Tumor necrosis factor alpha (TNF-α) is a cytokine that can potently stimulate the synthesis of a range of pro-inflammatory mediators in macrophages. The underlying epigenetic mechanism, however, is underexplored. Here we report that the transcriptional modulator megakaryocytic leukemia 1 (MKL1) is associated with a histone H3K4 methyltransferase activity. Re-ChIP assay suggests that MKL1 interacts with and recruits WDR5, a component of the COMPASS complex responsible for H3K4 methylation, to the promoter regions of pro-inflammatory genes in macrophages treated with TNF-α. WDR5 enhances the ability of MKL1 to stimulate the promoter activities of pro-inflammatory genes. In contrast, silencing of WDR5 attenuates TNF-α induced production of pro-inflammatory mediators and erases the H3K4 methylation from the gene promoters. Of interest, the chromatin remodeling protein BRG1 also plays an essential role in maintaining H3K4 methylation on MKL1 target promoters by interacting with WDR5. MKL1 knockdown disrupts the interaction between BRG1 and WDR5. Together, our data illustrate a role for MKL1 in moderating the crosstalk between BRG1 and WDR5 to activate TNF-α induced pro-inflammatory transcription in macrophages. Macrophage-associated inflammation is generally believed to be a double-edged sword serving both protective and pathogenic roles. When orderly programmed, it helps maintain tissue homeostasis. When aberrantly constructed, it disrupts physiologic integrity and contributes to a host of human diseases such as metabolic syndrome, atherosclerosis, and cancer[4–. In macrophages, inflammation-related transcriptional programs are dictated by a handful of conserved transcriptional factors (TF) that include NF-κB, AP-1, and STAT. The activities of these sequence-specific TFs are modulated by various co-factors, which may either enhance or suppress the inflammatory response. We have previously identified megakaryocytic leukemia 1 (MKL1) as a co-factor for NF-κB. MKL1 interacts with NF-κB and stimulates NF-κB-dependent trans-activation of target genes to promote the pathogenesis of pulmonary hypertension and colitis[8–. The mechanism whereby MKL1 mediates NF-κB-dependent pro-inflammatory transcription, however, is not completely defined.Macrophages constitute a major source of inflammation in both health and disease states. Typically, transcriptionally active chromatin is marked by methylated histone H3K4 while transcriptional repression is synonymous with methylated H3K9 and H3K27. In eukaryotes, histone H3K4 methylation is catalyzed by the COMPASS complex (short for complex proteins associated with SET1). COMPASS operates in a modular mode: whereas catalytic subunits, including MLL1, MLL2, MLL3, MLL4, and SET1 are exchangeable, structural/regulatory subunits, including ASH2, WDR5, WDR82, and RpBP5, are invariable. How individual COMPASS components regulate pro-inflammatory transcription in macrophages remains to be clearly sorted out.Engagement of the epigenetic machinery represents a paradigm of transcriptional regulation in eukaryotes[14–. Several histone methyltransferases, including MLL1, MLL4, and SET7/9, have all been implicated in the regulation of macrophage-derived inflammation by modulating NF-κB activity. Here we report that MKL1 regulates NF-κB-dependent pro-inflammatory transcription by moderating the crosstalk between WDR5 and BRG1.A range of epigenetic factors have been documented to participate in the regulation of pro-inflammatory transcription in macrophages. For instance, brahma-related gene 1, or BRG1, was reported to control temporal production of pro-inflammatory mediators induced by lipopolysaccharide (LPS) and free fatty acids. Procedures involving animals were approved by the intramural Ethics Committee on Humane Treatment of Experimental Animals.Human monocytic/macrophage-like cells  and human embryonic kidney fibroblast cells  were maintained in DMEM supplemented with 10% FBS. Mouse embryonic fibroblast (MEF) cells were isolated from wild type or MKL1 deficient mice. siRNAs for MKL1 and WDR5 were purchased from Dharmacon. Transient transfections were performed with Lipofectamine LTX (Invitrogen). Luciferase activities were assayed 24-48 h after transfection using a luciferase reporter assay system (Promega). All experiments were repeated three times.Expression constructs for MKL1, WDR5, as well as promoter constructs have been described before[20–. Aliquots of lysates containing 200 μg of nuclear protein were used for each immunoprecipitation reaction with anti-MKL1 (Santa Cruz), anti-BRG1 (Abcam), anti-WDR5 (Bethyl Laboratories), anti-dimethylated H3K4 (Millipore/Upstate), and anti-trimethylated H3K4 (Millipore/Upstate). For re-ChIP, immune complexes were eluted with the elution buffer , diluted with the re-ChIP buffer , and subject to immunoprecipitation with a second antibody of interest. All experiments were repeated three times.ChIP assays were performed essentially as described beforeRNA was extracted using an RNeasy RNA isolation kit (Qiagen). Reverse transcriptase reactions were performed using a SuperScript First-strand synthesis system (Invitrogen). Real-time PCR reactions were performed on an ABI STEPONE Plus (Life Tech) with primers and Taqman probes purchased from Applied Biosystems. All experiments were repeated three times.In vitro histone methyltransferase assay was performed as described before. Briefly, precipitated immune complex was mixed with histone H3 (Millipore), S-adenosyl methionine , BSA, and MAB buffer . After incubation at 37°C overnight, SDS loading buffer was added to stop reactions, and the methylation of histone H3 was determined by Western blotting.P values smaller than 0.05 were considered statistically significant (*).One-way ANOVA with post-hoc Scheffe analyses were performed using an SPSS package. . We asked whether MKL1 might be in a complex with an catalytically capable H3K4 methyltransferase in macrophages. To this end, we performed In vitro histone methyltransferase (HMT) assay. MKL1 was isolated from THP-1 cells by immunoprecipitation and incubated with recombinant histone H3 in the presence of the methyl donor SAM; RbBP5, a known component of the H3K4 methyltransferase complex, was included as a positive control whereas pre-immune IgG was included as a negative control. As shown in Fig. 1A, MKL1 was co-precipitated with an activity sufficient to catalyze H3K4 methylation. Re-ChIP assay showed that in response to TNF-α stimulation MKL1 was incorporated into a complex with WDR5, a well-defined component of the mammalian H3K4 methyltransferase complex, on the promoter regions of several pro-inflammatory mediators . Indeed, TNF-α treatment enhanced the occupancies of WDR5 on gene promoters in macrophages; MKL1 depletion with siRNA, however, significantly downregulated the binding of WDR5 , suggesting that MKL1 might play a role in recruiting WDR5 to the promoter regions of pro-inflammatory mediators. Of interest, we also observed that MKL1 was responsible for recruiting BRG1 , the catalytic subunit of the mammalian chromatin remodeling complex, and, to a lesser extent, BRM  to the gene promoters. Taken together, we conclude that MKL1 is associated with a histone H3K4 methyltransferase complex that includes WDR5, BRG1, and possibly BRM.We have previously shown that MKL1 mediates LPS-induced inflammatory response by influencing histone H3K4 methylationFig. 2A, WDR5 was a relatively weak activator of pro-inflammatory genes in reporter assays. In the presence of MKL1, however, WDR5 markedly enhanced the trans-activation of pro-inflammatory genes by TNF-α. Additionally, WDR5 cooperated with MKL1 to upregulate the activity of a κB reporter constructed by fusing two tandem repeats of the NF-κB response element to the luciferase gene . On the contrary, WDR5 knockdown by siRNA  attenuated the induction of endogenous pro-inflammatory mediators by TNF-α . Collectively, these data suggest that WDR5 might play a role in mediating the activation of pro-inflammatory genes in response to TNF-α treatment in macrophages.Next, we probed the involvement of WDR5 in TNF-α induced trans-activation of pro-inflammatory mediators in macrophages. As shown in Fig. 3A) and tri-methylation , two prominent markers for transcriptional activation, surrounding the proximal promoter regions of pro-inflammatory genes. Of note, BRG1 also appeared to play a role in the regulation of histone H3K4 methylation surrounding gene promoters because BRG1 knockdown achieved similar effects as WDR5 knockdown , suggesting of a possible interplay between WDR5 and BRG1 in the regulation of pro-inflammatory transcription. Indeed, Re-ChIP assays showed that WDR5 formed a complex with BRG1 on the proximal promoter regions of pro-inflammatory genes in macrophages treated with TNF-α . Therefore, WDR5 might regulate TNF-α induced pro-inflammatory transcription by interacting with BRG1 to promote histone H3K4 methylation.Next, we tackled the potential mechanism whereby WDR5 contributes to the regulation of pro-inflammatory transcription. Knockdown of WDR5 almost completely erased histone H3K4 di-methylation . Similarly, the interaction between WDR5 and BRG1 on the proximal promoter regions of pro-inflammatory genes was much stronger in bone-marrow derived macrophages (BMDM) isolated from wild type (WT) mice than from MKL1 knockout (KO) mice . Finally, we found that co-expression of WDR5 and BRG1 enhanced the 2xκB reporter activity in WT mouse embryonic fibroblast (MEF) cells but not in KO MEF cells; re-introduction of ectopic MKL1 restored the cooperation between WDR5 and BRG1 . In summary, we propose that MKL1 could moderate the crosstalk between different epigenetic factors to stimulate TNF-α induced