Mesenchymal stem cells  are multipotent stem cells that can differentiate into cells of at least three mesodermal lineages, namely adipocytes, osteoblasts, and chondrocytes, and have potent immunomodulatory properties. Epigenetic modifications are critical regulators of gene expression and cellular differentiation of mesenchymal stem cells (MSCs). Epigenetic machinery controls MSC differentiation through direct modifications to DNA and histones. Understanding the role of epigenetic machinery in MSC is crucial for the development of effective cell-based therapies for degenerative and inflammatory diseases. In this review, we summarize the current understanding of the role of epigenetic control of MSC differentiation and immunomodulatory properties. Major transcription factors determining MSCs’ are epigenetically controlled.CEBPA depends on HDAC1 activity. Opening of PPARG promoter region depends on SWI/SNF complex and simultaneous acetylation of histone H3 residues.C/EBPα (encoded by CBPA) and PPARγ (encoded by PPARG) are recognized as master regulators of adipocyte differentiation. The expression of RUNX2 and OSX major orchestrators of osteogenic differentiation are controlled by, methylation at histone H3 residues, acetylation at histone H3 and H4 residues, and DNA methylation signatures.Chondrogenic differentiation depends on MSCs condensation to form 3D structures. SOX9 is a major transcription factor in early chondrogenesis, and its expression is controlled by histone H3/H4 acetylation.MSCs licensing with proinflammatory cytokines, hypoxia conditions, and bioactive molecules induces their immune-modulatory activities.Do differentially sourced MSCs differ in the epigenetic landscape, showing higher environmentally defined potential for differentiation or immune modulation (trained effect)?What is the role of ncRNAs in the regulation of epigenetic machinery in MSCs?What are the epigenetic mechanisms controlling MSCs immunomodulatory properties after licensing with proinflammatory cytokines?How stress microenvironment is changing the epigenetic landscape of MSCs?How to modulate MSCs epigenetic landscape to improve their therapeutic properties?Mesenchymal stem cells  were first identified in the bone marrow by Alexander Friedenstein et al. in the late '60s , 2. TheyMSCs gained considerable interest due to their promising therapeutic potential in degenerative and inflammatory diseases. The lack of immunogenicity offers the possibilities of their applications both in autologous and allogeneic systems . NotablyAll cellular processes and functions depend on the expression of genes encoding proteins or regulatory molecules. In fact, gene expression is a fundamental process that enables the decoding of the DNA sequences into the final functional gene product (cellular proteins). Notably, DNA is tightly packed within the nucleus of eukaryotic cells in highly organized and compact structures, making the gene hardly accessible to transcription machinery. Nucleosomes represent the first level of DNA packing, formed by DNA wrapped around histone proteins. Histones are nucleosome core proteins providing structural support to form chromatin. Five types of histones were identified. The nucleosome is formed by core histones, namely H2A, H2B, H3, and H4, while H1 and H5 are involved in higher-order chromatin structures . Gene exEpigenetics refers to the heritable changes in gene expression and long-term alteration in the transcriptional potential at the chromatin level  Fig. 1)1). The f3) from S-adenosylmethionine (AdoMet/SAM) to cytosine in a dinucleotide sequence CpG . Approx in ref. . The can in ref. . At the  in ref. , 19. DNA in ref. , 21.Chromatin structure is dense and hardly accessible for enzymes involved in transcription processes. Therefore, histones undergo post-transcriptional modifications to control DNA accessibility. To date, four mechanisms of histone modifications are best described and functionally characterized, namely acetylation, methylation, ubiquitination, and phosphorylation .Histone acetylation is controlled by acetyltransferases (HATs) that transfer acetyl groups from acetyl co-enzyme A to lysine residues of the N-terminal H3 and H4 tails , 24. ConSimilarly, to DNA methylation, methyl groups can be transferred to amino acids of histone proteins by methyltransferases; however, this process is independent of DNA methylation. Histone methylations can both repress or activate transcriptional processes depending on the methylation region, and a number of modifications occurred . HistoneUbiquitination refers to modifying the epsilon amino group of lysine by attaching one (monoubiquitinylation) or more (polyubiquitination) ubiquitin monomers. Ubiquitin is a small protein found in the vast majority of human tissues. Similarly, to the processes mentioned above, ubiquitination occurs in lysine residues of the N-terminal histone tails. Histones H2A and H2B seem to be the most ubiquitinated proteins in the nucleus , 31. HowContrary to the abovementioned processes, histone phosphorylation occurs on serine, thyronine, and threonine. Their role in the regulation of transcription remains not fully elucidated, although a number of proteins with phosphor-binding domains recognizing phosphorylated histones have been characterized , 41. It Adenosine triphosphate (ATP)-dependent chromatin alternations are mediated by multi-subunit chromatin-remodeling complexes utilizing the energy from ATP hydrolysis. These ATP-dependent chromatin remodeling enzymes were classified within the RNA/DNA helicase 2 superfamily and, based on their ATPase domain sequence similarities, may be divided into four groups/subfamilies, namely chromodomain helicase DNA-binding (CHD), SWItch/Sucrose non-fermentable (SWI/SNF), imitation SWI/SNF (ISWI), and INO80 (reviewed extensively in ref. ). Their ncRNAs are widely present in the eukaryotic genome and in contrast to messenger RNA do not translate into proteins. They are classified into housekeeping or regulatory ncRNAs. Housekeeping ncRNAs include ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and telomerase RNA (TERC). They are involved in fundamental cellular processes, including mRNA translation , pre-mRNA splicing (snRNA), RNA modifications (snoRNA), and telomeric DNA synthesis (TERC). In contrast, regulatory ncRNAs are considered functional components of gene expression, which are usually classified into two subclasses, namely small ncRNAs including microRNA miRNA; 18-22 nt), small interfering RNA , piwi RNA , small cajal body-specific RNA , and long ncRNAs including long intergenic ncRNA , circular RNA , and natural antisense transcript . The importance and function of ncRNAs in cellular biology were extensively reviewed in ref. . In fact8-22 nt, As mentioned, MSCs present the plasticity to differentiate into mesodermal lineage cells, namely adipocytes, osteocytes, and chondrocytes. Nowadays, it has become clear that epigenetic reprogramming in response to the mediators regulating MSCs’ fate  plays a vital role during differentiation. Here we will summarize our understanding of epigenetic control of MSCs differentiation.The adipogenic differentiation of MSCs required changes in the epigenetic landscape, allowing activation of the specific transcription machinery inducing cellular plasticity and further adipocyte maturation. Differentiation toward adipocytes is usually considered a two-step process, namely determination (described as a commitment to the adipocyte lineage) followed by differentiation (adipogenesis) Fig. .Fig. 2EpCEBPA) and the peroxisome proliferator-activated receptor gamma  are recognized as master regulators of adipocyte differentiation that control both stages of the process. The PPARG expression is activated after a few hours of differentiation induction by CCAAT/enhancer-binding proteins beta and delta  binding to the PPARG2 promoter [The CCAAT/enhancer-binding protein alpha  residue of histone H3 (described as H3K9ac mark) and H3K27ac mark [CEBPA gene is initially blocked by HDAC1 activity, which is further downregulated upon accumulation of PPARγ through 26 S proteasome degradation, allowing C/EBPα expression [From the perspective of the epigenetic machinery, the opening of the 7ac mark . SWI/SNF7ac mark . Howeverpression , 58. Morpression . C/EBPα pression .th lysine (K4me2) residue of histone H3 (described as H3K4me2 mark) in the promoter regions of late adipogenic genes such as adiponectin (apM1), glucose transporter type 4 (glut4), g-patch domain protein 1 (gdp1), and leptin (lep) [PPARG expression [Committed cells remain undifferentiated and possess the phenotypic characteristic of MSCs. During the determination step, DNA demethylation in the promoter regions of genes critical for cell fate decision occurs. This process is accompanied by di-methylation of the 4in (lep) . Interesin (lep) , 63. Hisin (lep) , 65. Thepression . In addipression . Despitepression –70. Howepression .PPARG expression and, in consequence, block adipogenic differentiation [PPARG gene. In contrast, the noncanonical Wnt pathway members such as Wnt5a promote adipogenesis at the initial stage of differentiation [The differentiation process leads to the maturation of adipocytes and results in acquiring morphological and phenotypical characteristics as well as gene expression profiles of mature adipocytes. Importantly, once the adipogenic program is activated, MSCs lose their ability to differentiate towards other lineage cells and, therefore, are referred to as preadipocytes. Likewise, osteogenic differentiation inducers associated with the Wnt signaling pathway repress ntiation . The canntiation .Indian Hedgehog Signaling Molecule (Ihh), Gli Family Zinc Finger 1 (Gli1), and Patched 1 (Ptch1)), fibroblast growth factor , Wnt family member (Transcription Factor 7 (Tcf7), Wnt1 and Wnt10b), and parathyroid hormone-like hormone ) signaling genes [In contrast to adipogenesis, osteogenic differentiation is characterized as a three-step process starting with the proliferative phase, then matrix maturation, and mineralization Fig. . Runt-reng genes . In adding genes , 80. Howng genes , 82.Fig.Notably, transcription of both mentioned orchestrators of osteogenic differentiation undergoes epigenetic control. To date, described mechanisms are associated with changes in i) methylation at histone H3 residues; ii) acetylation at histone H3 and H4 residues; and iii) DNA methylation signatures , 84. AllHOXB7 increased expression influence ON, OCN, BSP, and COL1A2 gene expression and further RUNX2 [ALP expression increases through Runx2 influence [The process of bone formation decreases with aging and during inflammatory bone diseases. High levels of IL-1β or TNF pro-inflammatory cytokines suppress osteogenesis by BMP/Smad signaling inhibition , 104. Hoer RUNX2 . Importaer RUNX2 , 108. Fuer RUNX2 . Howeverer RUNX2 . Moreovenfluence .MSCs differentiation toward chondrocytes strictly depends on cellular density  and growth factors stimulation (tumor growth factor beta (TGFβ) family members are well established in this process, namely TGFβ1, TGFβ2, and TGFβ3) Fig.  112, 11.Fig. 4EpSOX9) is a key transcription factor in early chondrogenesis [COL2A1, COL9A1, COL11A2, and Aggrecan (ACAN)) [. SOX9 expression is directly regulated by miR-145 and miR-495, and indirectly by miR-574-3p