CviPII, 50 µl of NEB buffer 2 and adjusted the reaction volume to 500 µl with water and incubated for 16 h at 37 °C. Nt. CviPII enzyme in the reaction was heat-inactivated at 65 °C for 15 min. These processes ensured that accessible chromatin fragments in the solution for streptavidin magnetic bead enrichment as described below. For formaldehyde fixed cells and tissue nuclei, the decrosslinking step was performed by adding 20 µl of 20% SDS, 20 µl of proteinase K and the reaction was incubated at 65˚C o/n, and processed as described above. Please refer to supplementary text The isolated genomic DNAs (~200 ngs/25 K cell) were sonicated into 150 bp fragments (Covaris) and the entire reaction product was mixed with 20 μL of Streptavidin magnetic beads  in 1 mL of B&W buffer . Biotin-labeled open chromatin DNA was captured by streptavidin at 4 °C for 2 h with end-over-end rotation. The beads were washed four times with B&W buffer plus 0.05% of Triton X-100 followed by one-time wash with TE. The beads were resuspended in 50 μL of TE. The DNA was end-repaired, washed twice with B&W buffer plus 0.05% of Triton X-100, dA-tailed, and washed with B&W buffer plus 0.05% of Triton X-100. And finally, NEB Illumina adaptor  was ligated and washed twice with B&W buffer plus 0.05% of Triton X-100. A final wash of the bead-bound DNA was performed with TE, and the bound DNA was resuspended with 20 μl of TE. Ten to 20 μL of bound DNA was used for library amplification using PCR . Routinely, 8–10 PCR cycles were used to generate enough amount of library DNA for sequencing. Low input cell numbers between 250 and 500, the library was amplified 12–13 cycles. The library was examined and quantitated with a high-sensitive DNA chip .http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) with the following setting: --clip_R1 4 --clip_R2 4 --three_prime_clip_R1 4 --three_prime_clip_R2 4. Trimmed read pairs were mapped to the reference genome  using Bowtie2 [Adaptor and low-quality sequences were trimmed from paired-end sequencing reads using Trim Galore , we down-sized the mapped reads from different experiments to the same number of mapped fragments  through random sampling. Peaks were called using the same parameter with MACS2, as mentioned above. Bigwig files of normalized reads per million read in 10 bp non-overlapping windows across the genome were displayed in the Integrated Genomics Viewer . All anaThe FRiP score was calculated using the deepTools plotEnrichment function . Called Peaks called from different experiments were compared using the Bedtools . First pCorrelation analysis of UniNicE-seq open chromatin signals was performed with the DiffBind  package Functional annotation of called peaks was performed with HOMER  annotateTo investigate enrichment of open chromatin signals over sets of genomic regions with epigenetic significance, we first calculated normalized read coverage ) in tiling bins of 100 bp across the entire genome from the bam file of properly aligned fragments  using the deepTools . Then, hhttp://lcbb.swjtu.edu.cn/TiED/) and EnhancerAtlas [TSS of mouse (mm10) and human (hg38) genomes were extracted from the NCBI RefGene gene table downloaded from the UCSC Table Browser. ChIP-seq datasets of cell-specific and tissue-specific CTCF binding, RNA polymerase II binding, and histone marks  were downloaded from the mouse and human Encyclopedia of DNA Elements (ENCODE) projects (Supp. Table cerAtlas . The oriATAC-seq (SRX2717891 & SRX2717892) and OmniATAC-seq (SRX2717893 & SRX2717894) datasets of the mouse kidney were downloaded from the NCBI SRA database. The DNase-seq dataset of mouse kidney (ENCSR000CNG) was acquired from the ENCODE project. The human HCT116 ATAC-seq and WGBS datasets were downloaded from the NCBI GEO database (ATAC-seq: GSE101966 and WGBS: GSE97889). The DNase-seq dataset was acquired from the ENCODE project (ENCSR000ENM).All the UniNicE-seq data generated in this study are deposited in NCBI Gene Express Omnibus (GEO) under the accession GSE140276.Additional file 1: Supp Fig. 1 Optimization of universal NicE-seq (A) A schematic diagram of accessible chromatin labeling using dCTP or 5-mdCTP in the labeling reaction along with biotinylated-dCTP in the nucleotide mix. On-bead and off-bead represented presence of streptavidin magnetic beads for DNA capture and library preparation. (B) FRiP comparison between all 4 methods generated library that map to TSSs (+/-500 bp of TSS) and distal elements (>500 bp from TSS) from HCT116 cells. C and 5mC represents use of dCTP and 5-dCTP in the reaction mix. Supp Fig. 2: Optimization of accessible chromatin sequencing and comparison between UniNicE-seq, ATAC-seq and DNase-seq. (A) IGV screen shot of the normalized read density of the four NicE-seq conditions in HCT116 cells. (B) Distribution of the number of normalized HCT116 NicE-seq reads at transcription start sites (TSS) of human genes and the surrounding 2 Kb (- and +) regions. (C) Pearson correlation of normalized read densities in UniNicE-seq peaks of the 2 technical replicates in HCT116 demonstrating reproducibility. (D) IGV screen shot of the normalized read density of UniNicE-seq (top track), ATAC-seq (middle track) and DNase-seq (bottom track) in HCT116 (F) Overlap of HCT116 peaks called from 15 M unique alignments using UniNicE-seq, ATAC-seq and DNase-seq. Supp Fig. 3: Venn Diagram showing common and cell-type specific UniNicE-seq peaks between the three cell types. (A) HCT116, K562 and MCF7 accessible chromatin regions were analyzed. Peaks are called from 11 million random sampled deduplicated alignment pairs. Supp Fig. 4: UniNicE-seq of mouse T cells cells. (A) IGV screen shot of the normalized read density of the technical duplicates of UniNicE-seq libraries of HCT116 cells at different cell numbers. (B) Pairwise comparison between all Universal NicE-seq reads between different T cell numbers from 500, 5 and 25 K. Pearson’s correlation is indicated. Supp Fig. 5: Comparison between UniNicE-seq, ATAC-seq, Omini ATAC-seq and DNase-seq of mouse kidney cells. (A) Venn diagram of accessible chromatin regions derived from UniNicE-seq, ATAC-seq, Omini ATAC-seq and DNase-seq of mouse kidney cells. (B) Distribution of fold change (FC) values (derived from MACS2) of the common accessible chromatin peaks of UniNicE-seq, ATAC-seq, Omni ATAC-seq and DNase-seq. (C) FRiP score of UniNicE-seq 25 K, 0.5 K and 0.25 K compared with data obtained from ATAC-seq and OmniATAC-seq using 50 K cells, and DNase-seq. (D) Heatmap showing comparison of normalized RPKM of 25 K fixed, 0.5 K nonfixed and 0.25 K mouse kidney UniNicE-seq, 50 K omni ATAC-seq, 50 K ATAC-seq and DNase-seq data at TSS, PolII and random. (E) Similar comparison like (D) along with chromatin features including CTCF, H3K4me3, H3K27Ac and random fragments. Supp Fig. 6: Comparison between accessible chromatin sequences two liver FFPE tissue section (A) Venn diagram demonstrating common accessible regions in two different human 5-10 μm lung normal tissue sections. (B) Pearson’s correlation analysis of total reads between two different human 5-10 μm lung normal tissue sections. (C) Pearson’s correlation analysis of common reads between two different human 5-10 μm lung normal tissue sections demonstrating quality of accessible peaks. (D) Principle Component Analysis and heat map of TSS across fetal and adult tissue for normalized read density of the consensus peaks between the samples from fetal and adult tissue. (E) Heat map of TSS (-/+ 2 kb) between various tissue samples. Supp Fig. 7: UniNicE-seq of normal human liver FFPE tissue section. Pearson’s correlation analysis by pairwise comparison between two different liver samples, R1 and R2. Supp Fig. 8: GC content of HCT116 peaks called from 15 M unique alignment pairs using UniNicE-seq, ATAC-seq and DNase-seq. The last box represents the GC content of random genomic regions sampled from the human reference genome (hg38).Additional file 2: Supplementary Table 1: HCT116 UniNicE-Seq matrix for library with different amounts of enzyme. Note 25 and 50 U NicE-seq labeling reactions were incubated at 37 °C at 800 RPM. Supplementary Table 2: Quality control metrics of UniNicE-seq libraries applied to two human cell lines K562 and MCF7 in comparison to libraries made on and off beads with either with 5mdCTP or dCTP in the dNTP mix. We examined percentage of mitochondrial reads (“%mito”), number of total peaks and promoter peaks (+/- 500 bp of TSS) and enrichment of signal at TSSs (“FRiP (TSS peaks)”). Two technical replicates were conducted for each sample. All the values were calculated from a subsample of 11 million de-duplicated alignment pairs. Supplementary Table 3. Quality control metrics of UniNicE-seq libraries applied to mouse kidney tissues. 25 K fixed cells were compared with 25 K, 10 K, 1 K, 0.5 K and 0.25 K unfixed cells. Supplementary Table 4. a. Quality control metrics of UniNicE-seq libraries applied to human adult lung tissues. b. Quality control metrics of UniNicE-seq libraries applied to different human adult and fetal tissues.**Here the replicates are merged together and downsized to 50 M aligned pairs for the downstream analysis. Supplementary Table 5. Quality control metrics of UniNicE-seq libraries applied to human FFPE liver tissue sections. Supplemental Table 6. External ChIP-seq data sets of various human and mouse tissue and cell types in this work.Additional file 3: Supplementary text 1. Detailed stepwise protocol: universal NicE-seq (25000-250000 HCT116 cells).