pro-inflammatory transcription.Finally, we asked whether MKL1 could broker the communication between WDR5 and BRG1 to regulate TNF-α induced pro-inflammatory transcription. Depletion of MKL1 with siRNA significantly weakened the interaction between WDR5 an BRG1 on the proximal promoter regions of pro-inflammatory genes  gene promoter, likely through interacting with WDR5, in response to angiotensin II stimulation. These data clearly establish a role for BRG1 in regulating locus-specific H3K4 methylation. It is not known at this point how genomewide H3K4 methylation patterns would be influenced by BRG1. In human embryonic stem cells, BRG1 has been found to co-occupy with monomethylated H3K4, catalyzed by the MLL3/4-containing COMPASS complex, on early developmental enhancers. In cancer cells, loss-of-function mutation of BRG1 appears to influence genomewide distribution of H3K4 trimethylation although the mechanism is not clear. Future studies should make an effort in deciphering the connection between BRG1 and H3K4 methylation in a cell- and context-dependent manner.Epigenetic regulation of gene expression during the pathogenesis of human diseases remains an intriguing and everlasting area of research and SMAD-dependent transcription, respectively. WDR5 can also collaborate with PRMT5, a histone arginine methyltransferase, to activate the transcription of class II major histocompatibility complex (MHC II) genes. Of interest, the interaction between WDR5 and GClnc1, a long non-coding RNA (lncRNA), ensures its recruitment to a subset of pro-metastasis genes to activate transcription in gastric cancer cells, suggesting that non-protein factors could also contribute to the tethering of WDR5 to selective promoters although it is not clear whether these processes are independent of each other or act in concert. The dynamic interplay between WDR5 and other epigenetic factors likely reflect the extricate nature of transcriptional regulation, which deserves further investigation.We show that WDR5 cooperates with BRG1 to stimulate NF-κB dependent pro-inflammatory transcription. Of note, WDR5 has been reported to form extensive communications with other epigenetic factors to regulate transcription. For instance, WDR5 can interact with the acetyltransferase p300 in cancer cells and renal epithelial cells to regulate p53-dependent[8–10,21–22,34,37–. In keeping with our finding, Yu et al. have demonstrated that MKL1 is responsible for the dissemination of H3K4 trimethylation on the promoter regions of genes involved in inflammation across the genome. It remains unanswered whether this function of MKL1 relies on WDR5 and/or BRG1. ChIP-seq analysis of MKL1 in macrophages has found that in addition to NF-κB, MKL1 also preferentially binds to the target promoters of SRF, STAT, and IRF1, all of which are key regulators of inflammatory response. It would be interesting to determine whether MKL1 exploits a similar strategy to stimulate the transcription of those target genes.Our data suggest that MKL1 contributes to NF-κB dependent pro-inflammatory transcription by moderating the crosstalk between WDR5 and BRG1. Mounting evidence suggests that MKL1 relies heavily on the epigenetic machinery to regulate transcription. It is possible to employ this strategy to interfere with the MKL1-WDR5-BRG1 interaction in order to control cellular inflammatory response in an attempt to treat inflammation-related diseases.In summary, we present evidence to show that MKL1 mediates TNF-α induced pro-inflammatory transcription by bridging the crosstalk between BRG1 and WDR5. A recent study has found that small peptides that disrupt the interaction between WDR5 and MLL1/SET1 specifically inhibits the activity of MLL1/SET1-containing COMPASS


TORC2 gene is a member of the transducer of the regulated cyclic adenosine monophosphate (cAMP) response element binding protein gene family, which plays a key role in metabolism and adipogenesis. In the present study, we confirmed the role of TORC2 in bovine preadipocyte proliferation through cell cycle staining flow cytometry, cell counting assay, 5-ethynyl-2′-deoxyuridine staining (EdU), and mRNA and protein expression analysis of proliferation-related marker genes. In addition, Oil red O staining analysis, immunofluorescence of adiponectin, mRNA and protein level expression of lipid related marker genes confirmed the role of TORC2 in the regulation of bovine adipocyte differentiation. Furthermore, the transcription start site and sub-cellular localization of the TORC2 gene was identified in bovine adipocytes. To investigate the underlying regulatory mechanism of the bovine TORC2, we cloned a 1990 bp of the 5’ untranslated region (5′UTR) promoter region into a luciferase reporter vector and seven vector fragments were constructed through serial deletion of the 5′UTR flanking region. The core promoter region of the TORC2 gene was identified at location −314 to −69 bp upstream of the transcription start site. Based on the results of the transcriptional activities of the promoter vector fragments, luciferase activities of mutated fragments and siRNAs interference, four transcription factors  were identified as the transcriptional regulators of TORC2 gene. These findings were further confirmed through Electrophoretic Mobility Shift Assay (EMSA) within nuclear extracts of bovine adipocytes. Furthermore, we also identified that C/EBPγ, XBP1, INSM1 and ZNF263 regulate TORC2 gene as activators in the promoter region. We can conclude that TORC2 gene is potentially a positive regulator of adipogenesis. These findings will not only provide an insight for the improvement of intramuscular fat in cattle, but will enhance our understanding regarding therapeutic intervention of metabolic syndrome and obesity in public health as well.The  Transducer of regulated cAMP response element-binding protein (CREB) 2 (TORC2) is one of the main inhibitors of lipolysis by regulating PKA and HSL activities . The TORC2 gene is responsible for nutrient metabolism, gluconeogenesis, myogenesis and adipogenesis through the phosphoinositide 3-kinase-Akt (PI3K-Akt), adenosine monophosphate-activated protein kinase (AMPK), glucagon and insulin resistance signaling pathways via promotion of the anabolic and inhibiting catabolic processes within the cells [TORC2 is a coactivator gene that plays a key role in glucagon-mediated activation of gluconeogenesis through a synchronized mechanism of glucocorticoid receptor and glucagon-CREB pathways coordinated with PEPCK and G6P genes [PPARγ), coactivator 1α (PGC1α) and nuclear receptor subfamily 4 group A (NR4A), TORC2 promotes gluconeogenesis and adipogenesis [There are three members of this gene family: he cells . Moreove6P genes ,19,20,21ogenesis .TORC2 [TORC2 may perform a crucial role in the regulation of bovine adipogenesis. Intramuscular fat (marbling) is one of the most important indicators of meat quality grading. Unfortunately, molecular mechanisms regulating the bovine intramuscular fat through TORC2 gene is still unexplained. Therefore, the present study was conducted to explore molecular function and regulatory mechanisms of TORC2 gene in bovine adipocytes.Moreover, lipogenesis and gluconeogenesis are well known contributors to the paradoxical effects of insulin resistance ,24,25. LTORC2 ,30. TherTORC2 gene, the relative expression level of TORC2 was identified in eight different tissues of Qinchuan cattle , pcDNA3.1-TORC2 (OE-TORC2), siTORC2, and siNC. First, the transfection efficiency was confirmed through measurement of TORC2 expression in different cells transfected with OE-NC, OE-TORC2, siNC, and siTORC2 (TORC2 gene in cells transfected with OE-TORC2 was significantly increased (p < 0.01) as compared to OE-NC (TORC2 in cells transfected with siTORC2 was significantly decreased (p < 0.01) as compared to siNC  enriched cell cycle-related gene  expression, both at the mRNA and protein level (p < 0.01)  E,F. Furtmarkedly I–K. Nextith siNC H. CollecTORC2 in bovine adipocyte differentiation, we first performed an Oil red O staining analysis . Subsequently, these findings suggest that TORC2 is a positive regulator of bovine adipocyte differentiation.To elucidate the role of the analysis I,J and, 2 groups L. To furTORC2 gene transcription start site , while the RACE-R1-TORC2 primer revealed TSS tentatively to be located at 5 bp downstream of the NCBI reported TSS of the bovine TORC2 gene.The 5′rapid amplification of cDNA ends (RACE) was performed to identify the art site E. Two reTORC2 gene, seven serial reporter constructs were generated in the luciferase reporter vector through unidirectional deletions from the 5′ end. Transcriptional activities of the respective fragments were assessed in the bovine preadipocytes through Dual Luciferase Reporter Assay (TORC2-F1 (1990 −1800/+190) and the pGL3-basic was (44.6) (p < 0.001). When the sequence was deleted from −1800 to −1500 bp the mean difference between the pGL3 basic and the TORC2-F2 (1690 −1500/+190) reduced to 32.07 (p < 0.05). However, with further deletion from −1500 to −1180 bp, the transcriptional activity of the TORC2-F3 (1370 −1180/+190) construct increased significantly (p < 0.001). Further deletion from -1180 to 857 bp caused a reduction in the transcriptional activity with the mean difference of (31.65) (p < 0.05). However, the highest transcriptional activity was found in the cells transfected with TORC2-F5 (801 −611/+190) with a mean difference value of (81.60) (p < 0.001) as compared to the pGL3-basic vector. Further unidirectional deletion in the 5′ end sequence of the TORC2-F5 (801 −611/+190) fragments to TORC2-F6 (504 −314/+190) caused a reduction in the transcriptional activity with significant (p < 0.05) mean difference (71.54). When deletion was extended to −69 bp from −314 bp in TORC2-F7 (259 −69/+190), the transcriptional activity and mean difference (10.45) reduced significantly (p < 0.001). These results indicated that the functional minimal promoter of the TORC2 gene is located in the position −314 to −69 upstream of the transcription start site.To identify the core promoter region of the er Assay . The meahttp://www.genomatix.de/index.html accessed on 3 September 2019) was used for the identification of the transcription factor (TF) binding sites in the core promoter region of the bovine TORC2 gene with the cut off value of 90%. Various TF binding sites were found as shown in C/EBPγ, XBP1, INSM1 and ZNF263 were selected for further confirmation. The in silico analysis further revealed the absence of consensus TATA and CCAAT boxes in vicinity of the transcription start site (TSS) (http://www.urogene.org/methprimer/ accessed on 3 September 2019) explored one CpG island located from (−159 to +134 bp) relative to the TSS in the TORC2 promoter region (Bioinformatics tool Genomatix Mathinspector software (Intrexon Bioinformatics Germany GmbH available online: te (TSS) A. However region D. The seC/EBPγ, XBP1, INSM1 and ZNF263 were significantly reduced (p < 0.01 and p < 0.05). To further validate the roles of these TF binding sites in the core promoter region of the TORC2 gene, double TF binding sites mutation plasmid were constructed. p < 0.01 and p < 0.05) in the transcription activities of all the double mutated plasmids compared with the non-mutated plasmid fragment TORC2-F6 (504 −314/+190). These results suggest that the transcription factors C/EBPγ, INSM1, XBP1 and ZNF263 are positive activators of the TORC2 gene.For validating the regulatory roles of the selected transcription factors, mutated fragment plasmids were constructed. Bovine preadipocytes cells were transiently transfected with the mutated and non-mutated plasmids of the respective TF binding sites. The dual luciferase reporter assay explored a significant variation in the transcriptional activities of different mutated transcription factor binding sites B. Transchttps://genemania.org/ on 3 September 2019) and STRING database (Swiss Institute of Bioinformatics available online: https://string-db.org/ accessed on 3 September 2019) were applied for the identification of genetic interaction among the TORC2 gene and its four transcription factors. The in silico analysis showed a close interaction among these genes. Genetic interaction among the target genes were denoted by colors  revealed a 67.64% physical interaction. The co-expression, co-localization and shared protein domains structures were 13.50%, 6.17%, and 0.59%, respectively.Online bioinformatics tools GeneMANIA . The transfection efficiency was checked at 12- and 24-h intervals (p < 0.01) the mRNA expression levels of the XBP1, ZNF263, C/EBPγ and INSM1 as compared to the negative control (NC)  B–E. ThesTORC2 gene both at the mRNA and protein levels (TORC2 gene mRNA and the protein levels significantly reduced (p < 0.05) with the down regulation of the C/EBPγ, XBP1, INSM1 and ZNF263 transcription factors as compared to the NC-group.The interference of the selected transcription factors significantly altered the expression of the n levels A–F. The C/EBPγ, XBP1, INSM1, ZNF263 and NC  [TORC2 is an essential and conserved step in the activation of cAMP-responsive genes [TORC2 may regulate adipogenesis. Moreover, in the present study, the role of TORC2 in bovine adipocyte proliferation was confirmed through cell cycle staining flow cytometry, cell counting assay, 5-ethynyl-2′-deoxyuridine staining (EdU), and mRNA and protein expression analysis of proliferation-related marker genes. In addition, Oil red O staining analysis, immunofluorescence of adiponectin, mRNA and protein level expression of lipid related marker genes confirmed the role of TORC2 gene in the positive regulation of bovine adipocyte differentiation. The present study has identified the TORC2 gene as a potential positive regulator of adipogenesis. These findings imply that TORC2 is a novel hallmark for dealing with the problem of low intramuscular fat in cattle breeding program.Adipogenesis is a complex and precisely orchestrated process, characterized by an increased number of adipocytes and increased lipid storage in adipocytes. Adipocytes are derived from the existing pool of preadipocytes, which are differentiated in response to the proper signal ,39. Therogenesis ,40,41. In et al. . They co in pigs . Moreove in pigs ,43,44, w in pigs ,20,21 to in pigs ,46. Addis (SIKs) ,48. Howes (SIKs) ,50,51,52s (SIKs) . This trve genes . These fTORC2 gene in bovine adipogenesis, an attempt was made to explore the transcriptional regulation of the TORC2 gene in preadipocytes. Transcriptional regulation is a crucial biochemical regulatory mechanism in all living organism. It is organized by the orchestration of regulatory proteins and transcription factors. Therefore, the RNA level can be tuned precisely through various mechanisms, such as the copy number of transcribed RNA and spatiotemporal regulation of gene transcription. Transcriptional regulation allows the cell or organism to respond to various extracellular and intracellular signals and adopt accordingly. The promoter region is a DNA fragment, which may bind transcription factors, RNA polymerases, and other proteins for the transcription initiation of the particular gene [C/EBPγ, XBP1, ZNF263 and INSM1 genes were selected to investigate their roles in the regulation of the bovine TORC2 gene. These TFs were located in the core promoter region (−314 to +190) relative to the TSS. One CpG island was found in the promoter region of TORC2 gene. However, neither a TATA nor CCAATT box was identified in the proximal promoter region. These observations are consistent with the findings where the TATA box is present only in 10% to 20% of the eukaryotic promoter sequence and is not a dispensable transcription factor. [TORC2 gene in DNA methylation through regulation of DNA mismatch repair system [TORC2 gene explored the region between −314 to +190 bp as the core promoter region, which contained the consensus transcription factor motifs for the C/EBPγ, XBP1, ZNF263 and INSM1. Bioinformatics analysis showed location of these TFs within the GC-rich CpG island of TORC2 gene—which suggests that the function of the TORC2 is under epigenetic regulation.After elucidating the function of lar gene . Generallar gene . Therefo factor. ,58,59. Pr system . DNA metr system ,62,63. Dr system . In the C/EBPγ is a member of this family, which is expressed ubiquitously in tissues and has the binding affinity with regulatory motifs of immunoglobulin long chain enhancer and promoter. The C/EBPγ modulates the C/EBPs activity in a cell-specific manner [CEBPγ with the CEBPβ causes cell proliferation and inhibition of cell senescence [XBP1 transcription factor is a major regulator of endoplasmic reticulum (ER) and unfolded protein response (UPR) or stress response, activated by certain stressors including protein and fat overloading, which causes increase endoplasmic reticulum capacity. XBP1 regulate terminal stage differentiation of preadipocytes to adipocytes, and consequently elevate the secretion of the unfolded protein response (UPR) as a stress response in the endoplasmic reticulum to increase the rate of protein and lipid biosynthesis [XBP1 is main regulator of cell growth and survival; the expression of XBP1 is directly proportional to up-regulation of P13/mTOR-TORC2 signaling pathway, which is vital for cell proliferation and growth [INSM1 regulates the chromatin modifying factors of histone deacetylases; histone modification performs vital roles in various biological processes and the expression and regulation of genes [INSM1 is a zinc finger transcription factor, which performs a vital role in the regulation of target genes or indirectly through the stimulation of growth factor signaling pathways [INSM1 with the interaction of the RACK1 gene promotes P13/AKT signaling pathway as well [ZNF263 is a member of the Zinc finger motifs, which is a C2H2 protein that contains nine zinc finger domains, one SCAN domain and one KRAB repression domain. Genome-wide ChIP sequencing identified more than 5000 binding sites for this transcription factor in the promotion of multiple genes that regulate their target genes either as a repressor or activators [C/EBPγ, XBP1, INSM1 and ZNF263 significantly reduced the transcriptional activity of the TORC2 gene. Moreover, interference through siRNA caused a significant (p < 0.05) reduction in the expression of the TORC2 gene both at the mRNA and protein levels. Lipid droplets quantity and diameter was decreased in adipocytes transfected with siRNAs against the selected TFs as well, as compared with negative control group. Furthermore, EMSA assay additionally confirmed the binding of C/EBPγ, XBP1, INSM1 and ZNF263 in the nuclear extract of adipocytes in the promoter region of the TORC2 gene. From these findings, we can conclude that C/EBPγ, XBP1, INSM1 and ZNF263 regulate TORC2 gene as activators in the core promoter of TORC2 gene. Although we identified the role of C/EBPγ, XBP1, INSM1 and ZNF263 in the regulation of TORC2 gene further study is however needed to investigate the roles of these TFs in adipogenesis.The transcription factors CCAAT/enhancer binding proteins (C/EBPs) belong to the leucine zipper transcription factors family, which plays a vital function in cellular proliferation and differentiation. The c manner ,66. The nescence . The XBPynthesis . In the d growth . INSM1 rof genes . INSM1 ipathways . INSM1 w as well . The ZNFtivators ,74. In tThe procedures for animal handling for