inhibiting retinoid x receptor alpha (RXRα) an inhibitor of chondrogenesis [Col2a1 promoter, upregulate Sox9 expression during chondrocyte differentiation and lead to increased expression of Col2a1, Col11a2, and ACAN [Col2a1 gene promoter [Col2a1, a major component of cartilage surrounding extracellular matrix, by acetylation of histones H3 and H4 in the enhancer regions [Chondrogenesis commences with mesenchymal condensations (morphogenetic transformation mediated by cell adhesion), chondrocyte differentiation, and maturation. SRY-box containing gene 9 (ogenesis . Togethe (ACAN)) . SOX9 exogenesis –119. Morogenesis . Additioand ACAN . AT-richpromoter . Importapromoter , 124. Inpromoter , 126. p3 regions . Similar regions .Col2a1, Col9a1, and Acan, as well as cartilage oligomeric matrix protein (a non-collagenous cartilage matrix protein) [Runx2 expression, thus significantly reducing chondrocyte differentiation and hypertrophy [COL2A1 gene [On the other hand, HDAC1- and HDAC2-mediated histone deacetylation significantly lowers the expression of extracellular matrix genes, including protein) –131. HDAertrophy . In addi2A1 gene .Runx2 expression. This repression mark may be erased by the involvement of AT-rich interactive domain 5b (Arid5b) recruited histone demethylase Phf2 to promote chondrogenesis [Unfortunately, our understanding of histone methylation in this process remains elusive. Similarly, our understanding of the role of ncRNAs in the regulation of epigenetic machinery during MSCs chondrogenic differentiation needs significantly more attention in the future. Some reports indicate an important role of H3K4me3 and H3K36me3 active marks , 135. Moogenesis .MSCs acquire immunosuppressive functions in a proinflammatory microenvironment  , 138, hyThe MSCs stimulation with pro-inflammatory cytokines leads to an increase in indoleamine 2,3-dioxygenase (IDO) levels, high IL-10 and prostaglandin E2 (PGE2) secretion, and induction of immunoinhibitory checkpoint (coinhibitory) molecule expression; all of these activities are recognized as hallmarks of their immunomodulatory functions , 155. InCXCR2, CXCR4, and CXCR6 promoters, which enhances MSCs migration potential in response to IL-8, C-X-C Motif Chemokine Ligand (CXCL1), and SDF-1 gradient [IDO and IL10. These licensed MSCs exerted a regulatory effect on Th1 and Th17 cell differentiation and significantly decreased IFNγ, IL-2, and IL-17 levels [COX2 and PTGES, and migration factors CXCR2 and CXCR4, subsequently contributing to the increased PGE2 production by MSC and suppress lymphocyte proliferation [5-aza is a chemical analog of cytidine, which possess hypomethylating activity and direct cytotoxicity in higher doses. In clinical practice, it is used to treat some types of malignancies, such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) . In MSCsgradient . The com7 levels . Microarferation .via the increase in IDO expression [Cyclooxygenase (COX2), IL-4, Hepatocyte Growth Factor (HGF), TNF-stimulated gene 6 (TSG-6), C-C Motif Chemokine Ligand 5 , C-X-C Motif Chemokine Ligand ) [DKK1 promoter hypermethylation and a decrease of DKK1 expression, leading to the activation of the Wnt/b-catenin signaling pathway and modulation of Fas ligand (FasL) expression. Consequently, MSCs acquire the ability to induce T cell apoptosis and increase the frequency of Tregs [Deng L. et al. recently proposed chlorzoxazone for improving MSCs immunosuppressive properties in rat acute kidney injury model. Chlorzoxazone is a benzoxazole derivative used in clinical practice as a muscle-relaxing drug. Chlorzoxazone, in contrast to IFNγ stimulation, does not induce changes in the biological characteristic of MSCs and their MHC I and MHC II expression while similarly inhibiting the T cell activation and proliferation pression . Moreove(IP-10)) . At the (IP-10)) . Adminis(IP-10)) . In addiof Tregs .Ido1 gene seems to be associated with a combination of hypomethylating agents and histone deacetylation inhibitors (HDACIs) [Similarly, to the above-mentioned drugs, stimulation of MSCs with IFNγ causes an increase in IDO enzyme level, resulting in the accumulation of toxic tryptophan metabolites that in turn, hamper T cell proliferation . The exp(HDACIs) . The inc(HDACIs) .Taking together, differentially sourced MSCs may vary in their immunosuppressive properties, and distinct strategies for empowering therapy should be assessed with a focus on the changes in biological characteristics, immune privilege, and functionality of MSCs –172. TheThe approaches utilizing infusion or transfer of MSCs into patients have begun to develop in the 90’s of the last century. Over the past decades, the advances in MSCs administration procedures and safety strategies allowed for registering more than 950 clinical trials involving ~10,000 treated patients . The conTherefore, further efforts should be focused on enhancing the stability and effectiveness of MSCs transfer regardless of various conditions affecting the replicability of individual transplants. Given the complexity and diversity of the isolation procedures and expansion methods of MSCs, along with donor diversity and the heterogeneity in the inflammatory profiles of the recipient microenvironment, the achievement of the desired therapeutic outcome is very difficult. The advancement of two strategies might be useful in order to minimize currently defined issues, namely: (1) optimization of the MSC-related effects in controlled in vitro production conditions containing the standardized procedures using effective stabilizing/enhancing agents ; or (2) prediction suited properties and modification the MSC for personalized clinical indication (extensively reviewed in ref. ).Recently developed methods, including single-cell sequencing (with multi-omics approaches) and further exploring of next-generation technologies, may progress our understanding of MSCs heterogeneity being the result of donors’ diversity, culturing conditions , and isolation tissue sources . Those aThe improvement and substantial progress in the development of stem cell-based therapies require extensive research in the field of epigenetic machinery driving immunomodulatory activities and the differentiation potential of MSC. It is tempting to speculate that a better understanding of the role of epigenetic machinery and dynamic changes of the epigenetic landscape during fate decision, differentiation, and induction of immunomodulatory activities will allow us to better control the stability and enhance the therapeutic potential of MSCs.


The mammalian spermatozoon has a unique chromatin structure in which the majority of histones are replaced by protamines during spermatogenesis and a small fraction of nucleosomes are retained at specific locations of the genome. The sperm’s chromatin structure remains unresolved in most animal species, including the pig. However, mapping the genomic locations of retained nucleosomes in sperm could help understanding the molecular basis of both sperm development and function as well as embryo development. This information could then be useful to identify molecular markers for sperm quality and fertility traits. Here, micrococcal nuclease digestion coupled with high throughput sequencing was performed on pig sperm to map the genomic location of mono- and sub-nucleosomal chromatin fractions in relation to a set of diverse functional elements of the genome, some of which were related to semen quality and early embryogenesis. In particular, the investigated elements were promoters, the different sections of the gene body, coding and non-coding RNAs present in the pig sperm, potential transcription factor binding sites, genomic regions associated to semen quality traits and repeat elements. The analysis yielded 25,293 and 4,239 peaks in the mono- and sub-nucleosomal fractions, covering 0.3% and 0.02% of the porcine genome, respectively. A cross-species comparison revealed positional conservation of the nucleosome retention in sperm between the pig data and a human dataset that found nucleosome enrichment in genomic regions of importance in development. Both gene ontology analysis of the genes mapping nearby the mono-nucleosomal peaks and the identification of putative transcription factor binding motifs within the mono- and the sub- nucleosomal peaks showed enrichment for processes related to sperm function and embryo development. There was significant motif enrichment for Znf263, which in humans was suggested to be a key regulator of genes with paternal preferential expression during early embryogenesis. Moreover, enriched positional intersection was found in the genome between the mono-nucleosomal peaks and both the RNAs present in pig sperm and the RNAs related to sperm quality. There was no co-location between GWAS hits for semen quality in swine and the nucleosomal sites. Finally, the data evidenced depletion of mono-nucleosomes in long interspersed nuclear elements and enrichment of sub-nucleosomes in short interspersed repeat elements.These results suggest that retained nucleosomes in sperm could both mark regulatory elements or genes expressed during spermatogenesis linked to semen quality and fertility and act as transcriptional guides during early embryogenesis. The results of this study support the undertaking of ambitious research using a larger number of samples to robustly assess the positional relationship between histone retention in sperm and the reproductive ability of boars. Current research is showing evidences that, in addition to the paternal genome, sperm also carries other molecular information to the zygote. This includes a wide repertoire of RNAs, proteins and epigenetic marks in the form of DNA methylation, retained nucleosomes and histone modifications that can play a role in spermatogenesis, fertility, early embryo development and even the transmission of inter-generational information . During Nucleosome positioning in the genome and chromatin accessibility is critical in the regulation of gene expression and the alteration of this epigenomic architecture has been linked to multiple phenotypes in a range of tissues and cell types . NucleosSus scrofa), research has been carried to study the sperm RNA  and sub-nucleosomal (SN) chromatin fractions were characterized and their correlation with sperm RNA levels and co-location with genomic regions associated to semen traits was assessed.TM  to remove potentially present somatic cells and processed as described in TM diluted to a final ratio of 60% (v/v) with BoviDiluteTM  and centrifuged at 300 × g for 20 min at 20 °C with slow acceleration and deceleration rates. The resulting cell pellet was washed with PBS and resuspended in 1 mL of RNase-free PBS and further pelleted by centrifugation. The pellets were stored at −80 °C until further use.Two ejaculates, each from a different healthy Pietrain boar, MN_1 (16 months of age) and MN_2 (9 months old), from two artificial insemination centres were obtained by specialists during their routine sample collection using the gloved hand method  and were2 was added to a final concentration of 0.6 mM. At this time, 5 U of MNase (Sigma-Aldrich) were added and the samples were incubated for 7 min at 37 °C. Digestion was stopped by the addition of ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) to a concentration