The shift from maternal to embryonic control is a critical developmental milestone in preimplantation development. Widespread transcriptomic and epigenetic remodeling facilitate this transition from terminally differentiated gametes to totipotent blastomeres, but the identity of transcription factors (TF) and genomic elements regulating embryonic genome activation (EGA) are poorly defined. The timing of EGA is species-specific, e.g., the timing of murine and human EGA differ significantly. To deepen our understanding of mammalian EGA, here we profile changes in open chromatin during bovine preimplantation development. Before EGA, open chromatin is enriched for maternal TF binding, similar to that observed in humans and mice. During EGA, homeobox factor binding becomes more prevalent and requires embryonic transcription. A cross-species comparison of open chromatin during preimplantation development reveals strong similarity in the regulatory circuitry underlying bovine and human EGA compared to mouse. Moreover, TFs associated with murine EGA are not enriched in cattle or humans, indicating that cattle may be a more informative model for human preimplantation development than mice. Preimplantation embryos undergo extensive transcriptomic and epigenomic remodeling. Here the authors assay open chromatin in bovine oocytes, embryos, and embryonic stem cells, and compare the transcriptomes and epigenomes of cattle, human and mouse embryos, revealing species-specific regulation of genome activation. This shift from maternal dependence to self-sufficiency serves at least three functions: elimination of oocyte-specific messages, replenishment of transcripts that are common to both the oocyte and the embryo, and generation of novel embryonic-specific transcripts4.Preimplantation development encompasses several critical milestones as embryos progress from fertilization to implantation. Fusion of the transcriptionally quiescent oocyte and sperm results in a zygote with two haploid pronuclei. Gametic syngamy is completed after the first round of DNA replication, when pronuclear membranes dissolve, allowing maternal and paternal chromosomes to intermingle on the metaphase plate. Subsequent rounds of cleavage ultimately lead to formation of a blastocyst. However, the cleavage-stage embryo must first complete the maternal-to-zygotic transition (MZT), wherein the embryo assumes control over its own continued development by degrading oocyte-derived products and initiating its own transcriptional program. This dramatic change in gene expression proceeds gradually; minor embryonic genome activation (EGA) results in low levels of transcription in early cleavage-stage embryos5 and epigenetic information are simultaneously restructuring the embryonic epigenome. Chromatin remodeling is necessary to erase gamete-specific signatures and establish an open chromatin landscape that supports embryonic transcriptional programs. Specifically, chromatin structure defines the genomic context within which transcriptional machinery can operate, thereby determining the cell-specific gene expression patterns that confer cell identity and function.While embryos undergo this massive shift in transcriptional program during the MZT, wide-spread changes in chromatin structure6, and this loss of DNA methylation coincides with global decreases in repressive histone modifications7. Overall, epigenetic factors linked to relaxed chromatin are more abundant in mouse zygotes, whereas factors implicated in chromatin compaction become more prevalent during EGA8, pointing to a more permissive chromatin landscape in pre-EGA embryos. Indeed, mouse zygotes present increased histone mobility9, highly dispersed chromatin10, and lack chromocenters (congregations of pericentromeric heterochromatin)12. Moreover, in mouse, 3-D chromatin architecture is largely absent after fertilization, and is gradually established throughout preimplantation development, which facilitates long-distance chromatin interactions in later stage embryos13, and indicates increasing chromatin compaction and organization. The co-occurrence of genome activation and chromatin remodeling raises an interesting causality dilemma, namely, whether chromatin remodeling is necessary for transcription activation or whether transcription activation leads to chromatin remodeling. Several maternal products prompt transcription initiation by altering chromatin structure16, and some chromatin compaction occurs in the absence of embryonic transcription13. However, inhibiting embryonic transcription pervasively disrupts establishment of open chromatin during EGA18, suggesting that EGA and chromatin remodeling are likely interdependent.Following fertilization, the zygotic genome is globally demethylated22; however, these accessible sites demonstrate different motif enrichment patterns, implicating distinct sets of transcription factors (TFs) in either murine 17, or human EGA 20. Although some TFs appear to regulate EGA in multiple species, i.e., Krüppel-like factors (KLFs)23, double homeobox (DUX)27, ZSCAN429, and CTCF18, the level of conservation across mammals remains unclear. In fact, the timing of genome activation is highly species-specific. RNA-seq studies indicate that the major wave of EGA in mice occurs during the 2-cell stage30, in humans during the 4-to-8-cell stage31, and in cattle during the 4-to-8-cell stage32. The relative timing at which mice activate their genomes could indicate that the mechanism behind murine EGA differs significantly from other species’, which would have significant implications for modeling human preimplantation development. In particular, the timing of bovine EGA more closely resembles that of human EGA, as do global changes to histone post-translational modifications. For instance, the active mark trimethylation of lysine 4 on histone 3 (H3K4me3) decreases in global abundance during human33 and bovine EGA34, but increases during murine EGA36. However, the chromatin remodeling events that underscore bovine preimplantation development have yet to be cataloged, and it remains unclear whether the regulation and execution of the MZT in cattle resembles what has been observed in humans.In mouse and human preimplantation embryos, enrichment for chromatin accessible sites is gradually established as development progressesHere we report progressive gains in ATAC-seq peaks throughout bovine preimplantation development. Peaks established prior to major EGA are predominantly enriched for binding sites of maternally provided factors , whereas homeobox TF binding  becomes more prevalent during major EGA, and depends on embryonic transcription. Comparison of the open chromatin landscape during preimplantation development in cattle, human, and mice reveals that the regulatory circuitry underlying bovine and human EGA is very similar and distinct from mouse. Moreover, TFs that appear to regulate murine EGA  demonstrate no enrichment in cattle or humans, suggesting that cattle are a more informative model for human preimplantation development than mice.37  were notably enriched for the binding motifs of RFX factors and folliculogenesis specific bHLH factor (FIGLA)  Fig. . Four-ceLA) Fig. , which wded Fig. , they arOTX1, OTX2, and PITX2 were appreciably expressed during the 8-cell stage   Fig. . Interge54) Fig. . Footpri Fig. . I51. Therefore, enrichment of CTCF binding in maintained peaks could indicate a gradual establishment of a stable 3-D chromatin architecture in preparation for major EGA. This interpretation is consistent with 3-D chromatin architecture in mouse embryos, which is greatly diminished after fertilization and then gradually established throughout preimplantation development, facilitating long-distance chromatin contacts, i.e., promoter-enhancer interactions, in later stage embryos13. Indeed, transcription during minor EGA in mouse is primarily driven by proximal promoters, whereas enhancers are dispensable for transcription until major EGA52. The global reorganization of 3-D chromatin architecture in the mouse has also been observed in bovine embryos: the nonradial establishment of chromosome territories, when gene-poor chromosomes tend to localize to the nuclear periphery more often than gene-rich regions, is first established at the 8-cell stage53. Collectively, these results suggest that gradual establishment of 3-D chromatin architecture may be a conserved feature of pre-EGA embryos, although the specific mechanisms and factors regulating this restructuring remain unknown.Although many ATAC-seq peaks were stage-specific, a stable open chromatin landscape was also progressively established after fertilization. As early as the 2-cell stage, ATAC-seq peaks appeared and were maintained post-EGA until at least the morula stage. Maintained ATAC-seq peaks were enriched for CTCF footprints Fig. , especiaKLFs were maternally provided  elements demonstrated both increasing expression  and short interspersed elements —are increasingly transcribed during human preimplantation developmention Fig.  and acceion Fig.  startingage Fig. , althougage Fig. , indication Fig.  