experiments were approved by EAMC  at Northwest Agriculture and Forestry University, China. Moreover, all applicable rules and regulation of the organization and government were followed regarding the ethical use of experimental animals.TORC2 gene, eight tissues including rumen, reticulum, abomasum, small intestine, large intestine, liver, muscular fat and adrenal fat were aseptically collected in liquid nitrogen. The total RNA was extracted from the tissue using TRIzol™ Reagent . The integrity  of the extracted total RNA was checked through an optical density of 260 and ratio of the optical density (OD) of 260/280 using the Nano Quant plate TM , and was further verified through 1% agarose gel. cDNA libraries were constructed using PrimeScriptTM RT reagent kit with gDNA eraser . Quantitative real time (RT-PCR) was performed following the manufacturer’s protocol of Sybr Premix EX Taq Kit  using thermocycler 7500 system SDS V 1.4.0 . Bovine β-Actin and GAPDH  genes were used as an endogenous control. The thermocycling conditions were: pre heating at 95 °C for 5 min, a total 34 cycles of denaturation at 95 °C for 30 s, annealing temperature at 60 °C for 30 s and extension temperature at 72 °C for 30 s. The relative mRNA expression levels were calculated using 2−ΔΔCt method [For tissue collection, three mature Qinchuan cattle (24 months old) were selected from the National Beef Cattle Improvement Research Centre. The animals were dressed in a local abattoir under standard procedure of animal stunning, exsanguinations, and skinning. To measure the relative expression of g for 10 min, and the cell pellet was washed twice with DMEM-F/12 medium  without serum. The cell pellet was then resuspended with DMEM-F/12 medium supplemented with 10% FBS and seeded in 60 mm collagen coated cell culture plates and incubated at 37 °C and 5% CO2 for 1 h. One hour later, the medium was changed, and washed three times with 1XPBS to remove the debris and free-floating cells.Bovine preadipocyte cells were collected from a healthy newborn calf (5 days old) of the Qinchuan cattle breed using the methods previously described ,77,78. T4), calcium and magnesium, with pH 7.0 to 7.2 . After washing with 1× PBS the cells were permeabilized with 0.2% Triton X-100 for 15 min. For immunofluorescence, the cells were incubated with the primary anti-CRTC2 antibody  and anti-adiponectin antibody  overnight at 4 ˚C. The cells were washed with 1XPBS and incubated with secondary anti-rabbit IgG H&L antibody (Alexa Fluor® 555) , and protected from light at 37 ˚C for 1 h. The nuclei were stained under dark with DAPI  at room temperature for 15 min. DAPI was used at the final concentration of 1 μg/mL. Immunofluorescence images were taken with an Olympus IX71 microscope .Bovine preadipocyte cells were cultured in a 24-well culture plates. The cultured cells were fixed with 4% paraformaldehyde for 15 min. The fixed cells were washed with phosphate buffer saline (1× PBS) which contains 0.0067M (POThe 5-ethynyl-2′-deoxyuridine (EdU) assay was performed using a cell light EdU DNA proliferation kit . Bovine preadipocytes were seeded in 24-well cell culture plates and transfected at 50% to 60% density with OE-NC, OE-TORC2, siNC, or siTORC2. After 24 h, the cells were incubated with 5-ethynyl-2′-deoxyuridine medium for 2 h. After 2 h incubation, the EdU assay was performed according to the manufacturer’s protocol.Bovine preadipocytes were seeded in six-well cell culture plates. The cells were transfected with OE-NC, OE-TORC2, siNC, or siTORC2. After 24 h, the cells were harvested, washed with 1XPBS, and re-suspended with 1× PBS containing 1 mL DNA staining solution and 10 μL permeabilization solution . The suspension was vortexed for 15 s and incubated for 30 min in the dark at RT. The cell cycle was analyzed through flow cytometry  by counting 20,000 cells.2 cell incubator. The absorbance was measured using a Nano Quant plate TM  reader at a wavelength of 450 nm.Bovine preadipocytes were seeded in 96-well plates and transiently transfected with OE-NC, OE-TORC2, siNC, or siTORC2. The cell proliferation was detected after 0, 18 and 24 h of transfection using a TransDetect CCK kit , according to the manufacturer’s protocol. Cells were added 10 µL of CCK solution and to each well, and incubated for 3 h at 37 °C in a 5% COTORC2 gene was identified through RACE- Kit   as per the manufacturer′s instructions . Total RNA was extracted using TRIzol™ Reagent  from the muscular fat of Qinchuan cattle. RACE ready 5′-end first strand cDNA was synthesized through SMARTer RACE 5′ Kit  PCR was performed for the amplification of RACE ready cDNA using two different gene specific reverse primers  was used as forward primer. Nested PCR was conducted with the conditions as mentioned previously [Transcription start site (TSS) of  primers  located eviously ,80 usingTORC2 gene (NCBI accession No NC_037330.1 from 16477661 to 16487214). Qinchuan cattle genomic DNA was used as a template for the PCR amplification of TORC2 gene promoter, using KOD plus neo enzyme . Thermocycling (PCR) was performed using three-step cycle conditions with pre-denaturation at 94.0 °C for 5 min followed by 34 cycles of denaturation at 97 °C for 30 s, annealing  and DNA extraction kit . The extracted DNA was diluted to make the final concentration as 50 ng/μL and stored at −20 °C. Gene specific primers . The CpG Island in the prompter region of the TORC2 gene was predicted through an online website Meth Primer (The Li lab Home page available online: http://www.urogene.org/methprimer/ accessed on 3 September 2019). Based upon the predicted transcription factor binding sites and position of the CpG Island, different fragment primers were designed. In total, seven different fragments were amplified through gene specific primers TORC2-F1 (1990 −1800/+190), TORC2-F2 (1690 −1500/+190), TORC2-F3 (1370 −1180/+190), TORC2-F4 (1047 −857/+190), TORC2-F5 (801 −611/+190), TORC2-F6 (504 −314/+190) and TORC2-F7 (259 −69/+190) with unidirectional deletion of the bovine TORC2 gene promoter . The DNA was extracted from the gel with a gel extraction kit  and ligated through T4 ligation  into pGL3 basic (luciferase reporter vector). Ligated pGL3 basic vector with inserts of the target fragments were then cloned into DH5α . Five single colonies were selected for each fragment and then sequenced . Each fragment plasmid was extracted by Endo free Plasmid DNA Mini Kit II . Concentration of the extracted plasmid DNA was measured through Nano Quant plate TM  and stored at −20 °C.The putative transcription factor binding sites were identified through online software Genomatix suit  with pRL TK plasmid as an internal control vector to standardize transfection efficiency [Bovine preadipocyte cell culture was maintained in Dulbecco’s Modified Eagle Medium (DMEM-F/12)  containing 10% FBS , 1% antibiotics (100 IU/mL penicillin and 100 μg/mL streptomycin) and incubated at 37 °C with 5% COficiency ,82. FortC/EBPγ, XBP1, INSM1 and ZNF263 motifs were mutated through site directed mutagenesis with the overlapping pairs of primers . The details of the sequences have been mentioned in The transcription factors The Qinchuan cattle preadipocyte cells were lysed with RIPA lysis buffer  which contains protease inhibitor Cocktail  for the extraction of total protein. The cellular protein was then mixed with protein loading buffer. Equal volumes of each protein samples were loaded into a 12% SDS-PAGE gel for electrophoresis. The band from the gel was then transferred to a nitrocellulose membrane (PVDF), blocked with 5% (w/v) skim milk through incubation at room temperature for 2 h. After blocking, the membrane was incubated with primary antibodies of anti-TORC2 rabbit polyclonal antibody  and β-actin antibody  overnight. The IgG-Goat anti-Rabbit HRP antibody  was used as a secondary antibody. Finally, chemical luminescence signals were identified by exposing the membrane photographed using Chemi Doc System Bovine preadipocyte cells were cultured, maintained and transfected with siRNAs as described earlier. After 48 h of transfection, the cells were induced to differentiation, with first differentiation media supplemented with 0.5 mM hydro cortisol, IBMX (3. isobut-1-methylxanthine), dexamethasone 1 μM, and insulin 167 nM as described previously . Two days later, the media was changed to second differentiation medium containing DMEM-F/12 with 10% fetal bovine serum and insulin 167 nM. The cells were stained with Oil red O staining at day 9 of differentiation. Briefly, the stock solution for Oil red O was prepared by dissolving the staining powder in isopropanol under dark. Stock solution was filtered to prepare a working solution, diluted with 60% deionized water, and then filtered again. First, the cells were washed for three times with 1XPBS and then fixed with 4% paraformaldehyde solution for 30 min, at RT. After fixation, the cells were washed again three times with 1XPBS, and then, Oil red O staining (working solution) was applied and placed for 30 min at RT. Cells images were captured using the Olympus IX71 microscope  and lipid droplets were observed. Lastly, the stain was diluted with 100% isopropanol and lipids droplets were quantified through absorbance at 500 nm.C/EBPγ, XBP1, INSM1 and ZNF263 transcription factor motifs were synthesized  and stored at −80 °C. The 5′-biotinated, un-biotinated and site directed mutated probes for thesized  (Sangon,p < 0.05, *** p < 0.01, and **** p < 0.001.The data are presented as mean ± SEM. SPSS 20.0 software was used for statistical analysis and variation between the groups was determined through the Student’s t-test. Variations were considered statistically significant at * TORC2 positively regulates both bovine adipocyte proliferation and differentiation. Moreover, we identified the core promoter region of TORC2 gene spanning from −314 to −69 bp upstream of transcription start site. In addition to that, we identified that C/EBPγ, XBP1, INSM1 and ZNF263 regulate TORC2 gene as activators transcription factors in the promoter of the TORC2 gene as well. These findings will not only provide an insight for the improvement of intramuscular fat in cattle, but will additionally provide a clue to intervene the issue of metabolic syndrome and obesity in human beings.We can conclude from the present study that