of 5 mM. The resulting digested fraction corresponding to nucleosome-bound chromatin (soluble) was separated from the undigested protamine-bound fraction (pellet) by centrifugation at 20,000 rpm for 10 min. An aliquot of the digested DNA fraction (which included both the MN and SN fractions) was treated with sodium dodecyl sulfate (SDS) and proteinase K and evaluated in a 1.5% agarose 1x tris-acetate-EDTA (TAE) gel electrophoresis. Another aliquot of the nuclease digested DNA was used to extract DNA with a phenol-chloroform based protocol and was directly used for library prep after purification with the Agencourt AMPure XP beads . The MN and SN fractions were separated after sequencing and read mapping, using bioinformatics tools as described below. Purified DNA was subjected to quality control including quantification with the QubitTM DNA HS Assay kit  and Nanodrop  as well as size and concentration assessment with a 2100 Bioanalyzer and the Agilent DNA 1000 Kit . gDNA from the two sperm samples was also extracted as done by Hammoud and colleagues . Then, 1.6 µl RNase A-20 mg/mL -, 1.6 µl of 1 M DTT and 6.5 µl of 5M NaCl was added to each sample. This cell suspension was kept at 37 °C for 30 min. The cells were then dispersed with gentle vertexing and subsequently, 40 µl of 10% SDS and 20 µl of Proteinase K (20 mg/µl) were added. Samples were then incubated at 50 °C for 60 min. Following this step, 1 mL of 100% Isopropanol was added and the tubes were inverted several times until a gel-like DNA precipitate appeared. Then, 0.6 mL of NaI solution was added , and the tubes were inverted until DNA formed a white precipitate. The DNA pellet was washed three times with 70% EtOH with a final wash with 100% EtOH. The DNA pellet was solubilized with 300 µl of H2O. The two resulting gDNA samples were pooled to be used as input. Pooled gDNA were sheared to obtain 100 bp long fragments using a Covaris S2 instrument , according to the manufacturer’s instructions. Sequencing libraries of the two MNase treated samples and the input gDNAs were prepared with the PrepX™ DNA Library Kit . The libraries were used as a template to generate 50 bp paired end (PE) reads in an Illumina HiSeq2500 system.Chromatin digestion was performed as previously described  with minlleagues . Brieflyhttp://picard.sourceforge.net). Finally, for both fractions, peak calling was performed with MACS2 v.2.1.0  with Trimmomatic v.0.36 . Trimmed v.2.1.0  with “-q v.2.1.0  computeMPeaks were categorized by their position in relation to gene features as annotated in Ensembl (v96) using BEDtools v.2.29.2  intersecP-value < 0.001 and FC ≥ 1.5 or FC ≤ 0.66 were classified as enriched or depleted, respectively.In order to determine any potential positional preference or avoidance of the peaks within the gene features, 1,000 iterative permutations of the peak genomic locations with the same number of peaks following the same peak width distribution but with randomized genomic locations, were carried using BEDtools  shuffle.P-values were considered.The genes mapping within or less than 5 kbp apart from the identified peaks were extracted from Ensembl v96 with BEDtools  closestBMotif enrichment analysis was carried out with the software HOMER v.4.10.0  findMotiP-value < 0.001 and FC ≥ 1.5 or FC ≤ 0.66, was set to determine enrichment and depletion, respectively.A comparable human sperm MNase-Seq dataset that sequenced the mono-nucleosomal fraction (GSE15690) from The same approach was used for two cattle (GSE47843) comparable MNase-Seq datasets that sequenced the mono-nucleosomal fractions from two bulls (samples s_1_1_bovine and s_5_1_bovine) from the Holstein breed . Genome PRJNA520978) was used. These datasets provided a list of 4,120 protein coding RNAs  or FC ≤ 0.66 (depletion), respectively, was set to consider co-location enrichment or depletion.The positional enrichment of circRNAs or piRNAs at MNase peaks was also studied. First, the genomic regions of the MN and SN peaks were iteratively randomized 1,000 times using BEDtools  shuffle.P-value < 0.001 and FC = 1.5 (enrichment) or FC = 0.66 (depletion), respectively, was set to consider co-location enrichment or depletion.MNase profiles were also evaluated against the genomic regions showing genetic association with sperm quality traits in Pietrain pigs. These associations were identified through a Genome-Wide Association Study (GWAS) conducted by our group, which included the two samples used in this study . This GWP-value < 0.001; FC ≥ 1.5) or depletion  of MN and SN peaks at repeat elements.The list of repeat elements in the pig genome from RepeatMasker was downloaded from the UCSC genome browser. The same approach as described for the other genomic features was followed to evaluate enrichment or depletion of MNase peaks at the whole set of repeat elements, long interspersed nuclear elements (LINEs) or short interspersed nuclear elements (SINEs). The genomic locations of the MN and the SN peaks were randomized 1,000 times and the overlap with the different sets of repeat elements was assessed with BEDtools v.2.29.2  intersechttps://doi.org/10.6084/m9.figshare.21997523.v1.All scripts, including R, bash and software parameters used are available on Figshare: The MNase protocol yielded 95.2 and 75.6 ng of DNA for sample 1 and sample 2, respectively. The extraction of gDNA provided 1227.6 ng/ul. The MNase treatment of the pig sperm samples generated ~147 bp fragments corresponding to the MN fraction and an additional ~100 bp fragment corresponding to the SN fraction .The sequencing of the libraries yielded more than 43 million PE reads for each MNase biological replicate and 41.8 million PE reads for the input sample . In averP-value < 0.001, FC = 5.05), TSS  , coding sequences  and 3′-UTR    , 5′-UTR  = 1.56) . This re = 0.47) . A total = 0.47) . The two = 0.47) , which cetc.) and cell and organ morphogenesis (etc.) and other TFs related to embryo development and implantation . They also included TFs related to spermatogenesis or sperm function which have also been related to embryo development such as CUX1, CTCFL, PAX5 and Smad2. Finally, this list also contained Znf263 and FOXA1, two TFs that provide a direct link between the paternal gamete and the embryo , compared to randomization  within or near the MN peaks when compared to the catalog of absent genes , moderate  and highly abundant  genes, when compared to the set of absent genes  and SN  peaks was significantly higher than the average abundance of all the genes annotated in the pig genome  and exhibited an overlap of 65 MN peaks  for the set of piRNA genomic regions involving 1,355 piRNAs , while the SN sites presented enrichment at SINEs   .Xenopus laevis sperm  and reduced amounts of H2A and H2B thereby leading to the hypothesis that SN are partially unwrapped nucleosomes that have lost one or the two H2A and H2B dimers  could be indicating that the SN fraction identified in our study may correspond to partially unwrapped nucleosomes generated during spermiogenesis or by the MNase digestion protocol.The extraction of nucleosomal DNA was less efficient than in experiments carried in sperm from other species , and it The nucleosome-associated DNA spanned ~0.3% of the porcine sperm’s genome, which indicates a nearly fifteen, three and 45 -fold decrease in nucleosome retention when compared to what has been described in human , mouse , and catAn increasing body of evidence in other animal species points towards a programmatic nucleosome retention in the sperm genome . In our The concordance with previous findings in other species regarding the predominant location of nucleosome peaks in gene promoters  and genee.g., SOD2; , capaci, TRPV1; ) and cheg., CA6; ) and celg., CA6; .ATF4 , of genes exhibiting paternal preferential expression in the human early embryo that correspond to pig orthologs mapping near a MN peak with predicted ZNF263 binding motif in sperm , a largIn light of these results, and considering that nucleosome positioning can modulate gene expression , it was e.g., promoters, enhancers or genes) that could be overlapping with nucleosomes. Regarding the circRNAs, although the FC did not reach our threshold, none of the random permutations reached the same number of overlaps between MN peaks and the catalog of circRNAs indicating that, although modest, there might be a trend of co-location between circRNAs and MN peaks  (~ 147 bp) and one sub-nucleosomal (SN) (<100 bp) bands. Left: 100 bp DNA ladder; Right: ~ 300 ng of MNase digested sperm chromatin. b. Bioanalyzer electropherogram profile of a MNase digested sample showing the SN and MN DNA fractions. x-axis: bp fragment length; y-axis: FU  of the DNA fragments.Click here for additional data file.10.7717/peerj.15520/supp-2Supplemental Information 2Scatterplots showing the Pearson’s correlation of the normalized MNase-Seq signals between the two biological replicates.Click here for additional data file.10.7717/peerj.15520/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.15520/supp-4Supplemental Information 4P-value) and fold enrichment.For each peak, the list shows, chromosome, start position, end position, width, −og10 and fold enrichment.For each peak, data includes chromosome, start position, end position, width, −log10 and their associated genes.GO biological process terms with significant Bonferroni corrected Click here for additional data file.10.7717/peerj.15520/supp-8Supplemental Information 8P-values  and their associated genes.GO biological process terms with significant Bonferroni corrected Click here for additional data file.10.7717/peerj.15520/supp-9Supplemental Information 9P-value, percentage of targets sequences with motif, comments on the cognate transcription factor. Comments on the cognate transcription factor contains comments for these transcription factors with direct relevant functions in spermatogenesis or embryo development mostly obtained from searches in the GeneCards® database (https://www.genecards.org) and PubMed. Some of these binding motifs are from studies in Arabidopsis, yeast, Drosophila or C. elegans and were not included in the searches.Motif name, Click here for additional data file.10.7717/peerj.15520/supp-10Supplemental Information 10The list of human orthologous genes showing paternal preferential expression in human early embryos was obtained from the article “Single-Cell Transcriptome Analysis of Uniparental Embryos Reveals Parent-of-Origin Effects on Human Preimplantation Development” .Cells in clear green correspond to data from the parent-of-origin RNA expression in human embryo . Cells in clear grey indicate data from our study on genes mapping less than 500 bp away from MN peaks with a predicted Znf263 binding motif.Click here for additional data file.10.7717/peerj.15520/supp-11Supplemental Information 11P-value, percentage of targets sequences with motif, comments on the cognate transcription factor. Comments on the cognate transcription factor contains comments for these transcription factors with direct relevant functions in spermatogenesis or embryo development mostly obtained from searches in the GeneCards® database (https://www.genecards.org) and PubMed. Some of these binding motifs are from studies in Arabidopsis and were not included in the searches.Motif name, Click here for additional data file.