and incrion Fig. . These pMounting evidence suggests that specific LTR retrotransposons, especially intact elements, play pivotal roles in early development. During bovine preimplantation development, the most expressed retrotransposons were ERV1-1_BT and ERV1-2_BT Fig. . Upon fu26. Specifically, in human embryos DUX4 binds MLT2A1 elements21. Considering that intergenic open chromatin was especially enriched for DUX binding sites in 8-cell bovine embryos, it seemed likely that these sites would also correspond to retrotransposons. Indeed, several MLT elements were enriched in 8-cell open chromatin harboring DUX motifs . Only COCs with healthy layers of cumulus cells were selected for maturation. These were washed in collection medium 6:4 M199 (Sigma M7653): SOF-Hepes, supplemented with 2% fetal bovine serum  and transferred to maturation medium modified M199 medium (Sigma M2154) supplemented with ALA-glutamine (0.1 mM), sodium pyruvate (0.2 mM), gentamicin (5 μg/ml), EGF (50 ng/ml), oFSH (50 ng/ml), bLH (3 μg/ml), cysteamine (0.1 mM), and 10% FBS.2PO4, 0.49 mM, MgCl2, 1.17 mM CaCl2, 5.3 mM sodium lactate, 25.07 mM NaHCO3, 0.20 mM sodium pyruvate, 0.5 mM fructose, 1X nonessential amino acids, 5 μg/ml gentamicin, 10 μg/ml heparin, 6 mg/ml fatty acid-free BSA) and transferred to drops of SOF-IVF medium under mineral oil. Frozen semen from a Holstein bull was thawed, and 106 sperm/ml were added to drops with MII oocytes, which were incubated at 38.5 °C for 12–18 h. Zygotes were then removed from fertilization medium, and cumulus cells were removed by vortexing for 5 min in SOF-Hepes medium. Zygotes were then transferred to culture media (KSOMaa supplemented with 4 mg/mL BSA) under mineral oil, and incubated at 38.5 °C in 5% CO2, 5% O2, and 90% N2. If embryos were to be transcriptionally inhibited, the culture medium was supplemented with α-amanitin (50 μg/ml) on day-one. On day-three, the culture medium was supplemented with 5% stem-cell qualified FBS (Gemini Bio 100–525). Blastocyst development (minimum 30%) was evaluated at 7 days post-insemination (dpi).After COCs matured for 24 h, MII oocytes were washed in SOF-IVF medium  supplemented with 20 ng/mL human FGF2 , 2.5 μM IWR-1 , and 100 U/mL penicillin–100 μg/mL streptomycin  on irradiated mouse embryonic fibroblast (MEF)  at 37 °C and 5% CO2. Cells were sub-cultured every 4–5 days at 1:10 split ratio using TrypLE Express . Culture medium was changed daily between passages. To increase cell survival, bESC culture medium was supplemented with 10 μM ROCK inhibitor Y-27632  at seeding. Before cell lysis and library preparation CTFR-bESCs were trypsinized and separated from MEF feeders by plating the cell suspension in CTFR culture medium supplemented with 10 μM ROCK inhibitor Y-27632 for 1 h in a 60 mm cell culture dish  at 37 °C and 5% CO2. After incubation, cells that did not adhere to the culture dish were recovered for downstream processing. For each library, 50,000 cells were centrifuged at 500 rcf, 4 °C, for 5 min. Pellets were then washed once with 50 µl cold 1x PBS buffer and centrifuged at 500 rcf, 4 °C, for 5 min. Pellets were then resuspended in 50 µl cold ATAC-seq lysis buffer  and centrifuged at 500 rcf, 4 °C, for 10 min to isolate nuclear pellets.CTFR-bESC lines derived from ICM through Wnt-pathway inhibitionzona pellucida and washed with SOF-Hepes on a warming plate. Cells were then transferred to 1 ml cold SOF-Hepes, and centrifuged at 500 rcf, 4 °C, for 5 min. Morulae were vortexed for 3 min in cold ATAC-seq lysis buffer. Cell pellets were then resuspended in 1 ml cold ATAC-seq cell lysis buffer and centrifuged at 500 rcf, 4 °C, for 10 min, and supernatant was aspirated to isolate nuclear pellets. To isolate ICMs by immunosurgery, blastocysts were treated with pronase (10 mg/ml) and washed in SOF-Hepes medium, then washed in 20% antibovine whole serum , and transferred to individual drops of 20% antibovine whole serum solution under mineral oil (20 ul per drop). Blastocysts were incubated for 1 h at 38.5 °C, washed in SOF-Hepes medium, then washed in 20% guinea pig complement  and transferred to individual drops of 20% guinea pig complement solution under mineral oil (20 μl drop). Blastocysts were again incubated for 1 h at 38.5 °C. ICM were isolated by pipetting, treated with trypsin for 2–3 min, and pipetted several times before adding 10% fetal bovine serum to stop trypsinization. ICM cells were then washed in 1 ml cold SOF-Hepes medium and centrifuged at 500 rcf, 4 °C, for 5 min. Cell pellets were then resuspended in 1 ml cold ATAC-seq cell lysis buffer and centrifuged at 500 rcf, 4 °C, for 10 min, and supernatant was aspirated to isolate nuclear pellets.Pools of oocytes and embryos were collected for ATAC-seq library preparation from three separate collections per developmental stage. Embryos intended for collection at the 2-cell, 4-cell, or 8-cell stages were divided into two groups, one of which was supplemented with α-amanitin, and cultured simultaneously. GV oocytes were collected for ATAC-seq prior to maturation. Preimplantation embryos were collected at the 2-cell (30–32 h post-insemination), 4-cell (2 dpi), 8-cell (3 dpi), and morula stages (5 dpi). For ICM collection, blastocysts were collected at 7 dpi and subjected to immunosurgery. Oocytes or embryos (a minimum of 500 cells) were treated with pronase (10 mg/ml) to completely remove the 2O) and incubated for 60 min at 37 °C, shaking at 300 rpm. The transposase, which is loaded with Illumina sequencing adapters, cuts DNA where it is not sterically hindered and simultaneously ligates adapters, effectively producing a library in one incubation step. Transposed DNA was purified with the MinElute PCR purification kit  and eluted in 10 μl buffer EB. Libraries were then PCR amplified: 50 μl reactions , 0.6 μl 25 μM custom Nextera PCR primer 1, 0.6 μl 25 μM custom Nextera PCR primer 2  were cycled at 72 °C for 5 min, 98 °C for 30 s, and then thermocycling at 98 °C for 10 s, 63 °C for 30 s and 72 °C for 1 min. Libraries from GV oocytes, 2-cell, and 4-cell embryos were thermocycled for 13 cycles, and 8-cell and morulae libraries were thermocycled for 11 cycles. PCR-amplified libraries were again purified with the MinElute PCR purification kit  and eluted in 10 μl buffer EB. Libraries were then evaluated for DNA concentration and nucleosomal laddering patterns using the Bioanalyzer 2100 DNA High Sensitivity chip . Expected nucleosomal laddering was evidenced by the presence of both small fragments, corresponding to hyper-accessible DNA that was frequently transposed, and larger fragments, corresponding to DNA that was wrapped around one or more nucleosomes. This study focused on mapping open chromatin; therefore, the sub-nucleosomal length fraction of each library (150–250 bp) was size selected using the PippinHT system  with 3% agarose cassettes. Size-selected libraries were run on a Bioanalyzer DNA High Sensitivity chip to confirm size-selection and determine DNA concentration. Final libraries were then pooled for sequencing on the Illumina NextSeq platform to generate 40 bp paired end reads.Nuclear pellets were then resuspended in 50 μl transposition reaction mix , 2.5 μl TDE1 enzyme , 22.5 μl nuclease-free H71, to remove residual Illumina adapter sequences and low quality (q < 20) ends, keeping unpaired reads and reads 10 bp or longer after trimming. Trimmed reads were then aligned to either the GRCm38 (mouse), GRCh38 (human), or ARS-UCD1.2 (cattle) assemblies using BWA aln (-q 15) and sampe72. PCR duplicates were removed with PicardTools, and mitochondrial and low-quality alignments (q < 15) were removed with SAMtools73. Alignments from biological replicates from each stage were merged and randomly subsampled to equivalent depth with SAMtools for detection of open chromatin. To determine which regions of the genome demonstrated significant enrichment of ATAC-seq signal, broad peaks were called with MACS274, using a q value cutoff of 0.05, and settings --nomodel --shift -100 --extsize 200.Raw sequencing reads were trimmed with Trim_Galore, a wrapper around Cutadapt (v0.4.0)75. Low-quality leading and trailing bases (3 bases) were clipped, and illumine adapter sequences were removed, allowing 2 seed mismatches, a palindrome clip threshold of 30, and a simple clip threshold of 10. Sliding window trimming was conducted with a window size of 4 bases, and a quality threshold of 15. Reads 36 bases or longer were retained after trimming. Trimmed reads were aligned to either the GRCm38 (mouse), GRCh38 (human), or ARS-UCD1.2 (cattle) assemblies with STAR (v2.7.2a) with options –outFilterScoreMinOverLread 0.85 and –seedSearchStartLmax 30. Low quality alignments (q < 5) were removed with SAMtools. Raw counts were calculated for genes in the Ensembl 96 annotations for each species with summarizeOverlaps, from the R package GenomicAlignments (v1.18.1)76, using ‘Union’ mode and allowing fragments for paired end data. The DESeq2 R package (v1.22.2)77 was used to identify differentially expressed genes, which were required to demonstrate a logFC >2 and an adjusted p value < 0.05. Variance stabilizing