PRKN (PARK2) is a key gene involved in both familial and sporadic Parkinson’s disease that encodes parkin (PK). Since its discovery by the end of the 90s, both functional and more recently, structural studies led to a consensual view of PK as an E3 ligase only. It is generally considered that this function conditions the cellular load of a subset of cytosolic proteins prone to proteasomal degradation and that a loss of E3 ligase function triggers an accumulation of potentially toxic substrates and, consequently, a neuronal loss. Furthermore, PK molecular interplay with PTEN-induced kinase 1 (PINK1), a serine threonine kinase also involved in recessive cases of Parkinson’s disease, is considered to underlie the mitophagy process. Thus, since mitochondrial homeostasis significantly governs cell health, there is a huge interest of the scientific community centered on PK function. In 2009, we have demonstrated that PK could also act as a transcription factor (TF) and induces neuroprotection via the downregulation of the pro-apoptotic and tumor suppressor factor, p53. Importantly, the DNA-binding properties of PK and its nuclear localization suggested an important role in the control of several genes. The duality of PK subcellular localization and of its associated ubiquitin ligase and TF functions suggests that PK could behave as a key molecular modulator of various physiological cellular signaling pathways that could be disrupted in pathological contexts. Here, we update the current knowledge on PK direct and indirect TF-mediated control of gene expression. Parkin (PK) has been initially described as a ubiquitin ligase , and it TP53 gene expression and that it physically interacted with the human promoter of TP53  and chromatin immunoprecipitation assay (ChIP) of endogenous TF candidates are routinely considered as necessary and sufficient to delineate DNA-binding properties of a putative TF candidate. We used these two complementary approaches to demonstrate that PK downregulated  of TP53 . Thus, E context . To defiA probes . In thisproaches .In 2013, we have identified and validated by both approaches, two additional PK transcriptional targets linked to Alzheimer’s disease (AD), namely, presenilins 1 (PS1) and 2 (PS2) . PSs arein vitro by EMSA approach was dependent upon the presence of the mitochondrial TF TFAM  have shown an importance of zinc to its function -containing protein family. Crystal structure determination of full length PK  and its The delineation of PK structure has been rightly taken as the definitive evidence that PK acted as a ubiquitin ligase. It has also sometimes led to the empirical refuting of other evidences, even when they were derived from technically sound studies. That PK could have additional functions and indeed could behave as a pleiotropic protein is not consensually accepted. This dogmatic view based on structural grounds is particularly puzzling since close examination of the literature in the field indicates that the RING finger motif can also mediate protein–DNA interactions.in vitro binding to oligonucleotides in a zinc-dependent manner since the addition of EDTA and DTT blocked the formation of specific bands. This observation raised a question. Besides its ubiquitin ligase function, could PK DNA binding be mediated by its RING1 domain? Interestingly, we firmly demonstrated that this domain was indeed involved in both p53 and PS2 transrepression and PS1 transactivation  indicate the presence of potential RING1 independent DNA-binding motifs in the PK protein sequence, the functionality of which remains to be firmly established.Importantly, in vitro and in vivo approaches (reviewed in One important property of a TF stands in its ability to recognize and bind short specific sequences (6–12 bases) in the promoter region of a gene target. Usually, TFs bind to their preferred sequences referred to as consensus motif with a 1000-fold higher affinity than for other sequences . Thus, tTP53, PSEN1, and PSEN2) have been experimentally validated by both ChIP and EMSA approaches  in the chipped sequence. This supports the view of specific PK DNA binding properties and reinforces the reliability of the PK consensus motif signature for identifying direct TF targets.Despite a considerable number of genes directly or indirectly regulated by PK , only thproaches . Of mostgulation . Second,gulation . This negulation  will be  by ChIP  indicate by ChIP . Our in TP53 promoter , XBP1 (X box-binding protein). XBP1 directly binds to DJ-1 promoter and regulates DJ-1 transcription  PK-regulated genes awaiting definitive demonstration. These targets include all PK targets identified by ChIP on ChIP approaches . Of noteA levels . A RNAseA levels .Group 4 highlights the possible dual regulation of some genes and their translation products by both PK TF and E3 ligase activities, respectively. Thus, in this group, one can find p53, mitofusin 1, mitofusin 2, and voltage-dependent anion channel 1 (VDAC) that are validated E3 ligase substrates and potential PK TF targets .TP53 is also a TF target of PK suggesting that both TF and ubiquitin ligase functions may be implicated in transcriptional and post-traductional control of a given target. Conversely, some of the above-cited proteins could well behave as PK E3 ligase substrates only, without being transcriptionally regulated by PK.Group 5 gathers PK E3 ligase substrates that harbor a TF function like p53, NFkB, HIF1α, and PARIS (ZNF746) . Given tIt is clear that PK harbors ubiquitin ligase and TF functions, which could be both involved in gene regulation. Delineating the respective contribution of these functions in physiological and pathological conditions should help to understand the cellular defects underlying both neurodegenerative and cancer pathologies and should allow the development of innovative diagnostic and therapeutic tools centered on its direct and/or indirect gene control.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Setdb1, Ncor2, Rnf2, Kat5, Prmt5, Uhrf1, and Rrp8 caused widespread changes in TE expression and chromatin accessibility. These effects were context-specific, with different chromatin modifiers regulating the expression and chromatin accessibility of specific subsets of TEs. Our work reveals the complex patterns of epigenetic regulation of TEs.The majority of mammalian genomes are devoted to transposable elements (TEs). Whilst TEs are increasingly recognized for their important biological functions, they are a potential danger to genomic stability and are carefully regulated by the epigenetic system. However, the full complexity of this regulatory system is not understood. Here, using mouse embryonic stem cells, we show that TEs are suppressed by heterochromatic marks like H3K9me3, and are also labelled by all major types of chromatin modification in complex patterns, including bivalent activatory and repressive marks. We identified 29 epigenetic modifiers that significantly deregulated at least one type of TE. The loss of  Transposable elements (TEs) fulfill essential but poorly understood roles in genome organization and gene expression control. Here the authors show that the regulation of TEs occurs through overlapping epigenetic mechanisms that control the expression and chromatin signatures at TEs. TEs were originally thought of as genetic parasites with roles in human disease2, but TEs are now understood to contribute to normal biological processes3. There are many examples of exapted TEs that have become host cell genes, such as the RAG enzymes which are essential for antibody and T cell receptor recombination, or the Syncytin genes which are involved in placental development4. TEs can be transcribed to produce RNA, and have contributed to the evolution of long non-coding RNAs, microRNAs, and circular RNAs5. In addition, TEs often mimic host cell functions by incorporating cis-regulatory elements6, which can recruit transcription factors (TFs) to promote TE activity. This was initially observed for the TF repressor REST7, but has since been observed for a wide-range of TFs10. TEs copy themselves in the genome and are co-opted to form new regulatory elements12, and contribute to the rewiring of gene regulatory networks6. However, much of this data on exaptation of TEs is derived from genomic data and there is argument over how much is functional6.The modification of histones is an elaborate system to regulate gene expression, and provides an epigenetic landscape for the cell-type-specific interpretation of the genome. Yet, the major class of genomic elements in the cellular genome are not genes, but transposable elements (TEs), including endogenous retroviruses (ERVs)15, mRNA editing16, and DNA methylation17. DNA methylation is thought to be the dominant suppressive mechanism in somatic tissues17. However, DNA is globally demethylated during early embryonic development, and TEs are released from repression in a controlled, stage-specific manner19. The TEs are then free to compete with the epigenetic suppression mechanisms to duplicate themselves and enter the germ line20. Consequently, there is a delicate balance