Understanding their individual contributions to key developmental processes and identification of their genome-wide transcriptional targets has been hampered by a number of factors including their essential roles during embryonic development, and co-expression in several tissues. siRNAs were designed to target isoform-specific exons that code for the unique N-terminal region of either PntP1 or PntP2. The efficacy and specificity of the siRNAs were examined by co-transfection of isoform specific siRNAs with plasmids encoding epitope tagged PntP1 or PntP2 inDrosophila S2 cells. All P1-specific siRNAs were demonstrated to knockdown PntP1 protein level to greater than 95%, while having nominal impact on PntP2 level. Similarly, PntP2 siRNAs while ineffective at eliminating PntP1, were shown to reduce PntP2 protein level by 87-99%.Alternate splicing of the As a result, PntP1 and PntP2 have unique N-terminal regions. However, the isoforms possess identical C-terminal regions including the ETS DNA binding domain, and have been proposed to activate the same target genes. PntP1 does not require MAPK dependent phosphorylation and is a constitutive transcriptional activator, whereas, PntP2, like its vertebrate orthologs ETS1 and ETS2, must be phosphorylated by MAPK to be fully functional .Pointed (Pnt), an ETS family transcription factor, is a nuclear effector of the Receptor Tyrosine Kinase (RTK)/Ras/Mitogen-Activated Protein Kinase (MAPK) signaling pathway . Pnt function is critical for the differentiation of several cell types during embryonic and post-embryonic development . Transcription from alternate promoters and differential splicing produce two major isoforms ofin vivo has not been extensively investigated. This issue could be addressed using RNA interference (RNAi) to knockdown individual Pnt isoforms followed by RNA-seq analysis to identify genome-wide transcriptional targets. Such knockdown could be accomplished in the tissue of interest by crossing transgenic RNAi lines with tissue-specific GAL4 drivers, thereby circumventing the lethality exhibited by loss-of-function null alleles. RNAi lines targeting Pnt have been generated by the Transgenic RNAi Project (TRiP) and the Vienna Drosophila Resource Center (VDRC)Since Pnt is a key transcription factor that mediates the effects of the RTK signaling pathway in several developmental tissues, understanding its function in post-embryonic development has necessitated the generation of mitotic clones. While PntP1 and PntP2 are predicted to regulate the expression levels of the same transcriptional targets, whether they do soDrosophila S2 cells were transfected with either Myc-tagged PntP2 (MycPntP2) or Flag-tagged PntP1 (FlagPntP1) expressing plasmids. MycPntP2 was expressed in S2 cells transfected with the PntP2 expressing plasmid but not in untransfected control cells  used long dsRNAs that were designed to target mutually exclusive exon 4 variants to perform the isoform-specific knockdown ofDscammRNA inDrosophilacultured cellsEcdysone Receptor(EcR), or theDscam gene, during larval stagesEcR gene codes for three isoforms, EcR-A, EcR-B1 and EcR-B2, which have unique N-terminal domains. Accordingly, the researchers designed long dsRNA to target the unique N-terminal exon of either EcR-A or EcR-B1 to perform isoform-specific knockdown of EcR, without affecting the expression levels of the untargeted isoformEukaryotic genomes have been shown to employ alternate splicing to increase the protein encoding capacity. Additionally, the co-expression of multiple protein isoforms can make it challenging to elucidate their individual contributions to the regulation of key developmental processes. While there have been several papers documenting the use of RNAi for isoform-specific knockdown in mammalian cellsDrosophilacultured cells. Recently, the role of a novel Pnt isoform, calledpntP3was uncovered during photoreceptor specificationpntP3is encoded by an N-terminal exon that is unique from that of eitherpntP1orpntP2.pntP3however shares the rest of the exons withpntP2including the exons that code for the SAM and the ETS domains. The isoform-specific N-terminal region of PntP1 is specified by a single exon, exon 1, while the N-terminal region of PntP2 is produced from 5 different exons, exons I-V, of which exons III-V are shared with PntP3 . All 4 PntP1-specific siRNAs target exon 1 of PntP1, while the PntP2-specific siRNAs target exon I, III, and V. P2 siRNA #3 therefore uniquely targets the PntP2 isoform, while P2 siRNA #1 and #2 target both PntP2 and PntP3. Given PntP1 and PntP2 are co-expressed in certain tissues such as the intestinal stem cells, somatic follicle cells and the eyeDrosophila eye development, PntP3 was demonstrated to function redundantly with PntP2 for the specification of a subset of photoreceptors, and to activate PntP1In this study, I have demonstrated that siRNAs targeting unique sequences can be used to perform isoform-specific knockdown of PntP1 or PntP2 inCell Linehttps://dgrc.bio.indiana.edu//stock/181; RRID:CVCL_Z992) and cultured in Schneider's medium supplemented with 10% FBS and Penicillin/Streptomycin at 25°C.The S2-DRSC cell line was purchased from the Drosophila Genome Resource Center  or pAFW-PntP1 , and Flag (D6W5B) tagged Pnt were purchased from Cell Signaling Technology, while the anti-Tubulin antibody was purchased from DSHB (12G10). The nitrocellulose membrane was incubated overnight at 4°C with the relevant primary antibodies. All primary antibodies were used at 1:1000. The HRP conjugated anti-rabbit and anti-mouse antibodies were purchased from Jackson ImmunoResearch and used at 1:5000. Image J was used to quantify the expression levels of the PntP1 or PntP2 bands with respect to Tubulin and expressed as a percentage. Statistical significance was determined by one-way ANOVA.


The sixth Japanese meeting on host–transposon interactions, titled “Biological Function and Evolution through Interactions between Hosts and Transposable Elements,” was held on August 24th and 25th, 2023, at the National Institute of Genetics as well as online. This meeting was supported by the National Institute of Genetics and aimed to bring together researchers studying the diverse roles of TEs in genome function and evolution, as well as host defense systems against TE mobility, TE bursts during evolution, and intron mobility in mammals, insects, land plants, yeast, protozoa, and bacteria. Here, we have presented the highlights of the discussion.Organizers: Kenji Ichiyanagi, Yoko Ikeda, and Kuniaki Saito. Transposable elements (TEs) constitute a significant proportion of the eukaryotic genome and play key biological and evolutionary roles. The interactions between TEs and their hosts were discussed in a series of meetings titled “Biological Function and Evolution through Interactions between Hosts and Transposable Elements” –3. This RdDM-independent CH methylation Establishment (RiCHE) [Arabidopsis. Yoko Ikeda  presented a DNA methylation regulatory mechanism in the liverwort, Marchantia Polymorpha, which is a member of a basal land plant lineage. Flowering plants have evolved a system for maintaining stable CpG methylation in the next generation. In contrast to that in mammals, active DNA demethylation does not occur in plant germ cell lines. However, in Marchantia, genome-wide changes in both CpG and non-CpG methylation were observed, suggesting the presence of different regulatory mechanisms for DNA methylation. MpMET, an ortholog of the maintenance CpG methylation enzyme, and two copies of orthologs of DNA demethylation enzymes were found in the Marchantia genome [Arabidopsis thaliana. Although this variation is often referred to as the epigenome and is speculated to result from past environmental effects, Dr. Sasaki and her colleagues have demonstrated that a significant portion of this variation is influenced by genetic variation in trans modifiers, especially epigenetic regulators including CHROMOMETHYLASE2 (CMT2), CMT3, and NRPE1, the largest subunit of RNA polymerase V [JMJ26 specifically controls CHG methylation in RdDM-targeted TEs. These alleles displayed a geographic cline and were strikingly associated with the regional climate, suggesting a form of local adaptation. Although the specific nature of this adaptation remains unclear, the results of population genetics provide evidence that combinations of trans-modifiers influence the mobilization of TEs by controlling DNA methylation levels under natural conditions.Epigenetic regulation involving DNA methylation, histone modification, and small RNA pathways is a pivotal mechanism for silencing the transcription and mobility of TEs in eukaryotes. Taiko Kim To  introduced recent findings on a novel de novo DNA methylation pathway named  (RiCHE) . In many (RiCHE) , 6, india genome . Based omerase V . She alsSaccharomyces cerevisiae, a long terminal repeat (LTR) retrotransposon named Ty1 exhibits an interesting characteristic regarding the regulation of transcription. Hiroshi Masumoto  demonstrated the silencing effect of Ty1, which has a sequence that can silence a transgene inserted between gag and pol [In budding yeast,  and pol . Dr. Mastorimochi” in BmN4 cells from silkworm ovaries [torimochi is a complete transposon with LTRs. Its transposition activity in BmN4 cells, assessed using a nanopore sequencer, was the highest among all the TEs. When the cells were in vitro differentiated into non-gonadal cells , the expression of torimochi decreased, whereas other TEs were derepressed. Taken together, they proposed that torimochi, an unfragmented active transposon, integrates with a BmN4-specific activation mechanism and serves as a model for nascent piRNA cluster formation [In animals, PIWI-interacting RNAs (piRNAs) play an important role in TE silencing in germ and gonadal somatic cells. These RNAs are generated from precursor transcripts. However, the evolutionary origin of piRNA source loci, called piRNA clusters, which are dispersed over TE-accumulated regions in the genome, remains elusive. Keisuke Shoji  presented a model for the origin of piRNA clusters in insects. His colleagues first identified a piRNA cluster named “ ovaries . Using aormation .S. cerevisiae and introduces mutations via retrotransposition. Retrotransposition intermediates are found in virus-like particles (VLPs), which are localized in the cytoplasm. Interestingly, Atg19-dependent selective autophagy eliminates Ty1 VLPs from the cytoplasm under starvation. In the absence of Atg19, the Ty1 transposition frequency continually increased during starvation [TE activity is restricted by mechanisms other than epigenetic regulation. Kuninori Suzuki  demonstrated that selective autophagy downregulates Ty1 retrotransposition in budding yeasts. Autophagy is induced under starvation conditions and plays a cytoprotective role by degrading unwanted cytoplasmic components. Ty1 is the most abundant retrotransposon in arvation . This suTetrahymena thermophila possesses two distinct nuclei within a single cell: the transcriptionally inactive germline micronucleus (MIC) and the transcriptionally active somatic macronucleus (MAC). During sexual reproduction, the MIC differentiates into the MAC of sexual progeny. In this process, more than 12,000 loci containing TEs and their remnants are heterochromatinized de novo and eventually eliminated from the MAC genome. Kensuke Kataoka  