transformation (VST) was used to normalize expression counts for visualization.Raw sequencing reads were trimmed with Trimmomatic (v0.33)78 with options –outFilterMultimapNmax 100, --winAnchorMultimapNmax 100, and --twopassMode Basic. Raw expression values for individual repetitive elements were calculated for repeats in the RepeatMasker annotation for the ARS-UCD1.2 assembly (downloaded from the UCSC Genome Browser) using TEtoolkit79 in ‘multi’ mode, which improves quantification of transposable elements transcripts by including ambiguously mapped reads. Raw expression values were normalized with the variance stabilizing transformation included in the DESeq2 R package (v1.22.2).Trimmed reads were aligned to the ARS-UCD1.2 genome assembly with STAR (v2.7.0)80, which binned the genome into windows (50 bp default size) and calculated normalized signal in each window (fragments per kilobase million (FPKM) for paired-end data, and reads per kilobase million (RPKM) for single-end data). These bigwig files were used for track visualization with the UCSC genome browser. To compare bigwig files from replicate libraries on a genome-wide scale, the multiBigWigSummary function from Deeptools was used in bins mode (bin size of 500 bp). The output was then piped to the plotPCA function from DeepTools to generate the PCA plot , and the plotCorrelation function to calculate the Pearson correlation coefficient between biological replicates. To assess average accessibility or expression at genomic intervals of interest, ATAC-seq normalized signal (FPKM in 50 bp bins) was visualized using the DeepTools computeMatrix  and plotHeatmap functions. Regions of interest were scaled to 500 bp, and signal was also plotted for 1 kb up- and downstream.For both ATAC-seq and RNA-seq data, alignments were converted to bigwig format using bamCoverage from the DeepTools suite81, requiring a minimum of 1 bp overlap to consider a peak shared by both sets. Similarly, peaks were classified as genic if they overlapped either the 2 kb region upstream of a transcription start site (TSS), exons, or introns by 1 bp. Otherwise, peaks were considered intergenic.Peak sets from different stages were compared using the BEDtools intersect function82, using the exact sizes of the input genomic intervals (-size given). The most significant known or de novo motifs were reported, based on p value. Known motifs that matched significantly enriched de novo motifs were reported if their match score exceeded 0.6. To compare motif instance in open chromatin at different stages and in different species, sets of ATAC-seq peaks were evaluated for the presence of HOMER’s set of known TF motifs, measured as the percent of ATAC-seq peaks containing any given motif. To compare these data, PCA was performed with the prcomp function from the stats R package (v3.6.3), with options center = T and scale = F.Genomic regions were evaluated for binding motif enrichment using the findMotifsGenome.pl script from HOMER (v4.8)38. Using the FIMO tool from the MEME suite (v5.0.4)83, TF motif locations (p < 1e–4) were predicted genome-wide in the ARS-UCD1.2 genome assembly.Position-weight matrices were downloaded from the JASPAR database for TFs of interest84 was used to identify active TF binding sites from ATAC-seq data. When TFs are actively bound to DNA, transposase is sterically hindered, resulting in a small region, termed a “footprint,” where read coverage abruptly drops. To maximize ability to detect footprints, reads from biological replicates were pooled together for each developmental stage and treatment group, and broad peaks were called from pooled reads with MACS274 using a q value cutoff of 0.05, and settings --nomodel --shift -100 --extsize 200. For identifying footprints in an individual stage or treatment group, the rgt-hint command was used in footprinting mode to predict footprints within the corresponding ATAC-peaks, based on signal from pooled reads (commands –atac-seq and –paired-end). These footprints were matched to known motifs in JASPAR38 with the rgt-motifanalysis command in matching mode. For comparing TF binding activity between developmental stages, or between control and TBEs, ATAC-seq peaks called in each of the two stages or treatment groups were first combined with BEDtools merge. Footprints were then identified with rgt-hint in this merged set of ATAC-seq peaks based on signal from pooled reads in each of the two stages or treatment groups. Footprints were matched to known motifs in JASPAR38 with rgt-motifanalysis. Finally, the rgt-hint command in differential mode was used to compare the chromatin accessibility for each TF in each of the two stages or treatment groups. For each TF, the resulting binding activity score indicated whether the TF was more strongly bound in one group than the other, and the associated p-value indicated whether this change in binding activity between groups was significant .The HINT framework from the Regulatory Genomics ToolboxTo determine if repetitive elements  were enriched in open chromatin, the number of ATAC-seq peaks overlapping a set of repetitive elements was compared to randomized intervals (ATAC-seq peak locations shuffled with BEDtools shuffle function) overlapping the same set of repetitive elements, yielding a log ratio of random to observed.85. These Ensembl IDs were submitted to DAVID (v6.8)87 to identify enriched biological functions using medium stringency. Gene ontology terms  with a false discovery rate (FDR) <0.05 were reported.For gene sets of interest, the Ensembl gene IDs of human homologs were fetched using biomaRtFurther information on research design is available in the Nature Research Reporting Summary linked to this article.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Reporting Summary


Down syndrome (DS) or trisomy of chromosome 21 (Hsa21) is characterized by impaired hippocampal-dependent learning and memory. These alterations are due to defective neurogenesis and to neuromorphological and functional anomalies of numerous neuronal populations, including hippocampal granular cells (GCs). It has been proposed that the additional gene dose in trisomic cells induces modifications in nuclear compartments and on the chromatin landscape, which could contribute to some DS phenotypes. The Ts65Dn (TS) mouse model of DS carries a triplication of 92 genes orthologous to those found in Hsa21, and shares many phenotypes with DS individuals, including cognitive and neuromorphological alterations. Considering its essential role in hippocampal memory formation, we investigated whether the triplication of this set of Hsa21 orthologous genes in TS mice modifies the nuclear architecture of their GCs. Our results show that the TS mouse presents alterations in the nuclear architecture of its GCs, affecting nuclear compartments involved in transcription and pre-rRNA and pre-mRNA processing. In particular, the GCs of the TS mouse show alterations in the nucleolar fusion pattern and the molecular assembly of Cajal bodies (CBs). Furthermore, hippocampal GCs of TS mice present an epigenetic dysregulation of chromatin that results in an increased heterochromatinization and reduced global transcriptional activity. These nuclear alterations could play an important role in the neuromorphological and/or functional alterations of the hippocampal GCs implicated in the cognitive dysfunction characteristic of TS mice. Down syndrome (DS), the most frequent genetic cause of cognitive dysfunction  is causeIn DS and in the TS mouse, hippocampal-dependent learning and memory is particularly affected due to anatomical and functional alterations of this structure ,7. ReducThe cell nucleus is organized into structural and functional compartments involved in transcription, DNA replication and repair, and RNA processing. They include chromosome territories, the distinct nuclear sub-volumes occupied by each interphase chromosome, and the interchromatin region, which contains several nuclear structures with roles in gene expression and RNA processing, such as the nucleolus, Cajal bodies, and nuclear speckles of splicing factors ,16,17,18Chromosome territories are nonrandomly arranged within the interphase nucleus, and a precise 3D organization of the genome is required for establishing cell-specific gene expression programs ,19. UndeThe nucleolus is an archetype of nuclear compartment that is assembled around rDNA sequences called nucleolar organizer regions (NORs), where repeated rRNA genes are arranged in tandem ,21,22. EBecause of the essential role of the nucleolus in sustaining the protein synthesis machinery in developing and mature neurons, alterations in its structure and/or function can disrupt neurodevelopment, and may also be involved in the pathophysiology of several neurodegenerative disorders, including cerebellar ataxias, Alzheimer Disease (AD), Parkinson’s disease, and spinal muscular atrophy (SMA) ,27,28,29Cajal bodies (CBs) are dynamic nuclear structures of the interchromatinic region, discovered in mammalian neurons by Cajal ,31,32,33Recent advances in genomics and epigenomics reveal that the 3D spatial