between the beneficial effects of TEs, and their deleterious effects on genome integrity22.TEs/ERVs are silenced by a range of molecular mechanisms, including heterochromatin formation23, recruit the adaptor protein TRIM28/KAP1, and the histone H3K9me3 methyltransferase SETDB1 to silence TEs28. In addition to H3K9me3, there are other modes of epigenetic suppression of TEs29, such as the methylation of H4K20me330, H3K27me331, and H4R3me220, the biotinylation and sumoylation of H2A, H3, and H4 histones33, and the deposition of the histone variant H3.334. It is clear that the epigenetic system is regulating TEs38, however, there are at least 1100 distinct types of TE, comprising millions of genomic copies, for which the epigenetic regulation is unclear.TEs are suppressed in embryonic cells in a process that is well described for ERVs. Zinc-finger proteins (ZFPs) bind to specific sequences in ERVsSetdb1, Ncor2, Rnf2, Kat5, Prmt5, Uhrf1, and Rrp8 led to widespread changes in the expression of TEs, and a corresponding change in chromatin accessibility. Finally, we explore the consequences of these observations by showing that the loss of chromatin modifiers induces a gene expression program similar to totipotent 2 cell (2C) embryos, characterized by the upregulation of MERVL TEs and 2C-specific genes. Overall, our data suggests that the chromatin modifying system manages the expression of TEs by context-specific deposition of chromatin marks that regulate the chromatin state and expression level of TEs.Here, we reveal that TEs are marked by chromatin modifications in complex patterns. Of the 32 chromatin marks we explored, 22 were enriched on at least one TE type. We find evidence not only of repressive marks, but widespread marking of TEs by activatory marks, including bivalent marking of TEs by repressive H3K9me3, and activatory H3K27ac chromatin marks. When we knocked down a panel of chromatin regulators, 29 of 41 led to the deregulation of expression of at least one type of TE. The loss of To explore the chromatin code for the suppression of TEs, we uniformly reanalyzed mouse ESC ChIP/epigenetic-seq data for 32 chromatin marks, including histone methylation, acetylation, ubiquitination, and variant histones, along with DNA 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), and Assay for Transposase-Accessible Chromatin (ATAC)-seq , and a mixture of other ERVs, for example, BGLII_Mus  TEs42. We found that 87/679 TE types were > 2-fold-enriched with H3K9me3, and 72/679 were marked by H4K20me3. H3K9me3 and H4K20me3 tended to co-occur, and they both marked 58/679 TE types , and most types of DNA-binding motif are represented. We noticed that the YY1 and REST motifs were enriched in our analysis. The YY1 motif was enriched at LINE, IAP, and MERVL TEs, as previously reported46, and ChIP-seq data for YY1 showed that YY1 was bound to IAPLTR2b 30 and the CMs SETDB1, SUV39H1/2, TRIM28, and ZFP809 , if the chromatin mark or CM was enriched >2 fold on the TE type Fig. . One maj809 Fig. . Many ofs50 Fig. . The chr251 Fig. . We also52, REST7, or CTCF10. Amongst the Active/Open super-group were several subgroups which could be distinguished based upon their major members, such as the P300/mediator, or Pol II, along with groups that had patterns resembling enhancers (H3K4me1). One of the subgroups contained CHAF1A, a histone chaperone involved in depositing the variant histone H3.3, both of which have been implicated in TE control34. Finally, the Heterochromatin supergroup contained many factors specific for silencing, particularly SETDB1, MBD proteins and the chromatin marks H3K9me3 and H4K20me3. Overall, TEs were bound by a wide-range of CMs associated with both repressive and activatory chromatin , suggesting a higher level of TE-derived RNA , LTR (ERV), and DNA class TEs (See Methods). On average 1–5% of reads would map to TEs  that deregulated 20 or more types of TE for further analysis. Western blotting for the pluripotency proteins OCT4 and NANOG confirmed that the eight selected knockdowns were not inducing differentiation , they are also members of large multi-protein complexes involved in multiple regulations , or there is substantial redundancy . Consequently, the number of potential chromatin mark changes caused by the loss of any single CM can be large. To gain an overview of changes in chromatin in response to the knockdown of a CM, we used ATAC-seqRnf2 was knocked down , in developing mouse embryosIn conclusion, our study reveals the complexity of epigenetic regulation of TEs in ESCs. TEs are marked not only by repressive chromatin marks, but are also widely marked by activatory chromatin marks, including patterns of simultaneous marking by activatory and repressive marks. We show that the loss of CMs caused specific changes in TE expression and chromatin accessibility, and these effects are not limited to the formation of heterochromatin or DNA methylation, but encompass a large fraction of known chromatin modifications. This work suggests that the regulation of TEs is a function of overlapping epigenetic pathways, and that a major task of the epigenetic system is to manage the expression of TEs.All experiments were performed in mouse E14 ESCs (RRID:CVCL_C320), to avoid any differences in genetic background that may influence the number of TEs. Mouse ESCs were grown in serum + LIF medium: DMEM-HIGH GLUCOSE , 15% (v/v) fetal bovine serum , 1% (v/v) L-Glutamine , 1% (v/v) sodium pyruvate , 1% (v/v) non-essential amino acids , 0.5% (v/v) penicillin-streptomycin solution , 0.1 mM 2-mercaptoethanol , and 1000 U/ml mLIF .41. Briefly, pLKO.puro lentiviruses were added to ESCs and the medium was changed after 8 h. Cells were selected with 2 μg/ml puromycin (Gibco) for 2 days, and harvested after a further 2 days. Sequences for the shRNAs are in Supplementary Data CMs were knocked down in serum + LIF medium using pLKO.puro lentiviral system as previously describedg for 20 mins and the supernatant was discarded. The pellet was resuspended in 0.2 N HCl and left at 4 °C overnight. The mixture was vortexed, and the pH was neutralized with 1 M Tris-HCl pH 8.0. Western blots were performed using typical laboratory procedures with the antibodies: anti-H3K9me3 , anti-H3K4me3 , anti-H3K4me1 , anti-H3K27me3 , anti-H3ac , anti-H4K12ac , anti-H4ac , anti-H3K27ac , anti-H2AK119ub , anti-H3 , anti-OCT4 , anti-NANOG , and anti-GAPDH . Uncropped raw images of the Western blot membranes are in Supplementary Figure Histone proteins were extracted by acid precipitation. Briefly, cells from a 6-well dish were scraped, washed with PBS and resuspended in extraction buffer , 5 mM sodium butyrate ), and allowed to extract for 20 mins. The extract was centrifuged at 14,000× 69. Briefly, a 6-well dish of ESCs was scraped and lysed in TRIzol . RNA was extracted according to the manufacturer’s instructions, and 1 μg was submitted for sequencing. All RNA-seq was sequenced to a depth of 50 M tags, and 150 bp paired-end. For RT-qPCR RNA was extracted using TRIzol , cDNA was prepared with reverse transcriptase kit , and RT-qPCR was performed with SYBR Green Master kit . RT-qPCR primers are described in Supplementary Data RNA-seq was performed as described70. A total of ~50,000 cells were washed once with 50 μl of cold PBS and resuspended in 50 μl lysis buffer  IGEPAL CA-630). The suspension of nuclei was then centrifuged for 10 min at 500 × g at 4 °C, followed by the addition of 50 μl transposition reaction mix  of Nextera DNA library Preparation Kit (96 samples) . Samples were then PCR amplified and incubated at 37 °C for 30 min. DNA was isolated using a MinElute Kit (Qiagen). ATAC-seq libraries were first subjected to five cycles of pre-amplification. To determine the suitable number of cycles required for the second round of PCR, the library was assessed by quantitative PCR70, and the library was then PCR amplified for the appropriate number of cycles. Libraries were purified with a Qiaquick PCR (Qiagen) column and were quantified using a KAPA Library Quantification kit  according to the manufacturer’s instructions. Library integrity was checked by gel electrophoresis. Finally, the ATAC library was sequenced on a NextSeq 500 using a NextSeq 500 High Output Kit v2 (150 cycles)  according to the manufacturer’s instructions.ATAC-seq was performed as described71. Ten million cells were centrifuged and resuspended in Buffer 1 ), along with 2 ml of Buffer1 + 0.1% IGEPAL. The resulting 4 ml of nuclei were layered on top of 8 ml of Buffer 3  and centrifuged at 10,000 × g for 20 min at 4 °C unbraked. Nuclei were resuspended in Buffer A ) and digested for 10 mins at 37 °C with MNase (Affymetrix) in buffer A supplemented with 3 mM CaCl2. The reaction was stopped with 5 mM EGTA, centrifuged at 13,500 × g for 10 min, and chromatin resuspended in  and rotated at 4 °C for 2–4 h. The mixture was adjusted to 500 mM NaCl, allowed to rotate for another 45 min and then centrifuged at 13,500 × g for 10 mins. Chromatin supernatant was diluted to 100 ng/µl with buffer B  and incubated for 20 mins at 4 °C with 60 µl protein G beads . Antibody was added and rotated overnight at 4 °C. A volume of 100 µl of Protein G beads were added and the sample was rotated for a further 3 h at 4 °C. The beads were washed three times with buffer B, and once with buffer B without Tween 20. The DNA was eluted with 300 µl of elution buffer  and incubated for 4 h at 56 °C. The resulting samples were extracted with phenol-chloroform followed by purification using a Qiagen MinElute columns, according to the manufacturer’s instructions. Antibodies used: anti-H3K9me3 , anti-H3K27ac . qPCR was performed with SYBR Green Master kit . Primer pairs are described in Supplementary Data