studied the dynamics of heterochromatin and its effects on DNA elimination [Tetrahymena.Binuclear protozoans, ciliates, whose lineage is evolutionarily distant from animals or plants, also use epigenetic marks to transcriptionally silence TEs; however, they also eliminate the DNA sequences of marked TEs from their active genome. The ciliate mination , 14. KatV. marina and V. riukiuensis, that have adapted to coastal environments and acquired salt tolerance. The scaffolds obtained showed that the overall structure of the genome, including gene order, was highly conserved [V. marina and V. riukiuensis have accumulated many copies, with V. riukiuensis having more than 1000 copies, of WUSCHEL-related homeobox (WOX) gene family. The amplified WOX genes in the two species were mostly surrounded by identical LTR sequences at both ends, with typical target site duplications (TSDs). Although the contribution of WOX gene amplification to coastal adaptation remains unknown, their study revealed that LTR retrotransposons can burst rapidly and have the potential to affect gene content in certain species.In some species, certain TEs are amplified rapidly (TE burst), which provides their hosts with an opportunity to significantly change their phenotype. The genus Vigna is a reservoir of diversity consisting of more than 100 species, many of which have adapted to harsh environments, including coastal, desert, marsh, and limestone karst . Given tonserved . HoweverLotus japonicus. They found recent retrotransposition events of several LTR retrotransposons in three recombinant inbred-line populations (RILs) of L. japonicus [Eigo Fukai  and his colleagues have been working on TEs in the model legume aponicus . These LTpn1 family transposons (Tpn) belonging to the CACTA superfamily in Japanese Morning Glory (Ipomoea nil). Transposition of the Tpn1 family frequently accounts for most mutations affecting the phenotype. However, most genomic Tpn copies are nonautonomous elements that require a transposase encoded by an autonomous element for transposition [TpnA2, one of the autonomous elements, is present in only one copy in Asian strains including Japan, and maintains the transposase gene stably due to deletion of the 3′ terminal region [TpnA2 is a major transposase source. In Japan, numerous mutants have been recorded since the middle of the eighteenth century, suggesting that such mutations are caused by the activation of Tpn transposition. Analysis of the genomic sequences of 140 Japanese strains revealed that, in hypermutable strains, the number of Tpn copies has increased. Most of these strains share mutations caused by Tpn transposition in two flower color genes, suggesting that they were derived from a single plant with increased Tpn transposition activity. Taking advantage of the homogeneity of the genome, Mizunaru et al. identified candidate genes for Tpn activation in Japanese strains by comparing the genome data between two groups showing activated and silenced Tpn transpositions.Yuki Mizunaru  discussed the transposition of position . TpnA2, l region . Genetichttp://geve.med.u-tokai.ac.jp) of 19 mammalian genomes [Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus). RTOM genes encode reverse transcriptases and are expressed specifically in the testes of both the platypus and echidna. Such testis-specific reverse transcriptase genes have not been found in therian mammals, but their involvement in reproductive functions unique to monotremes has been previously discussed [Drosophila ovarian somatic cell line OSC, in which Piwi-piRNA-dependent TE silencing occurs. Although the expression of ERV-derived proteins in the ovary has been reported previously [TE sequences are known sources of novel protein-coding genes. So Nakagawa  has discussed the diversity and evolution of de novo protein-coding genes derived from endogenous virus-like elements (EVEs), including endogenous retroviruses (ERVs). Several protein-coding EVEs, primarily ERVs, are present in the genomes of various mammalian species. Most of these encoded proteins are truncated owing to nonsense and/or frameshift mutations and are epigenetically silenced. However, some are under purifying selection, suggesting that they provide new functions to their hosts, such as cell-cell fusion activity involved in placental development . However genomes . Various genomes . Koichi iscussed . He alsoeviously , their fcis-regulatory elements that have altered gene expression during evolution. Many LTR retrotransposons in the human genome are primate-specific and introduce novel cis-regulatory elements into the lineage. While the transcriptional activity of LTRs is restricted by the host through various mechanisms, the nucleotide changes in LTRs associated with their activity and expansion during primate evolution remain largely unknown. Fumitaka Inoue  et al. found that the MER11 family is one of the youngest simian-specific ERVs and is highly enriched with active epigenetic marks in human pluripotent stem cells. They analyzed the phylogenetic relationship between the MER11A/B/C subfamilies and classified them into multiple phyletic groups, which resolved the sequence and epigenetic heterogeneity of the MER11 subfamilies. To understand the functional divergence of MER11 sequences, massively parallel reporter assays (MPRAs) were used to systematically analyze MER11 sequence variants in human, chimpanzee, and macaque genomes to determine their regulatory activities [TEs also provide tivities , 27. TheCaldivirga maquilingensis, they found that tRNAGly(TCC) and tRNAGly(GCC) consist of three separate transcripts, a phenomenon termed “tri-split tRNA” [In Archaea, some tRNA genes have introns and some tRNA genes are split. Recently, an association between some intron-containing tRNA genes and TEs was reported. Akio Kanai  et al. discovered disrupted tRNA genes in various species by computational analysis of their complete genomes. Notably, within the hyperthermoacidophilic archaeon it tRNA” . Interesit tRNA” . In thisit tRNA” , which sIn preimplantation development in mice, the LTR retrotransposon MERVL is transiently expressed during zygotic gene activation (ZGA) at the 2-cell stage, which is essential for blastocyst formation . In embrIn contrast to the expression analysis of long interspersed elements (LINEs) and LTR retrotransposons, the expression of short interspersed elements (SINEs), which are transcribed by RNA Pol III and are typically < 500-bp long, is difficult to analyze by deep sequencing because many copies reside within other transcripts such as mRNAs. Kenji Ichiyanagi  recently established a deep sequencing method, melRNA-seq, to determine the RNA abundance, RNA length, and source loci of SINEs , 33. The


The promyelocytic leukemia (PML) protein organizes nuclear aggregates known as PML nuclear bodies (PML-NBs), where many transcription factors localize to be regulated. In addition, associations of PML and PML-NBs with chromatin are described in various cell types, further implicating PML in transcriptional regulation. However, a complete understanding of the functional consequences of PML association to DNA in cellular contexts where it promotes relevant phenotypes is still lacking. We examined PML chromatin association in triple-negative breast cancer (TNBC) cell lines, where it exerts important oncogenic functions. We find that PML associates discontinuously with large heterochromatic PML-associated domains (PADs) that contain discrete gene-rich euchromatic sub-domains locally depleted of PML. PML promotes heterochromatic organization in PADs and expression of pro-metastatic genes embedded in these sub-domains. Importantly, this occurs outside PML-NBs, suggesting that nucleoplasmic PML exerts a relevant gene regulatory function. We also find that PML plays indirect regulatory roles in TNBC cells by promoting the expression of pro-metastatic genes outside PADs. Our findings suggest that PML is an important transcriptional regulator of pro-oncogenic metagenes in TNBC cells, via transcriptional regulation and epigenetic organization of heterochromatin domains that embed regions of local transcriptional activity.  The PML (promyelocytic leukemia) nuclear protein provides a molecular scaffold for the nucleation of insoluble biomolecular condensates named PML nuclear bodies (PML-NBs). PML-NBs are generally described as platforms for the regulation of proteins therein associated upon cellular stress ,2. HowevThe molecular functions of PML are challenging to delineate due to a lack of enzymatic activities and well-defined functional domains and have been mostly assigned based on protein interactors. The predominance of transcription factors (TFs), transcriptional regulators and chromatin remodeling proteins in the PML interactome has long implicated PML in the regulation of transcription. Until recently, PML-mediated transcriptional regulation had been mostly linked to events occurring within PML-NBs, such as TFs post-translational modifications, and sequestration or assembly of transcriptional complexes . HoweverThe transcriptional functions of PML are of particular interest in cancer, where PML has been described as a tumor suppressor or oncogene depending on tumor contexts . We and Here, we mapped PML chromatin association in TNBC cells to evaluate its impact on transcriptional regulation. We find that PML associates with large heterochromatic regions outside PML-NBs, where it regulates deposition of H3K9me3. Importantly, these regions contain spatially restricted euchromatic domains where PML regulates expression of pro-metastatic genes. Our data suggest that in TNBC cells, PML acts as regulator of a metastasis gene expression program by modulating heterochromatin homeostasis.2. For in vitro treatment with arsenic trioxide (ATO), cells were incubated for 6 and 24 h with 1 μM arsenic trioxide (Sigma-Aldrich A1010).MDA-MB-231, BT549, MDA-MB-468 and MCF7 cells were purchased from ATCC and maintained in DMEM supplemented with 10% fetal bovine serum (FBS) (Euroclone) and 1% Penicillin/Streptomycin antibiotics (Lonza) at 37°C in a humidified atmosphere containing 5% COThird generation lentivirus (LV) stocks were prepared, concentrated and titrated as previously described ,19. Brie2 1.5 mM, KCl 10 mM, DTT 0.5 mM) and centrifuged at 1250 rpm 5 min at room temperature (RT). Resulting pellets were resuspended in 3 volumes of hypotonic buffer and incubated 10 min on ice. Triton X-100 was added to a final concentration of 0.5%, followed by 10 x Dounce homogenization with a tight pestle and 4°C 14 000 × g centrifugation for 1 min. Nuclear pellets were resuspended in extraction buffer  and incubated 30 min at 4°C on rotation. 2 mM MgCl2 and 75 U/ml benzonase (Sigma Aldrich e1014-25ku) were added for 40 min at 4°C on rotation. Nuclear extracts were obtained upon 16 000 × g centrifugations at 4°C for 30 min. 3000 μg of nuclear extract was immunoprecipitated with 4 μg of anti-PML (Santa Cruz Biotechnology 71910) or negative IgG control  overnight at 4°C on rotation. 