organization of the genome contributes to the regulation of gene expression through complex long-range intrachromosomal and interchromosomal interactions, which are often mediated by the formation of chromatin loops. In particular, newborn maturing postmitotic mammalian neurons that exit from cell cycle must acquire, in the early G1/G0 phase, the stable 3D genome organization characteristic of each neuronal type. This includes chromosome movements to achieve the spatial rearrangement of chromosome territories, the spatial tethering of NORs sequences of rDNA resulting in nucleolar fusion, and the acquisition of the epigenetic pattern of chromatin configuration with the specific heterochromatin  and euchromatin  pattern domains ,40,41.In this context, it has been demonstrated that the extra Hsa21 causes a displacement and higher compaction of other chromosome territories in trisomic cells . In addiBecause of its essential role in hippocampal learning and memory, alterations in the 3D reorganization of the genome and/or in the nuclear structure and function of hippocampal GCs could contribute to the neuromorphological or functional alterations observed in this neuronal population. In particular, these alterations may be implicated in the cognitive dysfunction that the TS mouse presents. The aim of this study is to analyze the effect that the 3D reorganization of the genome induced by the partial trisomy of Mmu16 in the GCs of the TS mouse has on the nuclear size and organization of the nuclear compartments involved in transcription and mRNA and rRNA processing. We will primarily focus on (I) the epigenetic changes in chromatin structure and its potential influence on global transcriptional activity, (II) the reorganization of NORs, and its effect on the fusion pattern of nucleoli, and (III) the behavior of CBs.The impact of alterations in the 3D-organization of chromosomes and their associated changes in the epigenetic programs in GCs was studied in TS mice. First, we analyzed variations in the nuclear size and the heterochromatin compartment. Dissociated neuronal preparations of the dentate gyrus GCL from wild-type (hereafter referred to as control) mice co-stained with DAPI and propidium iodide, two cytochemical markers for DNA and RNA, respectively, revealed the well-preserved morphology of whole GC perikarya. Thus, the cell nucleus showed several prominent masses of condensed heterochromatin and appeared to be surrounded by a narrow RNA-rich cytoplasmic band stained with propidium iodide A.Next, we performed immunolabeling for the repressive histone modification H4K20me3, a heterochromatin marker, which is involved in gene silencing and also contributes to the cellular senescence program ,52,53. AMecp2 and H1f0, encoding methyl-CpG-binding protein 2 (MeCP2), which has a great affinity for methylated DNA, and the H1.0 linker histone, respectively, were significantly increased in the hippocampal RNA extracts from TS mice relative to control animals , nucleophosmin/B23 (Npm1) and UBF (Ubft), we performed RT-qPCR on hippocampal RNA extracts from control and TS mice. Whereas 45S pre-rRNA is very short-lived and is considered to be an indicator of RNA pol I-dependent transcriptional activity of ribosomal genes [Ubft mRNA expression, encoding a key transcription factor of ribosomal genes, were found between control and TS hippocampal RNA extracts  the epigenetic regulation of chromatin structure and function, with increased heterochromatin and a global reduction of transcription, (II) the nucleolar fusion pattern, with increased number of nucleoli, and (III) the molecular assembly of the CBs involved in pre-mRNA splicing.per se their 3D genome organization. This could result in physical alterations in both gene positioning and the intrachromosomal and interchromosomal interactions mediated by chromatin loops. Given the closely intertwined relationship between genome topology and gene regulation, changes in the gene positioning and chromatin interactions in DS trisomy could lead to altered gene expression, resulting in a global downregulation of transcription observed here in the TS GCs. This effect may contribute to the neuromorphological and functional alterations found in the hippocampal GCs [Notwithstanding the influence of the dosage imbalance of gene expression in this mouse model of DS , we propmpal GCs ,12,13,14mpal GCs . Thus, tMecp2 and H1f0, involved in the organization of heterochromatin are upregulated in the hippocampi of TS mice [The hippocampal GCs of the dentate gyrus have a small nucleus with prominent heterochromatin masses of transcriptionally repressed heterochromatin and compact small nucleoli . The red TS mice ,57,67. I TS mice .The global reduction of the transcription rate in TS GCs detected with the in situ 5′-FU transcription assay is noteworthy. The decreased incorporation of 5′-FU into nascent RNA clearly occurs in active euchromatin domains, supporting the fact that downregulated genes mainly correspond to protein coding genes. However, the contribution of chromatin non-coding genes cannot be ruled out. Consistent with the run-on transcription assay, we have detected a reduction of the acetylated histone H4, a marker of “open” transcriptionally active euchromatin , by immuDespite the high complexity of gene expression in aneuploid mammalian cells, a common feature is decreased proliferative capacity . In partRegarding the particular impact of the Mmu16 trisomy on global gene expression in the central nervous system, Kahlem et al.  reportedHlcs catalyzes the biotinylation of some histones and induces chromatin condensation and the repression of genes [Dyrk1A and Runx1 codify proteins that may alter the epigenetic architecture of the nucleus [Additionally, the altered expression of several orthologous Hsa21 genes, triplicated in the TS mouse, may contribute to increased heterochromatinization, or the decrease in global transcription found in the GCs of this mouse model. Among them, of genes ,71. Othe nucleus ,73.In relation to the nucleolar behavior of GCs from the TS mouse, the increase in the number of nucleoli is remarkable. Two important factors may influence the determination of the number of nucleoli in maturing GCs: the number of NORs, and the nucleolar fusion pattern. In eukaryotic cells, beginning at telophase, the transcription of rRNA genes resumes and nucleoli start to reform around individual NORs, initially forming multiple nucleoli. They fuse together, resulting in the assembly of one or a few larger mature nucleoli, each containing several NORs ,24,40. IRegarding nucleolar fusion, its cellular and molecular mechanisms are not well known, but there is evidence that this cellular process must involve an important spatial reorganization of NOR-bearing chromosomes within the nucleus . Indeed,In this context, we propose that partial trisomy-associated alterations in nuclear architecture and the epigenetic state of chromatin in differentiating TS GCs interferes with the normal pattern of nucleolar fusion, resulting in an increase in the number of nucleoli. In addition to rRNA transcription, nucleolin plays a key role in nucleolar assembly ,62,77. WImportantly, whereas most large size mammalian neurons are mononucleolated, small size neurons with lower transcriptional activity tend to be multinucleolated. This suggests a negative correlation between transcriptional activity and the number of nucleoli in mammalian neurons ,38. It iSupporting the hypothesis of the spatial reorganization of NOR-bearing chromosomes in trisomic GCs, we have found changes in the normal distribution of Ag-NORs stained with a silver nitrate procedure that recognizes some argyrophilic proteins associated with rDNA arrays ,22. In aOur results show non-significant changes in the expression of both 45S pre-rRNA and mature 18S and 28S rRNAs in hippocampal TS mice compared with control ones, suggesting that ribosomal gene transcription is preserved in TS GCs. Consistent with these findings, no variations in the expression of genes encoding three key nucleolar proteins, UBF, fibrillarin and nucleophosmin, were found between control and TS hippocampal RNA extracts. The maintenance of ribosomal gene transcription in TS GCs may reflect a reactive response of the nucleolus to the global reduction of the transcription of protein-coding genes, in an attempt to preserve ribosome biogenesis and translational activity. A similar reactive compensatory nucleolar response has been reported in (i) SMA spinal motor neurons, (ii) sensory ganglion neurons under experimental conditions of proteotoxic stress and (iii) certain neuronal populations from AD patients ,29,79. IAnother important issue is the changes in the organization of CBs in TS GCs, with two crucial manifestations: a reduction in the number of canonical CBs, and an increased incidence of perinucleolar caps of coilin which are free of snRNPs. CBs are very dynamic transcription-dependent nuclear structures ,32,33,35The increased formation of perinucleolar caps of coilin suggests a defective assembly of CBs which is dependent on post-translational modifications