Native-ChIP-qPCR was performed as described69. Briefly, reads were aligned to the genome using bowtie272 and RSEM73, with the settings ‘—bowtie2—bowtie2-sensitivity-level very_sensitive—no-bam-output—estimate-rspd’ using an index built against the ENSEMBL v81 transcriptome, and normalized using EDAseq74 (v2.4.1) (which = ‘full’). Samples that did not closely correlate with other replicate samples, clustered closer to unrelated samples, or were outliers (R2 correlation < 0.6), were deleted from the analysis.As a quality control step the RNA-seq data was analyzed for normal gene expression, using the method described75, with the options:—readFilesCommand zcat—outFilterMultimapNmax 100—winAnchorMultimapNmax 100—outMultimapperOrder Random—runRNGseed 777—outSAMmultNmax 1—outSAMtype BAM—outFilterType BySJout—alignSJoverhangMin 8—alignSJDBoverhangMin 1—outFilterMismatchNmax 999—alignIntronMin 20—alignIntronMax 1000000—alignMatesGapMax 1000000. Tags mapping to genes and TEs were counted using TEtranscripts76 with the option:—mode multi. GTF files for gene annotation were downloaded from GENCODE (M9). GTF files for TE annotations were downloaded from the TEtranscripts website (mm10_rmsk_TE.gtf.gz). For the quantification of genes and TEs, samples were normalized using EDAseq74 (v2.4.1) (which = ‘full’). The GC content for TEs was generated by taking the average for all TEs. TEs were normalized at the same time as the genes. This has the effect of suppressing the magnitude of the fold-changes, however the fold-changes reported here are similar to those reported in other studies that used knockdowns40. Differential expression was called using DESeq275, with a P value < 0.05 (Benjamini-Hochberg-corrected) and an absolute fold-change > 2. Gene ontology analysis was performed using goseq (v3.2.5)77. Other analysis was performed using glbase78.For the analysis of TEs, reads were mapped to the mouse genome (mm10) using the STAR aligner11. Briefly, the average divergence between the TE repeats and the consensus (milliDiv from RepeatMasker UCSC track) was calculated for each TE type, and the Jukes-Cantor method with a substitution rate of 4.5e-9 per base per year was applied. This gave an estimate for TE age in years. The results were broadly consistent with other estimates of TE age11.The age of TEs was calculated as in60. A second approach was used to generate a 2C-associated gene signature, by taking the top 200 upregulated genes in each CM knockdown, and then measuring the fold-change of those genes in the 2C sample, relative to the ESCs.The list of 2C-related genes was generated by calculating the fold-change of 2C genes versus ESC genes, and taking the top 200 2C-associated genes. RNA-seq data was from61.MERVL-associated genes were defined as those genes that have a MERVL (either MERVL-int or solo-LTR MT2_Mm) within 10 kb up- or downstream of the gene body. A gene was annotated as the same orientation (same ori.) if it had a MERVL in the same strand flanking it, or in the opposite orientation (opposite ori.) if it had a MERVL element in the opposite strand, as described inMapping statistics and details of all the samples used in this study are contained in Supplementary Data 72 with the options: -p 20—very-sensitive—end-to-end—no-unal. To analyze repetitive sequences, only the best alignment is reported for multimapped reads, if more than one equivalent best alignment was found, then one random alignment was reported. Reads mapping to mitochondrial DNA or unassigned sequences were discarded. For pair-end sequence data, only concordantly aligned pairs were kept. Alignment bam files were transformed into read coverage files (bigwig format) using deepTools79 with the RPKM (Reads Per Kilobase per Million mapped reads) normalization method. For analysis of TEs, coordinates and annotations of TEs were downloaded from the UCSC Genome Browser (GRCm38/mm10) version of RepeatMasker. TEs shorter than 300 bp or those TE types with less than 50 copies were deleted from the analysis. For TE enrichment analysis, as many ChIP and ATAC signals were not evenly distributed across TEs, we divided the TEs into evenly spaced 500 bp bins. The coverage signal on each bin was extracted using deeptools, and the bin with the maximum signal was used as the observed value. For the expected background value, random genomic regions of the same number as the TE type and the same size as the TE were taken, the coverage signal was measured, and the maximal bin was taken as the randomly expected background. Enrichment was calculated by taking the observed over the expected enrichment, and is expressed as log2(fold-enrichment). Quality control was performed by measuring the reads per kilobase of TE per million library sequence reads (RPKM) at all TEs, and then combining replicates where the Pearson correlation was >0.6 (Supplementary Figure 79, motif discovery was performed using MEME80.ChIP-seq data for histone modifications, variant histones, DNA methylation, chromatin accessibility, and a selection of TFs and chromatin modifiers (Supplementary Data GRCm38/mm0 versionP < 0.05, unless indicated. The statistical test used is indicated in the figure legend.No statistical methods were used to predetermine sample size, and experiments were not randomized. The investigators were not blinded to the allocation of experiments, nor the outcome. The number of biological replicates are described in the figure legends, or in Supplementary Data Further information on experimental design is available in the Supplementary InformationDescription of Additional Supplementary FilesPeer Review FileSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Source DataReporting Summary


OUROBOROS or SAMSARA, in the brown alga Ectocarpus result in conversion of the sporophyte generation into a gametophyte. The OUROBOROS and SAMSARA proteins heterodimerise in a similar manner to TALE HD TF life cycle regulators in the green lineage. These observations demonstrate that TALE-HD-TF-based life cycle regulation systems have an extremely ancient origin, and that these systems have been independently recruited to regulate sporophyte developmental programs in at least two different complex multicellular eukaryotic supergroups, Archaeplastida and Chromalveolata.Three amino acid loop extension homeodomain transcription factors (TALE HD TFs) act as life cycle regulators in green algae and land plants. In mosses these regulators are required for the deployment of the sporophyte developmental program. We demonstrate that mutations in either of two TALE HD TF genes,  Brown algae and land plants are two groups of multicellular organisms that have been evolving independently for over a billion years. Their last common ancestor is thought to have existed as a single cell; then, complex multicellular organisms would have appeared separately in each lineage. Comparing brown algae and land plants therefore helps us understand the rules that guide how multicellular organisms evolve from single-celled ancestors.During their life cycles, both brown algae and land plants alternate between two multicellular forms: the gametophyte and the sporophyte. The gametophyte develops sexually active reproductive cells, which, when they merge, create the sporophyte. In turn, spores produced by the sporophyte give rise to the gametophyte. Specific developmental programs are deployed at precise points in the life cycle to make either a sporophyte or a gametophyte.Two proteins known as TALE HD transcription factors help to control the life cycle of single-celled algae related to land plants. Similar proteins are also required for the sporophyte to develop at the right time in land plants known as mosses. This suggests that, when multicellular organisms emerged in this lineage, life cycle TALE HD transcription factors were recruited to orchestrate the development of the sporophyte. However, it was not clear whether TALE HD transcription factors play equivalent roles in other groups, such as brown algae.Ectocarpus, which produce gametophytes when the non-mutated alga would have made sporophytes. Genetic analyses revealed that these mutated brown algae carried changes in two genes that encode TALE HD transcription factors, indicating that these proteins also regulate the formation of sporophytes in brown algae. Taken together, the results suggest that TALE HD transcription factors were originally tasked with controlling life cycles, and then have been independently harnessed in both land plants and brown algae to govern the formation of sporophytes. This means that, regardless of lineage, the same fundamental forces may be shaping the evolutionary paths that lead to multicellular organisms.To address this question, Arun, Coelho et al. examined two mutants of the brown alga Proteins similar to TALE HD transcription factors also regulate life cycles in other groups such as fungi and social amoebae, which indicates that their role is very ancient. It now remains to be explored whether such proteins control life cycles and developmental programs in other multicellular organisms, such as animals. Chlamydomonas, plus and minus gametes express two different HD TFs of the three amino acid loop extension (TALE) family called Gsm1 and Gsp1  locus has been shown to cause the sporophyte generation to be converted into a fully functional (gamete-producing) gametophyte , which diverged from the green lineage over a billion years ago  interval on chromosome 14 using a segregating family of 2000 siblings derived from an ORO x oro cross and a combination of amplified fragment length polymorphism (AFLP)   and micr factor. .samsara-1, samsara-2 and samsara-3, abbreviated as sam-1, sam-2 and sam-3). The sam mutants closely resembled the oro mutant