80 μl Sepharose beads  were used for 4 h at 4°C on rotation. Beads were washed 3× in Co-IP buffer  and elution was performed at 95°C for 5 min in 4X Laemmli buffer and β-mercapthoetanol. Input represents 3% of immunoprecipitated lysate. All buffers were supplemented with protease inhibitors . SDS-PAGE was performed at different polyacrylamide concentrations (4–15%) and transferred on polyvinylidene difluoride membrane through transBlot Turbo Transfer System (Bio-Rad). Membranes were blocked with 5% Milk or 5% BSA in PBS 0.1% Tween 20 and incubated with antibodies against PML , LMNB1 , H3K9me3 , H3K27me3 , H3K4me3 , H3K27ac , β-Actin  and histone H3 , SETDB1 . Proteins were detected with peroxidase-conjugated antibodies  using the ECL Western Blotting Detection Reagents .Proteins were extracted with RIPA buffer , supplemented with protease inhibitors  and sonicated. Protein concentration was determined via Bradford assay. 10 μg of proteins were diluted in 4× Laemmli buffer and β-mercapthoetanol and boiled 5 min at 95°. To visualize PML after ATO treatment, equal cell numbers were lysed in 4× Laemmli buffer and β-mercapthoetanol, sonicated and boiled. Nuclear fractionation of MDA-MB-231 cells was performed using the Nuclear Extract Kit . Immunoprecipitation was performed on nuclear extracts as follows: MDA-MB-231 cell pellets were washed with 5 volumes of hypotonic buffer  following the manufacturer's instructions. When PLA was coupled with IF, PLA was performed until the last wash in the Duolink buffer B 1×, then IF was performed as previously described. In order to test antibodies specificity, we included technical and biological controls: (i) omission of each probe separately; (ii) titration of primary antibodies; (iii) use of PML knockdown cells. Final antibodies concentrations: anti-PML , anti-H3K9me3  and anti-SETDB1 .Images were acquired with the GE HealthCare DeltaVision Ultra microscope (60× objectives) in z-stacks (stacks of 0.2 μm). Identical settings and contrasts were applied for all images of the same experiment to allow data comparison. Raw images were analyzed with Fiji software . ColocalGAPDH gene. Relative expression was calculated using the comparative threshold cycle method (2-ΔΔCt), except for assessing basal gene expression where the 2-ΔCt was used.Total RNA was isolated using RNeasy mini Kit (Qiagen). Equal amounts (1 μg) of RNA were reverse transcribed into cDNA with Advantage RT-for-PCR Kit (Clontech) and analyzed by qPCR using a 7900 Fast-Real Time PCR System (Applied Biosystems). Probes for TaqMan assays were purchased from Applied Biosystems and use as follows: PLOD2 HS01118190_m1; TNC HS011156665_m1; SPARC HS00234160_m1; GAPDH HS02758991_g1; LOX HS00942480_m1; ZEB2 HS00207691_m1; PML HS00971694_m1; CIITA HS00172106_m1. Data were normalized to the https://github.com/usadellab/Trimmomatic/releases/tag/v0.39)   and processed for Illumina library preparation. TruSeq stranded mRNA protocol (Illumina) was used for 5′/3′ library preparation. Libraries were then barcoded, pooled and sequenced on an Illumina Nova-Seq 6000 sequencing system. For each run, RNA sequencing experiments were performed generating 30M single end reads, 100 nucleotide long. Sequencing adapters were removed using trimmomatic v0.39 (g/v0.39) , and fas/2.5.3a) . Annotat/2.5.3a)  using th/2.5.3a)  and usinCells were seeded 24 h before chromatin isolation at 60–70% confluence in 15 cm plates. Cells were trypsinized and double crosslinking was performed in suspension. Cells were first resuspended in PBS containing 2 mM Di(N-succinimidyl) glutarate  for 45 min at RT on gentle rotation. Cells were then centrifuged at 1200 rpm at RT for 5 min. The resulting pellet was resuspended in PBS containing 1% Formaldehyde (Sigma-Aldrich 252549) for 10 min at RT on gentle rotation. After formaldehyde quenching with Glycine  for 5 min, cells were centrifuged at 1350 × g at 4°C for 5 min, and the supernatant was discarded. Chromatin extraction was performed as previously described  and soniAll primers have been manually designed and purchased by Eurofins. PAD_01 forward, ACTAAGCCACGAGCTGC; PAD_01 reverse, TATCTTCGAGCCTGGGA; PAD_02 forward, CAGGACTTGTAGGAGACCAG; PAD_02 reverse, GCACACAGGCCTGGTTAT; PAD_03 forward, GAGGTCATAAGGGAATGTTG; PAD_03 reverse, CGATTATGGTGGTATGGC; PAD_04 forward, TTCAGCACACAAGTTCAGG, PAD_04 reverse, AATCTGACCTCAGCCTGTC; HHAT forward, TGTGCTCCTGCACTGTG; HHAT reverse, AGTCACCAAGGACAGCTTC; CIITA forward, CACTGACATAGAAAGGGGTCC; CIITA reverse, TTATGCTGTTATGCAACTTGACTCT; SPARC forward, AGGCAAAGAGAGACTGTGAAAGA; SPARC reverse, CCAGTGTACCTGTCCTTGCT; LOX forward, AGGTCACACTGGAAATTTGTCT; LOX reverse, CAATGCCTGCTCTGTGTCCT.http://sourceforge.net/projects/bbmap/) with suggested settings (ktrim = r k = 23 mink = 11 hdist = 1), then mapped using BWA-MEM version 0.7.12-r1039 (http://broadinstitute.github.io/picard/). Further filtering was done on reads mapping in regions present in the ENCODE hg38 blacklist (ATAC-seq was performed as reported  and libr12-r1039  on the hlacklist  and regihttps://github.com/macs3-project/MACS/releases/tag/v2.2.7.1) (reads per genome coverage (RPGC) function in Deeptools v3.5.1 (https://github.com/deeptools/deepTools/releases/tag/3.5.1) (https://software.broadinstitute.org/software/igv/)  . ChIP ang/3.5.1) . Bigwig g/3.5.1) . Bigwig re/igv/) . To assere/igv/) .P < 0.05, FDR < 0.05, FC>/<0.5).Diffbind  was used to identify differentially accessible regions (peaks) from ATAC-seq peaks. Peaksets from MACS2 and associated metadata for each sample were read into Diffbind within R and EdgeR  was emplhttp://github.com/CollasLab/edd)   with authttps://github.com/arq5x/bedtools2/releases/tag/v2.30.0) (https://github.com/bedops/bedops/releases/tag/v2.4.41) (venn function (https://github.com/asntech/intervene/releases/tag/intervene-v0.5.8) . Mean Log2(ChIP/Input) or normalized counts of the PADs intersected and non-intersected regions were calculated using multiBigwigSummary from Deeptools.Intersects were determined using BEDTools v2.30 (v2.30.0)  and BEDOv2.4.41) . The num-v0.5.8) . Randomith GREAT , while, th GREAT . Mean Loreldist function)   using ATP-value <0.1. Enrichment of genomic features annotated in PADs or enhancer sequences was assessed via fisher's exact test using a collection of UCSC feature tables and transcription factors binding sites form ENCODE provided by the LOLAweb database (https://ggplot2.tidyverse.org/)   was usedwww.graphpad.com), and the R statistical environment. Permutation tests were performed on R using the Bioconductor packages RegioneR (version 3.14).Data were processed using GraphPad Prism version 9.0.2  , while nTo characterize the epigenetic profile of PADs, we mapped heterochromatic (H3K9me3 and H3K27me3) and euchromatic (H3K4me3 and H3K27ac) histone modifications by ChIP-seq . We founMany large H3K9me3-rich heterochromatic regions are defined by association with nuclear lamins (lamina-associated domains or LADs) . In mappInterestingly, although most PADs overlap with LADs, unique domains of LMNB1 or PML association with distinct epigenetic composition were identified . SpecifiFinally, as previously observed in MEFs , immuno-We conclude that PML associates with large heterochromatic domains enriched in LMNB1 and DNA repeats in TNBC cells. Further, although we cannot exclude that specific PADs are adjacent to PML aggregates, this association occurs largely outside PML-NBs.To elucidate the role of PML in heterochromatin maintenance and genome organization in PADs, ChIP-seq of H3K9me3, H3K27me3 and LMNB1 was performed in TNBC cells upon PML silencing . ReducinPML silencing also led to reduced LMNB1 enrichment in PADs Figure  and reduFinally, in search of a mechanistic function of PML in mediating heterochromatin deposition, we investigated the role of the H3K9 methyltransferase SETDB1, which plays oncogenic and pro-metastatic functions in TNBC  and inteHeterochromatin and euchromatin domains subsist in a state of reciprocal equilibrium in the genome, such that disruption of one compartment may lead to spreading and reorganization of the other . With thThe increase of euchromatin marks within PADs after PML silencing Figure  promptede.g. cell adhesion and epithelial to mesenchymal transition; EMT) along with regulation of the neuronal system .,59.e.g. 2 Figure  were alsT Figure . As expeT Figure , confirmT Figure , we obseT Figure , suggestTo gain broader insights into the regulation of gene expression by PML with respect to its role in chromatin organization, the PML-regulated transcriptome was defined by RNA-seq upon PML silencing. PML knock-down led to downregulation of 1104 genes (genes positively regulated by PML) and upregulation of 1051 genes  was confirmed in 3 TNBC cell lines ..ZEB2, PLTo obtain mechanistic insights into gene regulation by PML, we defined the regulatory regions of differentially expressed genes by analyzing chromatin accessibility upon PML silencing via ATAC-seq. A total of 4338 regions of differential chromatin accessibility were identified . These rLOX, SPARC and CIITA: Figure S9D) that are regulated by PML in TNBC cells and localize outside PADs . Thus, we implicate PML in STAT3 co-activating functions in TNBC cells.Of note, beside transcriptional regulation within PADs, most genes regulated upon PML silencing localize outside PADs Figure , suggestTaken together, our data indicate that in TNBC, PML regulates gene expression via a dual mechanism that involves indirect regulation of transcription and localized regulation of chromatin organization in regions of PML association.Here, we show for the first time that in TNBC cells PML contributes to chromatin organization and (in)directly regulates expression of metastasis genes both within and outside domains of PML association with chromatin.PML-associated domains (PADs) have also been described in MEFs . Here, wde novo PML aggregates that form in response to specific pathological triggers (PML-mediated SETDB1 activity may be coupled to H3.3 deposition in PADs by DAXX/ATRX, which was shown in MEFs  and was triggers . Therefotriggers .de novo generation of H3K9me3 domains. We suggest that this may reflect a global reorganization of chromatin triggered by reduced deposition of H3K9me3 at PADs. This interpretation fits into the concept that chromatin homeostasis is regulated via compartmentalization and interconnection of chromatin environments (Interestingly, we show that PML modulates the epigenome in regions that do not associate with PML. This occurs at different levels: reducing PML expression promotes not only a global increase in euchromatic marks outside PADs, but also ronments .Another relevant finding of our study is that embedded within the heterochromatic domains of PML association are euchromatic and gene-rich sub-domains that contain pro-metastatic genes positively regulated by PML and their regulatory regions. These regions are depleted of PML compared to remaining PADs area, thus PML does not regulate the expression of genes located therein via association to their regulatory elements. This observation may appear puzzling, but is coherent with published data demonstrating that PML regulates the expression of genes to which at large it does not associate ,12. AlsoFinally, in addition to this regulatory function on DNA, we find that PML promotes expression of genes that do not localize to PML-associated regions, suggesting indirect transcriptional regulation. This is in line with a larger body of evidence linking PML to the regulation of TFs via their expression, localization to PML-NBs and/or post-translational modifications. Examples of this literature include PML-mediated regulation of STATs , c-Jun ,81 and cgkad819_Supplemental_FilesClick here for additional data file.