of CB proteins such as SMN and coilin ,83. CoilIn conclusion, in this study we demonstrate that the three copies of a fragment of Mmu16 in the TS mouse alters the nuclear architecture  of their hippocampal GCs. The organization of CBs and nucleoli is emerging as a reliable nuclear indicator of neuronal homeostasis, and of its dysfunction under neurodegenerative conditions such as AD ,28,86. BThis study was approved by the Cantabria University Institutional Laboratory Animal Care and Use Committee and was performed in accordance with the Declaration of Helsinki and the European Communities Council Directive (86/609/EEC of 24 November 1986). TS and control mice were generated and karyotyped using real-time quantitative PCR (qPCR) as previously described . In all For immunofluorescence, at least three animals of each karyotype were anesthetized and perfused under deep anesthesia with paraformaldehyde 3.7% in PBS, and post-fixed for 1 h. Their brains were removed and coronally sliced in a vibratome (200 μm thick). Small fragments from the GCL of the hippocampus were dissected out and transferred to a drop of PBS on a siliconized slide. Squash preparations of dissociated GCs were performed following the protocol described by . The samConfocal images were obtained with a LSM510  laser scanning microscope using a 63x Oil (1.4 NA) objective. In order to avoid overlapping signals, images were obtained by sequential excitation at 488 nm and 543 nm to detect FITC or TexasRed, respectively. The images were processed using Adobe Photoshop CS4 software .http://imagej.nih.gov/ij). At least 100 GCs per animal were sampled in all the analyses. The nuclear area of the GC was determined using DAPI staining. Nuclear perimeters were traced by hand and the areas were measured with Image J default tools to calculate the shape descriptors. Acetylated histone-H4 fluorescence intensity was quantified within the nuclear perimeter of the GC. Quantification of the area occupied by tri-methylated histone-H4 was performed in single-plane images. First, the nuclear area was determined and then the regions occupied by tri-methylated histone-H4 were delimited by applying an automatic local threshold. For the quantitative analysis of the number of CBs and nucleoli, coilin and nucleolin positive spots were counted, respectively, on serial confocal sections of the whole nucleus.The following primary antibodies were used: polyclonal rabbit anti-acetyl histone H4 , polyclonal rabbit anti-tri-methylated histone H4 at K20 , anti-coilin 204/10 (204.3 serum), anti-TMG-cap  and anti-nucleolin . Morphometric and quantitative analyses were performed using ImageJ software  containing 0.5% Triton X for 10 min. Their brains were removed, post-fixed with a perfusion buffer for 1 h and washed in HPEM. Then, mechanical GCs dissociation was performed as described above. The incorporation of 5′-FU into nascent RNA was detected with mouse monoclonal anti-BrdU  for 1 h at 37 °C. Then, the samples were washed with 0.01% Tween 20 in PBS, incubated for 45 min with an anti-mouse FITC-conjugated secondary antibody, washed in PBS and mounted with Vectashield. Quantification of the intensity of 5′-FU fluorescence was determined within the nuclear perimeter in at least 100 GCs from three animals of each karyotype.Active transcription sites were labelled by incorporation of 5′-fluorouridine (5′-FU) into nascent RNA as previously described . The micTo analyze the ultrastructure of GCs, three TS and control mice were perfused with 3% glutaraldehyde in 0.1 M phosphate buffer pH 7.4. Their brains were removed and post-fixed overnight with the perfusion buffer. Then, coronal brain slices (300 μm thick) were obtained using a vibratome, and small fragments of the GCL of the hippocampus were dissected out. The processing of the hippocampal samples was performed following the procedure previously reported by .For immunoelectron microscopy of coilin, three CO and TS mice were perfused with 3.7% paraformaldehyde in 0.1 M phosphate buffer. Small fragments of the GCL of the hippocampus were isolated from 300 μm-thick slices. The samples were processed following the protocol previously described by Pena et al. . The samActive chromosomal NORs were revealed in squashes of GCs by a silver impregnation method following the protocol described by . Images Mice were euthanized by decapitation and their hippocampi were dissected and stored at −80° C. Six hippocampi from TS and CO mice were lysed and homogenized as previously described . The homProtein bands were detected using a LI-COR ODYSSEY IR Imaging system V3.0 (LI-COR Biotechnology) and quantified following the protocol described by .(Ncl), fibrillarin (Fbn1) and UBF (Ubtf) was determined by RT-qPCR using gene-specific SYBRGreen (TaKaRa)-based primers. Each individual sample was performed in triplicate. The threshold cycle (Ct) was determined and the results were normalized to the housekeeping gene GAPDH. Relative expression was calculated according to the 2-(ΔΔCt) equation [For qRT-PCR analysis, six control and TS mice were anesthetized and decapitated. Their hippocampi were removed, rapidly frozen and stored at −80 °C. RNA was isolated using RNeasy Plus Mini Kit . RNA was reverse-transcribed using RevertAid H Minus First Strand cDNA Synthesis Kit  and cDNA concentration was measured in a spectrophotometer (Nanodrop Technologies ND-1000) and adjusted to 1 µg/µL. The rRNA expression of 45S precursor, 18S 5′junction intermediary and mature 18S and mRNA expression of genes encoding nucleolar components nucleolin equation . SYBRGret-test. All analyses were performed using SPSS  for Windows.For comparisons, data was analyzed using Student’s


Leishmania major (LmBrf1), a parasitic protozoan that shows distinctive transcription characteristics, including the apparent absence of Pol III general transcription factors TFIIIA and TFIIIC. Although single-knockout parasites of LmBrf1 were obtained, attempts to generate LmBrf1-null mutants were unsuccessful, which suggests that LmBrf1 is essential in promastigotes of L. major. Notably, Northern blot analyses showed that the half-lives of the messenger RNAs (mRNAs) from LmBrf1 and other components of the Pol III transcription machinery (Bdp1 and Pol III subunit RPC1) are very similar (~40 min). Stabilization of these transcripts was observed in stationary-phase parasites. Chromatin immunoprecipitation (ChIP) experiments showed that LmBrf1 binds to tRNA, small nuclear RNA (snRNA), and 5S rRNA genes. Unexpectedly, the results also indicated that LmBrf1 associates to the promoter region of the 18S rRNA genes and to three Pol II-dependent regions here analyzed. Tandem affinity purification and mass spectrometry analyses allowed the identification of a putative TFIIIC subunit. Moreover, several proteins involved in transcription by all three RNA polymerases co-purified with the tagged version of LmBrf1.In yeast and higher eukaryotes, transcription factor TFIIIB is required for accurate initiation of transcription by RNA Polymerase III (Pol III), which synthesizes transfer RNAs (tRNAs), 5S ribosomal RNA (rRNA), and other essential RNA molecules. TFIIIB is composed of three subunits: B double prime 1 (Bdp1), TATA-binding protein (TBP), and TFIIB-related factor 1 (Brf1). Here, we report the molecular characterization of Brf1 in  Leishmania produce the three main forms of leishmaniases  in tropical and subtropical regions of the world. These protozoan parasites are transmitted to humans through the bite of infected female sandflies of the genera Phlebotomus and Lutzomyia. Leishmania and other trypanosomatids, such as Trypanosoma cruzi and Trypanosoma brucei, are ancient organisms that diverged early from the main eukaryotic lineage dCTP using the High Prime labeling system . The Brf1 probe was amplified with primers BRF-XmaI-5′ and BRF-XbaI-3′. The Bdp1 probe was amplified with oligonucleotides B’’-BamHI-5′ (5′–ATGGATCCATGGACGACAACGAGTTCGAA) and B’’-XbaI-3′ (5′–ATTCTAGACTCAAACGAGAAGTCCGAGTC) and the RPC1 probe with primers C160-1-5′ (5′–GATCATCAACGCCAACAAGAGC) and C160-2-3′ (5′–TTGAGCAGGATGTTGAAGCTGC). The α-tubulin probe was amplified with primers Alfa-tub-5′ (5′–AGAAGTCCAAGCTCGGCTACAC) and Alfa-tub-3′ (5′–GTAGTTGATGCCGCACTTGAAG), and the 18S rRNA probe with oligonucleotides Lm-rRNA18S 5′ (5′–CGGCCTCTAGGAATGAAGG) and Lm-rRNA18S 3′ (5′–CCCCTGAGACTGTAACCTC). Filters were washed to a final stringency of 0.1× SSC and 0.1% SDS at 65 °C. RNA signals from three independent experiments were quantified by densitometry using the MultiGauge software and plotted. Half-lives were determined from the linear regression estimates derived from the linear segment of the mRNA decay curves. For Southern blot analysis, 5 µg of genomic DNA was digested, separated by electrophoresis, and transferred by capillary to Hybond-N membranes. The radioactive probe corresponded to the 5′-targeting region of Brf1. Filters were washed as described above.For determination of the half-life of the different mRNAs, Northern blot experiments were performed with RNA isolated at several time points from cells incubated in the presence