Saccharomyces cerevisiae was one of the first global co-repressors of gene transcription discovered. However, despite years of study, a full understanding of the contribution of Tup1p and Cyc8p to complex function is lacking. We examined TUP1 and CYC8 single and double deletion mutants and show that CYC8 represses more genes than TUP1, and that there are genes subject to (i) unique repression by TUP1 or CYC8, (ii) redundant repression by TUP1 and CYC8, and (iii) there are genes at which de-repression in a cyc8 mutant is dependent upon TUP1, and vice-versa. We also reveal that Tup1p and Cyc8p can make distinct contributions to commonly repressed genes most likely via specific interactions with different histone deacetylases. Furthermore, we show that Tup1p and Cyc8p can be found independently of each other to negatively regulate gene transcription and can persist at active genes to negatively regulate on-going transcription. Together, these data suggest that Tup1p and Cyc8p can associate with active and inactive genes to mediate distinct negative and positive regulatory roles when functioning within, and possibly out with the complex.The Tup1-Cyc8 complex in  Saccharomyces cerevisiae, was one of the first global co-repressors of gene transcription discovered. However, despite years of study, a full understanding of this complex is lacking. We examined TUP1 and CYC8 single and double gene deletion mutants and show that the Tup1 and Cyc8 proteins can make distinct contributions to the regulation of Tup1-Cyc8 target genes. Furthermore, we show that Tup1p and Cyc8p can be found independently of each other to negatively regulate gene transcription and can persist at active genes to negatively regulate on-going transcription. Together, these data suggest that Tup1p and Cyc8p can associate with active and inactive genes to mediate distinct negative and positive regulatory roles when functioning within, and possibly out with the complex. This suggests the Tup1-Cyc8 complex should be considered more as a ‘regulator of transcription’ and not solely as a dedicated ‘repressor of transcription’.The Tup1-Cyc8 complex in the yeast,  Saccharomyces cerevisiae was one of the first global repressors of gene transcription discovered SUC2 mRNAtup1 mutant, the level of SUC2 mRNA in the repressed (high glucose) condition was similar to the SUC2 mRNA present in the wt strain under the de-repressed (low glucose) condition (tup1). This is consistent with the loss of glucose repression of SUC2 in the tup1 mutant due to disruption of the Tup1-Cyc8 complex [SUC2 mRNA levels were even greater when the tup1 mutant was grown under low glucose conditions.In the ondition , tup1. T complex . Howevercyc8 and the tup1 cyc8 double mutant strains grown on high glucose, SUC2 mRNA levels were higher than levels found in both the repressed and de-repressed tup1 cells  suggests that the lower levels of SUC2 mRNA in the tup1 mutant under either glucose condition is dependent upon CYC8. Finally, these data show that even in the absence of both Tup1p and Cyc8p, SUC2 is not fully de-repressed when glucose is present.Together, these data show that in the wt strain under conditions of low glucose, CYC8 makes the major contribution to SUC2 repression and can exert a repressive effect upon SUC2 transcription in the absence of TUP1. Furthermore, TUP1 and CYC8 continue to exert a repressive effect upon SUC2 transcription during the de-repressing conditions associated with a low-glucose environment.Thus, tup1, cyc8 and tup1 cyc8 mutants to determine if there were more widespread differences in gene transcription in strains deleted for TUP1 and/or CYC8.We next examined global transcription in the tup1 mutant, 809 genes were upregulated in the cyc8 mutant, and 851 genes were upregulated the tup1 cyc8 double mutant  ≤0.3). Secondly, upregulation of these genes should not be higher in the tup1 cyc8 double mutant compared to the cyc8 mutant. Thirdly, we excluded genes showing very low transcription levels in the mutants (cut off average transcripts per million (TPM) ≤60). This analysis uncovered 13 genes which we propose are uniquely repressed via CYC8 in wt  in each of the mutants were discarded, and genes showing any change in transcription in the tup1 mutant were also excluded. This revealed a cohort of 14 CYC8 repressed genes at which TUP1 was required for their transcription in the absence of CYC8 , tup1 (469) and tup1 cyc8 (851) strains revealed no difference in their average levels of de-repression , distinct levels of repression by TUP1 and CYC8, with CYC8, on average, making the greater contribution to their repression than TUP1.Comparison of the levels of de-repression of the total number of genes de-repressed in the pression . Howeverup1 cyc8 . Further compare  to 4A. Ttup1 cyc8 mutant  to determine if there were any unique characteristics that might explain why their repression was most dependent on either CYC8 or TUP1.In a large proportion of the 429 genes commonly upregulated in the tup1 see . An examTUP1 and CYC8 repressed genes were enriched near the ends of chromosomes (CYC8 and TUP1 (TUP1 and CYC8 repressed genes correlated well with the Hda1-affected sub-telomeric (HAST) domains located between 5 and 40 kb of a telomere  consensus sequences in the promoter regions of the two gene cohorts . This shThus, the two cohorts of commonly repressed genes differ in their distribution within the HAST domains of the sub-telomeric regions and are associated with different transcription factor binding sites.tup1, cyc8 and tup1 cyc8 mutants, gene transcription was de-repressed the least in a tup1 mutant compared to the de-repression in the cyc8 and tup1 cyc8 double mutants  analysis of Cyc8p and Tup1p occupancy using SUC2 as an example of this cohort of genes in the glucose-grown strains . However, we could detect significant enrichment of Cyc8p at the partially de-repressed SUC2 promoter in the tup1 mutant . Thus, Cyc8p was present at the partially de-repressed SUC2 promoter in the absence of Tup1p where it could contribute directly to negatively influencing SUC2 transcription.Surprisingly, we could not detect significant enrichment of Tup1p or Cyc8p at the repressed nditions ,37,47. CFLO1 gene which was representative of the smaller subset of the 429 commonly repressed genes . Most interestingly, Tup1p could be detected at the FLO1 promoter in the absence of Cyc8p (cyc8).We next looked at RNAP II, Tup1p and Cyc8p occupancy at the ed genes . De-reprCYC8 see . Firstly was off , wt [27,1 mutant , tup1. Mof Cyc8p , cyc8.FLO1 promoter. The data also revealed that Cyc8p could be detected at high levels at FLO1 and SUC2 in the absence of Tup1p, and that Tup1p could be detected at the FLO1 promoter in the absence of Cyc8p. Thus, Cyc8p could directly contribute to repression of SUC2 transcription independent of Tup1p, and Tup1p could directly contribute to FLO1 repression in the absence of Cyc8p.Therefore, ChIP analysis confirmed that Tup1p and Cyc8p could be detected at the repressed SUC2, following gene activation [SUC2 following its activation in response to low glucose conditions  contained a Tup1p peak  contained a site of Cyc8p occupancy in their promoters  some genes are uniquely repressed by either TUP1 or CYC8, (iii) other genes are subject to redundant repression by TUP1 and CYC8, and (iv), some TUP1 repressed genes require CYC8 for their de-repression and vice-versa  ce-versa . We alsoed genes .SUC2, whereby CYC8 makes the dominant contribution to repression (SUC2-type genes). A smaller cohort of commonly repressed genes behave like FLO1 and are subject to dominant repression by TUP1 (FLO1-type genes). These two cohorts of commonly repressed genes differ in their distribution within the HAST domains of sub-telomeric regions, in which they are enriched, and are associated with different transcription factor binding sites , Flo8p occupied the FLO1 promoter, and FLO1 was transcribed at levels similar to that in a cyc8 mutant  compared to the tup1 mutant (469), suggesting a wider role for Cyc8p in global gene repression. Of the genes upregulated in the tup1, cyc8, and tup1 cyc8 mutants, there was a cohort of 429 genes that were commonly de-repressed in all the mutants . Thus, the Tup1p that we have detected at the FLO1 promoter in the cyc8 mutant could mediate the reported retention of Hda1p which could contribute to the Cyc8p independent repression of FLO1 transcription shown here  binding and HDAC dependency. The HDACs might contribute most to gene repression, whilst the TFs might be more relevant to target genes when active. Importantly, we suggest that Tup1-Cyc8 persists at active target genes with the Tup1p and Cyc8p subunits interacting and influencing HDAC and TF occupancy and activity differently at the distinct gene cohorts to yield the transcriptional outcome.tup1 or cyc8 mutants  and viewed under 100x oil immersion magnification. Leica Application Suite (LAS) software was used to capture the images.8 exponentially growing cells were resuspended in 5 ml YPD and incubated for 1 hour at 30°C. Aliquots were exposed to ethanol (15%), hydrogen peroxide (5 mM), Amphotericin B (15 μg/ml), growth at 50°C, and growth at 30°C (control) for 1 hour. The percent survival compared to the control was calculated by counting colony forming units (CFUs).The survival assays were performed as previously described with minor adaptations ,70. AliqFLO1- and SUC2-type genes were retrieved from YEASTRACT [FLO1 and SUC2-type genes was omitted. A parameter of at least a difference of 10% in the proportion of genes with that motif in each of the groups (FLO1-type and SUC2-type genes) was categorised as a significant difference.The known DNA binding motifs for all transcription factors (TF) found in the sequences 1000 bp upstream of the EASTRACT . The numSUC2 analysis, exponentially growing YPD cultures were divided into two equal portions. Cell pellets were washed twice and resuspended in YP containing glucose at either 2% (repressed) or 0.05% (de-repressed). Cultures were incubated for a further 120 min [ACT1 RNA. Primers used are shown in RNA extraction, cDNA preparation and RT-qPCR analysis were performed as previously described . For SUC 120 min . Values Saccharomyces cerevisiae S288c reference genome available on ENSEMBL using the STAR aligner v.2.5.2b. and BAM files were generated. Unique gene hit counts were calculated by using featureCounts from the Subread package v.1.5.2. Genewiz preformed DGE analysis using the DESeq2 Bioconductor package. Using normalised counts, Log2 fold change and a Benjamini-Hochberg adjusted p-value was then calculated. Conversion from BAM into BigWig files was carried out using bamCoverage from the deeptools package [http://bioinf.gen.tcd.ie/jbrowse/?data=RNA-seq_Tup1Cyc8_merged. The RNA-seq datasets are available in the Gene Expression Omnibus (GEO) repository (accession code GSE230732). RNA was extracted from exponentially growing cells using the Hot Phenol method and purified using the RNeasy Minelute Cleanup Kit (Qiagen) . Total R package . The BigTel-VI for RNAP II, Int-V for Tup1p, Cyc8-Myc and Flo8-Myc) to give the ‘relative occupancy’. Tup1p and Cyc8-Myc relative occupancy were further normalised to similarly processed ChIP results from a tup1 deletion and an untagged (No tag) strain, respectively. Details to show the ChIP resolution and the normalisation pathway are shown in Locus-specific ChIP was performed as previously described ,54. The et al., 2021 [http://bioinf.gen.tcd.ie/jbrowse/?data=RNA-seq_Tup1Cyc8_merged.The global Tup1p and Cyc8p occupancy data used in l., 2021 , and is S1 AppendixThe Excel file contains the RNA-seq data used to construct the appropriate figures.(XLSX)Click here for additional data file.S2 Appendixet al., 2021 [Data retrieved from Rossi l., 2021  and used(XLSX)Click here for additional data file.S3 AppendixThe Excel file contains multiple tabs, with each tab containing the data for a single figure.(XLSX)Click here for additional data file.S1 FigACT1 mRNA and error bars reflect standard deviation from 3–4 biological replicates.mRNA values in the strains indicated were normalised to (TIF)Click here for additional data file.S2 FigSUR2 mRNA levels in wt, tup1, cyc8 and tup1 cyc8 strains. (B) RT-qPCR analysis of SUR2 mRNA levels in wt and each of the mutant strains. (C) JBrowse image of RNA-Seq data of PHO3 mRNA levels in wt, tup1, cyc8 and tup1 cyc8 strains. (D) RT-qPCR analysis of PHO3 mRNA levels. In both B and D, mRNA levels were normalised to ACT1 mRNA and error bars reflect standard deviation . (E) JBrowse image of RNA-Seq data of FIT2 mRNA levels in wt, tup1, cyc8 and tup1 cyc8 strains. (F) JBrowse image of RNA-Seq data of MET6 mRNA levels in wt, tup1, cyc8 and tup1 cyc8 strains.(A) JBrowse image of RNA-Seq data of (TIF)Click here for additional data file.S3 FigRNR3 open reading frame (ORF) in wt, tup1, cyc8 and tup1 cyc8 in glucose grown cells measured by chromatin immunoprecipitation (ChIP). RNAP II signals (IP/IN) were normalised to an internal negative control region (IP/IN at Tel-VI) (n = 3). (B) RNR3 transcript levels measured relative to ACT1 mRNA levels using RT-qPCR in the strains indicated (n = 3). In A and B, error bars reflect standard deviation. (C) JBrowse image of RNA-Seq data of RNR3 mRNA levels in wt, tup1, cyc8 and tup1 cyc8 strains.(A) RNA polymerase II (RNAP II) occupancy at the (TIF)Click here for additional data file.S4 FigFLO1-type (blue), SUC2-type (green), and the remaining commonly upregulated genes (pink). The percentage of genes in each group was calculated. (B) Distribution of Gcn5p and Rsc1p occupancy over the first 100 kb regions from the telomeres divided into 5 kb regions. Occupancy of each protein in each 5 kb region is shown as a % of total protein occupancy over this region. It is important to note that Gcn5p and Rsc1p do not co-localise with the sub-telomeric sites of enrichment of the FLO1- and SUC2-type genes, thus acting as negative controls to support the observation of the exclusive FLO1- and SUC2-type gene localization as being unique to Tup1p and Cyc8p regulated genes. Gcn5p and Rsc1p occupancy data were extracted from Rossi et al., 2021 [(A) Distribution of the 429 commonly upregulated genes over the first 100 kb regions from the telomeres divided into 5 kb regions and separated into l., 2021 .(TIF)Click here for additional data file.S5 FigFLO1- and SUC2-type genes. We assigned a cut-off of average TPM values ≤10 as ‘off’ in wt, represented by the dashed line.Scatterplot to show the average transcript per million (TPM) values from the three biological wt replicates for the (TIF)Click here for additional data file.S6 FigFLO1- and SUC2-type genes and the closest protein coding gene; also shown are the ORF lengths of FLO1- and SUC2-type genes (from SGD) (75). FLO1- and SUC2-type gene upstream intergenic region length with gene de-repression.Correlating  Graphs depicting the length of the upstream intergenic length (X axis) and the change in transcription compared to wt (Y axis) for the (C) FLO1-type and (D) SUC2-type genes in tup1, cyc8 and tup1 cyc8 mutant strains. A line of best fit is shown for each strain. For FLO1-type genes (C), a two tailed Spearman correlation showed a statistically significant correlation between the upstream intergenic length and upregulation of transcription in the tup1 cyc8 strain compared to wt (P = 0.0294). For SUC2-type genes (D), a two tailed Spearman correlation showed a significant correlation in the cyc8 strain (p = 0.0449).(A) Schematic depicting how intergenic length was calculated. (B) Scatterplot depicting the up- and downstream intergenic lengths of (TIF)Click here for additional data file.S7 FigFLO8 3’ ORF by homologous recombination. The resulting strain contains a genomic copy of FLO8 immediately followed by the marker. (B) A forward primer was designed with homology to the FLO8 5’ ORF that contained an A-G base-pair substitution corresponding to position +425 of the FLO8 ORF. This was used in conjunction with a reverse primer with homology to an intergenic region downstream of FLO8. Using genomic DNA from the strain with the selectable marker directly downstream of the FLO8 ORF; these primers were used to generate a PCR product that contained the majority of the FLO8 ORF, but with the A-G point mutation at position +425. This product also contained the selectable marker adjacent to the FLO8 3’ ORF. (C) A second transformation was carried out in a wild type BY4741 strain using the PCR product containing the point mutation at position +425 in the FLO8 ORF. This resulted in a strain with a genomic copy of FLO8 containing a G at position +425 in place of an A. This strain also contained a selectable marker immediately downstream of the FLO8 ORF. The resulting strain  was sequenced to confirm the point mutation. The restored FLO8 gene in YMC19 was subsequently tagged with a 9-Myc epitope to generate strain YMC34.(A) In the first round of mutagenesis, a PCR product containing a selectable marker integrates into the position immediately downstream of the (TIF)Click here for additional data file.S8 FigFLO8 gene in wt and cyc8, and from the restored FLO8 gene in YMC19 (FLO8+), was analysed by RT-qPCR . (B) Expression of Flo8-Myc in YMC34 was confirmed by western blot analysis. Wt and a Cyc8-myc strain were used as controls. Actin was used as a loading control. The proteins detected for Cyc8-Myc and Flo8-Myc were of the expected size. (C) FLO1 and (D) SUC2 mRNA levels detected by RT-qPCR in wt, cyc8 and the FLO8+ strain. The result shows that FLO1 is transcribed in the FLO8+ strain, whilst SUC2 is not transcribed . The data in C is the same data shown in FLO1 promoter, SUC2 promoter, and at a negative control region, Int-V. (F) Flo8-Myc occupancy data from (E) shown as ‘relative occupancy’ following normalisation to Int-V . The Flo8-Myc relative occupancy data shown here for FLO1 is the same data shown in FLO1; Flo8-Myc does not occupy the SUC2 promoter, and SUC2 remains repressed in the FLO8+ strain.(A) Transcription from the non-functional (TIF)Click here for additional data file.S9 FigTUP1  Venn diagram showing the overlap between global Tup1p occupancy and genes identified as being uniquely repressed by TUP1 see . (B) VenCYC8 see . Tup1p al., 2021 .(TIF)Click here for additional data file.S10 FigFLO1 promoter and at the Tel-VI negative control region. (C) Cyc8-Myc occupancy across the FLO1 promoter region to show specific enrichment at -585 bp in wt and tup1. Cross linked chromatin from a Cyc8-Myc strain (wt), tup1/Cyc8-Myc strain (tup1), and an untagged strain (No tag) were immunoprecipitated with antibodies against the Myc tag. IP/IN for each of the indicated amplicons, as well as a negative control region within the right arm of Tel-VI, are shown. Mean and standard deviation are shown, * = p≤0.05, ** = p≤0.005 obtained from One-Way ANOVA analysis (n = 3–4).(A) Representative agarose gel to show DNA fragment size before and after chromatin sonication. A 1 kb DNA ladder and a 100 bp ladder (NEB) are indicated. The gel shows results from two wt samples (wt-1 and wt-2). For each sample the DNA included was 1: pre-sonicated input genomic DNA (RNase treated); 2: pre-sonicated input genomic DNA (RNase and DNase treated); 3: sonicated input DNA (RNase treated); 4: sonicated input DNA (RNase and DNase treated). (B) Schematic of the amplicons used for ChIP analysis across the (TIF)Click here for additional data file.S11 FigFLO1 promoter and ORF. (B-C) RNA polymerase II (RNAP II) occupancy at the FLO1 ORF in wt, tup1, cyc8 and tup1 cyc8 mutant strains. (B) IP/ IN for RNAP II occupancy at the FLO1 ORF and the negative control region (Tel-VI) are shown. (C) RNAP II occupancy at the FLO1 ORF normalised to Tel-VI to yield ‘relative occupancy’. (D-F) Cyc8-Myc occupancy at the FLO1 promoter. Chromatin from a Cyc8-Myc strain (wt), a tup1/Cyc8-Myc strain (tup1), and an untagged strain (No tag), were immunoprecipitated with antibodies against the Myc tag. (D) IP/IN for Myc occupancy at the FLO1 promoter and the negative control region (Tel-VI), are shown. (E) Cyc8-Myc occupancy at the FLO1 promoter normalized to occupancy at Tel-VI to yield ‘relative occupancy’. (F) Cyc8-Myc relative occupancy following normalization to relative occupancy in the No Tag strain. In all graphs mean and standard deviation are shown, * = p≤0.05, ** = p≤0.005 obtained from One-Way ANOVA analysis (n = 3–6).(A) Schematic to illustrate the amplicons used for ChIP analysis at the (TIF)Click here for additional data file.S12 Fighda1::KAN mutant strain. Lane 1: 10 bp marker (NEB), lane 2, 3: PCR using primers for ACT1 (positive PCR control), lanes 4, 5: PCR using primers upstream of HDA1 (hda1DFconF) and internal to hda1::KAN (KanB), lanes 6, 7: PCR using primers downstream of HDA1 (hda1DFconR) and internal to hda1::KAN (KanC). (B) Western Blot analysis to confirm the deletion of CYC8 in a hda1 mutant strain (in duplicate). β-actin was used as a loading control. Bands detected were of the expected size. RT-qPCR analysis of transcription of (C) FLO1, (D) SUC2 and (E) PMA1 in the strains indicated. Values were normalised to ACT1 mRNA and error bars reflect standard deviation (n = 3–4). . This result is specific to FLO1. Transcription at SUC2 and PMA1 are not significantly affected.(A) PCR results from genomic DNA of the strains indicated to confirm the (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.