of the transcription inhibitor actinomycin D (10 µg/mL). Total RNA was extracted with TRI reagent , separated in agarose–formaldehyde gels and transferred to Hybond-N filters . Hybridization was performed with probes that were labeled with . N. NLmjF.2. brucei .The zinc ribbon fold in ScBrf1 consists of three antiparallel β strands . SimilarTo examine the subcellular localization of LmBrf1, a cell line where this protein is labeled with a C-terminal PTP tag was obtained to carry out indirect immunofluorescence assays. This tag is composed of Protein A (Prot A) and Protein C (Prot C) epitopes separated by a cleavage site of the TEV protease . The expIn order to determine the decay rate of the LmBrf1 mRNA, Northern blot experiments were performed with total RNA from mid-log parasites that were grown in the presence of actinomycin D (10 mg/mL) for different times (between 0 and 16 h). The half-life of the LmBrf1 mRNA was around 39 min . NotablyL. major, we planned to produce LmBrf1-null mutant parasites by replacing the endogenous genes with selectable marker genes. Since L. major Friedlin is a diploid organism [puromycin (pac) and hygromycin (hyg) resistance genes were bounded by 5′ and 3′ flanking regions of LmBrf1. To study the role of LmBrf1 on cell growth and transcription in organism , two conpac gene was demonstrated in one of the clones by Southern blot analysis using the 5′-targeting region as a probe, as the expected bands of ~1158 and ~5251 bp were observed when digesting with SalI and NotI, respectively  and in the intergenic region between this gene and the 5S rRNA gene (Inter 11.0930)  . Unexpec11.0930) .L. major ribosomal proteins, heat-shock proteins, translation elongation factors, actin, and α- and β-tubulins. These proteins are common contaminants in tandem affinity purifications [To identify proteins that interact with LmBrf1, tandem affinity purification experiments were performed with the cell line that expresses the PTP-tagged version of LmBrf1. The correct expression of LmBrf1-PTP was confirmed by Western blotting and immunofluorescence . LmBrf1-ications .LmjF.12.0560) . Pol III subunits RPC1, RPAC1 (shared with Pol I), and RPB5 (shared with Pol II) were also identified. Moreover, La protein, which binds to Pol III transcripts, co-purified with LmBrf1-PTP.In addition to LmBrf1, mass spectrometry analysis of the LmBrf1-PTP sample allowed the identification of multiple proteins that were divided in seven categories: transcription factors, Pol subunits, regulators of transcription and/or chromatin remodelers, DNA- or RNA-binding proteins, DNA replication, kinases or phosphatases, and other functions . As anti12.0560) . This isInterestingly, several proteins related to Pol II transcription were also purified. These include Pol II subunits RPB1 and RPB2, transcription elongation factors TFIIS and SPT6, some subunits from the PAF1 and the CCR4-NOT complexes, and FACT complex subunit SPT16 . MoreoveL. major. Bioinformatic analyses demonstrated that the N-terminal half of LmBrf1 contains the distinctive zinc ribbon, cyclin repeats, and homology block I , Bdp1 (from 36 to 72 min), and RPC1 (from 40 to 62 min) . The stant stage . A similo 2.25 h . Destabiishmania ,58. ConsL. major [LmjF.11.0930 gene, and to the SL RNA locus (transcribed by Pol II). The co-purification of proteins involved in Pol II and Pol I transcription with LmBrf1-PTP supports this finding  co-purified with LmBrf1-PTP. It is worth noting that our group identified the T. brucei ortholog (Tb927.1.3860) as an interacting partner of Pol III subunit RPC3 by tandem affinity purifications (manuscript in preparation). Thus, contrary to what was presumed [Tandem affinity purification experiments allowed the identification of several proteins that co-purified with LmBrf1-PTP, including TBP . It was T. brucei to maintain an open chromatin structure and facilitate their transcription [The multiple proteins identified by the mass spectrometry analyses , includicription .L. major, as has been reported in human cells [Several DNA- and RNA-binding proteins that could regulate gene expression were co-purified with LmBrf1-PTP, including a KD domain containing protein, putative ATP-dependent RNA helicase SUB2, and ALBA-domain protein 3 . Proteinan cells . A reducL. major. Nevertheless, the interaction of LmBrf1 with Pol I- and Pol II-dependent regions and their transcription regulators might be fortuitous. A genome-wide study showed that TBP is highly enriched at most SSRs, the SL RNA genes, and the rRNA transcription units [At the present time, we do not know if the association of LmBrf1 with some genes transcribed by Pol I and Pol II  and withon units . Thus, LIn this work, we showed that, despite sequence divergence, LmBrf1 possesses the six conserved domains of Brf1 orthologs. Moreover, the predicted three-dimensional structure of the N-terminal region of LmBrf1 is conserved. Our results also suggest that LmBrf1 is an essential nuclear protein. Northern blot assays showed that the mRNAs encoding LmBrf1, Bdp1, and RPC1 (involved in Pol III transcription) share similar half-lives. All three mRNAs were stabilized in stationary-phase parasites. ChIP experiments demonstrated that LmBrf1 associates to 5S rRNA, tRNA, and snRNA genes. However, unlike other species, LmBrf1 associates to the Pol I-dependent 18S rRNA gene promoter and to three regions transcribed by Pol II. Moreover, our results suggest the interaction of LmBrf1 with transcriptional complexes from all three RNA polymerases. Subunit Tau131 of TFIIIC was identified as one of the putative interacting partners of LmBrf1.


The fru promoter‐1 pre‐RNA generates a set of BTB‐zinc finger family FruM proteins expressed exclusively in the male neurons, leading to the formation of sexual dimorphisms in neurons via male‐specific neuroblast proliferation, male‐specific neural survival, male‐specific neuritegenesis or male‐specific arbor patterning. Such a wide spectrum of phenotypic effects seems to result from chromatin modifications, in which FruBM recruits Bonus, Histone deacetylase 1 (HDAC1) and/or Heterochromatin protein 1a (HP1a) to ~130 target sites. One established FruBM transcriptional target is the axon guidance protein gene robo1. Multiple transcriptional regulator‐binding sites are nested around the FruBM‐binding site, and mediate sophisticated modulation of the repressor activity of FruBM. FruBM also binds to the Lola‐Q transcriptional repressor to protect it from proteasome‐dependent degradation in male but not female neurons as FruBM exists only in male neurons, leading to the formation of sexually dimorphic neural structures. These findings shed light on the multilayered network of transcription regulation orchestrated by the master regulator FruBM.The  Drosophila Fruitless regulates proteolysis to produce sex‐specific Lola isoforms that specify neuronal sex types. The neural pathway for courtship behavior represents one of the best characterized circuitries in the fly. This is primarily due to the discovery of a courtship‐defective mutant, fruitless ,fru gene .2+ (signifying an excited state) when exposed to a live female or dummy targets with appropriate properties as courtship inducers.fru‐positive, reinforcing the idea that the core portion of the courtship neural circuit is built up by connections among fru‐positive neurons.fru orchestrates the formation of mutual connections between a specific set of fru‐positive neurons to organize the entire courtship circuit. To answer this question, we need to know the molecular modes of action of fru gene products.The fruitfly e Figure B, which s Figure A‐C. By us Figure D,E. With2fru gene displays an exceedingly complex organization , a female‐specific splicing regulator known to function as a feminizer protein in the sex‐determination cascade.fru primary transcript at sites locating distally in exon‐2, resulting in a long mRNA that includes the exon‐2 sequence containing a translation stop signal; Figure fru primary transcript is spliced at a more proximal site, yielding a shorter mRNA that lacks the exon‐2‐derived stop codon. As a result, fru mRNA in males can encode a large open reading frame for proteins  whereas that in females is unable to encode a protein.fru primary transcript, producing C‐terminal variants referred to as FruAM, FruBM, FruCM, FruDM and FruEM (FruM members), or their non‐sex‐specific counterparts (FruCOM members) FruA, FruB, FruC, FruD and FruE, at least in theory .fru isoform‐specific mutants have been generated, the functional differences of these proteins in neural sex differentiation were not fully elucidated.fru‐positive neurons express all three of these isoforms together, some neurons express only two or one isoform(s). It remains to be clarified whether the function or the expression of the isoforms is responsible for the observed phenotypic differences among isoform‐specific mutants.The n Figure B. It has3bon and HDAC1 (Rdg3) mutations agonistically act with fru mutations in abrogating male courtship activities, whereas HP1a (Su(var)205) mutations negatively modulate the effects of fru mutations. Possible effects of these mutations on sex‐specific neural development were examined by clonally labeling the mAL cluster of fru‐positive neurons, because this particular cluster displays conspicuous sex differences: the number of cells composing the cluster is 5 in females and 30 in males; the ipsilateral neurite is present in some male neurons but totally absent in all female neurons; the tip of the contralateral neurite in the subesophageal ganglion always bifurcates in females whereas it has a comb‐like appearance without a bifurcation in malesfru mutant males (Promoter‐1 null mutants).fru or HDAC1 gene activity increased the female‐type mAL neurons, whereas similar reductions in HP1a gene activity in fru hypomorphic males increased male‐type mAL neurons. A remarkable fact is that no single mAL neuron with intersexual characteristics was found in these genetic variants.grim, hid and reaper, affects primarily the sex difference in the number of mAL neuronsrobo1 (see below) specifically affects the sex‐dependent presence or absence of the ipsilateral neurite.fru‐positive neurons are also sexually dimorphic, as showed by the dedicated efforts to resolve the finer structures of individual neurons on the standardized brain. Some neural clusters are only found in the female or male brain as a result of sex‐specific cell death or sexually distinct proliferation patterns of a neuroblast. Thus, the mechanistic triad underlying the sexual dimorphisms found in mAL neurons would also be useful in understanding the mechanisms for sex‐type specification in other fru‐positive neurons.At the molecular level, the FruAM, FruBM and FruEM isoforms have been characterized in some detail. FruM, but not FruCOM, has an N‐terminal extension of 101 a.a., which is immediately followed by a common broad‐complex, tramtrack, and bric‐a‐bric (BTB) domain, whereas the C‐terminus has two zinc finger motifs unique to each isoform4fru‐transfected cultured S2 cells, and a complementary reverse transcription PCR (RT‐PCR) experiment with RNAs extracted from male flies. In this attempt, ~100 potential targets for each of the major three Fru isoforms were obtained; she found that ion channel‐encoding genes were the dominant group, biases existed for X‐chromosome‐linked genes and increased transcription was prevalent rather than downregulation upon Fru overexpression. Dalton et al.fru‐expressing neurons in vivo, so that the FruM moiety binds to a target DNA sequence, which in turn is methylated by the Dam moiety and thus tagged.fru knockdown for tens of genes locating close to the deduced FruM‐binding sequences on the genome led them to propose that CadN and lola are two most likely candidates for FruEM targets.Leucine‐rich repeat G‐protein coupled receptor 3 (Lgr3) gene is more intense in females than males, and this sexual dimorphism in Lgr3 reporter expression disappears when FruEM is knocked down. They identified several possible FruEM‐binding sites in an Lgr3 intron by electromobility shift assays (EMSA) in vitro; however, mutating all these sites in the GAL4 knock‐in allele failed to affect the sexually dimorphic Lgr3 expression in vivo,Lgr3 is a direct target for FruEM. Dauwalder et al.takeout gene that encodes a putative lipid transporter expressed in the fat body associated with the brain. Although both fru and dsx stimulate transcription of the takeout gene, it is unknown whether they coordinately act on the takeout regulatory sequence. We cannot exclude other possibilities; for instance, fru and dsx might regulate each‐others' transcription, thereby affecting expression of their target effector genes, such as takeout. Ito et al.fru‐expressing mAL neurons to obtain ones that convert the neural sex‐type, with a particular focus on the presence or absence of the male‐specific ipsilateral neurite upon knockdown. This search yielded robo1, a gene encoding a transmembrane receptor for the neurite guidance signal Slitrobo1 knockdown in females resulted in an ectopic formation of the male‐specific contralateral neurite in females yet without effect in malesrobo1 promoter to repress its transcription. Gel‐mobility shift and reporter assays showed that the FruBM binding requires a 42 bp region (named FROS) in the robo1 promoter encompassing a palindrome sequence, TTCGCTGCGCCGTGAA  not only increased the number of male flies with no male‐specific neurite but also impaired the male courtship posture, even in the flies carrying only one copy of the mutation, that is, heterozygotes. When they generate courtship songs, normal males vibrate only one wing at a time, and they alternate the wing to vibrate once every several seconds. However, males carrying robo1Δ1 alternate the wing to vibrate several times per second (the precocious wing‐switching phenotype). This observation suggests that leaky expression of robo1 occurs if FruM fails to bind to the robo1 promoter, with the consequence that both the neurons and behavior are de‐masculinized, and these changes manifest as dominant phenotypes as in the case of the robo1Δ1 heterozygotes.Which genes are transcriptionally regulated by FruM? Vernes5robo1 gene promoter offers an unparalleled opportunity to dissect the molecular mechanism by which the actions of FruM lead to the sex‐specification of individual neurons and ultimately to sex‐specific behavior. In fact, the robo1 promoter harbors additional motifs for binding of other transcription factors around the FruM‐binding site FROS. Located ~30 nt upstream of the robo1 transcription start site  is the DNA‐replication element association motif (DREAM), which is proximally flanked by the TCT motif ,robo1 promoter fragment with TCT and DREAM motifs binds TRF2. What is interesting, however, is the result of the reporter assay with the robo1 promoter fragment in transfected S2 cells: TRF2 activates transcription when applied singly, but when applied together with a low concentration of FruBM that is insufficient for transcription repression, TRF2 actually represses transcription—that is, TRF2 now potentiates the FruM repressor action.robo1 promoter might underlie the context‐dependent switching between activation and repression of robo1 transcription.The 6fru genetic modifier screens repeatedly identified lola,robo1 reporter transcription in S2 cells, whereas small Lola‐Q counteracts large Lola‐Q. EMSA assays identified tandem direct repeats of 18 nt (named DR1) as the Lola‐Q‐binding site, which are mostly included within FROS flanking 3′ to the Pal1 palindrome, that is, the core binding motif for FruBM (Figure robo1 promoter.fru loss‐of‐function; e.g., it restores the male‐specific neurite otherwise lost in fru mutant males (Figure fru mutant males is abrogated by simultaneous overexpression of small Lola‐Q (Figure fru‐positive sexually dimorphic neurons.DR1 in the robo1 promoter (robo1Δ4; shown in Sato et al.robo1Δ1.In addition to TRF2, s Figure C. This aQ Figure D, in keeDrosophila Ci and its mammalian homolog Gli3, Epstein‐Barr virus‐protein EBNA1, and the RNA polymerase degradation factor Def1 in yeast. Interestingly, mass spectrometric analysis of immunoprecipitates with an anti‐Lola‐Q antibody identified Cullin1, an E3 ligase that polyubiquitinates lysine residues, which are in turn targeted by the 26S proteasome for degradation. Knockdown of either Cullin1 or proteasome subunit beta 5 induced the male‐specific neurite in some mAL neurons, in accord with the hypothesis that the masculinizer large Lola‐Q is converted into the feminizer small Lola‐Q by partial proteasomal degradation in female, but not male, fliesPeptide sequencing by Edman degradation showed that small Lola‐Q lacks N‐terminal amino acid residues 1‐263 of large Lola‐Q, including the entire BTB domain.7fru‐positive neurons, forming approximately 50 clusters of somata each uniquely locating within the brain.fru. The robo1 promoter has served as a useful model in exploring the molecular bases for FruBM actions in the sex‐type specification of neurons. Based on the picture emerging from the investigation of FruBM actions on the robo1 promoter, we may need to reconsider the traditional view of the “master regulator” in a given developmental process. Turning ON and OFF middle management genes encoding transcription factors is not the sole job with which FruBM is charged. For example, FruBM also fine tunes the mode of action of a transcription complex to which it contributes, a complex that binds to the DNA motif on terminal effector genes encoding structural proteins. Strikingly, the FruBM core‐binding motif Pal1 in the robo1 promoter is surrounded by multiple putative binding motifs for different transcriptional regulators, two of which were identified as those for TRF2 and Lola‐Q. While TRF2 and Lola‐Q can bind to their own binding motifs on the robo1 promoter, both proteins are included in the FruBM‐containing protein complex. This could mean that the FruBM‐containing protein complex may induce altered folding of the DNA strand around the binding sites. As salivary chromosome immunostaining unraveled ~130 FruM‐bound sites distributed across the chromosomes,The fly brain contains ~2000