in that gamete-derived parthenotes did not adopt the normal sporophyte pattern of development but rather resembled gametophytes. Young, germinating individuals exhibited the wavy pattern of filament growth typical of the gametophyte and, at maturity, never produced unilocular sporangia  gametophytes and complementation analysis indicated that the mutations were not located at the same genetic locus as the oro mutation  meiotic daughter cells that carried the mutant sam allele. Note that no meiotic defects were observed in heterozygous sporophytes carrying the oro mutation.Genetic crosses confirmed that the mutation . Interes meiosis . This inEctocarpus sporophytes produce a diffusible factor that induces gametophyte initial cells or protoplasts of mature gametophyte cells to switch to the sporophyte developmental program (oro mutant is not susceptible to this diffusible factor (oro protoplasts regenerate as gametophytes in sporophyte-conditioned medium) indicating that ORO is required for the diffusible factor to direct deployment of the sporophyte developmental pathway .The 7_006660 . The ideORO and SAM transcripts were most abundant in gametes , which does not cause switching between life cycle generations . In Chlamydomonas, the Gsp1 and Gsm1 proteins are carried specifically by plus and minus gametes, respectively, so that dimerisation of the two proteins allows the organism to detect syngamy and therefore the transition from a haploid to a diploid state. Based on transcript detection  and at the opposite end of the life cycle, during the diploid to haploid transition (meiosis). However, these phenotypes make more sense when viewed from an evolutionary perspective, if the ORO SAM system originally evolved as a global regulator of diploid phase processes.Interestingly, diploid sporophytes heterozygous for There is now accumulating evidence for an ancient role for HD TFs in life cycle regulation in both the bikont and unikont branches of the eukaryotic tree of life . The ideSporophyte-conditioned medium, gametophyte-conditioned medium and protoplasts were produced as previously described . Protoploro mutation has been shown to behave as a single-locus, recessive, Mendelian factor . These reactions were diluted 1:150 for the selective amplifications. The selective amplifications used an EcoRI-anchored primer and an MseI-anchored primer, each with three selective nucleotides, in various different combinations. The PCR conditions for both steps were 94°C for 30 s, followed by 20 cycles of DNA amplification  and a 5 min incubation at 72°C except that this protocol was preceded by 13 touchdown cycles involving a decrease of 0.7°C per cycle for the selective amplifications. PCR products were analysed on a LI-COR apparatus. This analysis identified two flanking AFLP markers located at 20.3 cM and 21.1 cM on either side of the ORO locus. For 23 (12 oro and 11 wild type) of the 100 individuals, no recombination events were detected within the 41.4 cM interval between the two markers. Screening of these 23 individuals (11 wild type and 12 oro) with the microsatellite markers previously developed for a sequence-anchored genetic map  and an additional 11 microsatellite markers within the mapping interval  and 1.8 pmol of the fluorescence marked M13 primer. The PCR conditions were 94°C for 4 min followed by 13 touch-down cycles  and 25 cycles at 94°C for 30 s, 53°C for 1 min and 72°C for 30 s. Samples were genotyped by electrophoresis on an ABI3130xl Genetic Analyser (Applied Biosystems) followed by analysis with Genemapper version 4.0 (Applied Biosystems). Using the microsatellite markers, the oro mutation was mapped to a 34.5 kbp (0.45 cM) interval, which contained five genes. Analysis of an assembled, complete genome sequence for a strain carrying the oro mutation  in the wild type Ectocarpus reference strain Ec32 included one short region of uncertain sequence 1026 bp downstream of the end of the ORO open reading frame. The sequence of this region was completed by PCR amplification and Sanger sequencing and confirmed by mapping Illumina read data to the corrected region. The corrected ORO gene region has been submitted to Genbank under the accession number KU746822.The sequence of the 34.5 kbp mapped interval containing the SAM gene (sctg_251) with homologous supercontigs from several independently assembled draft genome sequences corresponding to closely related Ectocarpus sp. strains (SAM gene were missing. The complete SAM gene was therefore assembled and has been submitted to Genbank under the accession number KU746823.Comparison of the reference genome (strain Ec32) supercontig that contains the  strains  indicatesam-1 (Ec374) and sam-2 (Ec364) partheno-gametophytes using the Qiagen RNeasy Plant mini kit and any contaminating DNA was removed by digestion with Ambion Turbo DNase (Life Technologies). The generation marker genes analysed were Ec-20_001150 and Ec-26_000310 (sporophyte markers), and Ec-23_004240 and Ec-21_006530 (gametophyte markers), which are referred to as IDW6, IDW7, IUP2 and IUP7 respectively, in SAM gene was used to verify that there was no contaminating DNA  was performed using the software GraphPad Prism5.Total RNA was extracted from wild-type gametophytes and partheno-sporophytes (Ec32) and from Ectocarpus reference genome  of approximately 300 mg (wet weight) of tissue either using the Qiagen RNeasy plant mini kit with an on-column Deoxyribonuclease I treatment or following a modified version  of the pe genome  using Toe genome . The mape genome  to obtaihttp://colorbrewer.org).Differential expression was detected using the DESeq2 package . Blast2GO . In P. patens, GUS fusion experiments failed to detect expression of KNOX genes in the gametophyte but RT-PCR analysis and cDNA cloning has indicated that KNOX (and BEL) transcripts are expressed during this generation  by combining human influenza hemagglutinin (HA)-tagged and glutathione S-transferase (GST) fusion proteins. In vitro transcription/translation of HA-tagged ORO proteins was carried out using the TNT Coupled Wheat Germ Extract System . GST-tagged SAM proteins were expressed in Escherichia coli. Protein production was induced by adding IPTG to a final concentration of 2 mM and shaking for 20 hr at 16°C. After the capture phase, beads were washed four times with 400 μL of washing buffer  at room temperature. Beads were then recovered in SDS-PAGE loading buffer, and proteins analysed by SDS-PAGE followed by ClarityTM chemiluminescent detection . The anti-HA antibody (3F10) was purchased from Roche, and the anti-GST antibody (91G1) from Ozyme.Pull-down assays were carried out using the MagneGSTSaccharina japonica,Cladosiphon okamuranus,Nannochloropsis oceanica, Aureococcus anophagefferens, Phaeodactylum tricornutum, Thalassiosira pseudonana, Pseudo-nitzschia multiseries) and transcriptomes  using both Blast (Blastp or tBlastn) and HMMsearch with a number of different alignments of brown algal TALE HD TF proteins. As the homeodomain alone does not provide enough information to construct well-supported phylogenetic trees, searches for ORO and SAM orthologues were based on screening for the presence of the additional protein domains conserved in brown algal ORO and SAM proteins.Searches for homeodomain proteins from additional brown algal or stramenopile species were carried out against the NCBI, Uniprot, oneKP  and iMicORO and SAM orthologues within the brown algae, this information was also used to search for ORO and SAM orthologues in other stramenopile lineages. However, this analysis failed to detect any additional candidate ORO or SAM orthologues. These observations are consistent with a similar analysis of plant homeobox introns, which showed that intron positions were strongly conserved in recently diverged classes of homeobox gene but concluded that homeobox introns were of limited utility to deduce ancient evolutionary relationships  and consensus sequence logos were generated with WebLogo (http://weblogo.berkeley.edu/logo.cgi). Intrinsic disorder in protein folding was predicted using SPINE-D  lineage, such as the KNOX, ELK and BEL domains. Interestingly, both the ORO and SAM proteins possess regions that are predicted to be highly disordered . Intrins In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Christian Hardtke as the Senior Editor.Thank you for submitting your article "Convergent recruitment of life cycle regulators to direct sporophyte development in two eukaryotic supergroups" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Title: Please consider adding the name of the organism(s) studied; "two eukaryotic supergroups" is not very clear, and for that matter, "life cycle regulators" could probably be stated more precisely (e.g. add TALE-HD).Summary:ORO and SAM, act to promote diploid development in the brown alga Ectocarpus. These data are consistent with the hypothesis that the ancestral function of HD proteins is to activate diploid genetic programs following fusion of haploid gametes. Because the haploid-diploid life cycles of brown algae and the green lineage evolved independently , the authors conclude that TALE homeoproteins evolved convergently as key regulators of sporophyte development in these two groups of eukaryotes. This finding may have broader implications, given that homeoproteins are well known to govern zygogenesis in several fungi, and homeodomain-like proteins play a similar role in social amoebae, suggesting that these functions may have arisen very early in eukaryotic evolution.A highly interesting and timely set of results that represent a considerable step forward in our understanding of the evolution of eukaryotic lifecycle controls. The authors provide evidence that two TALE-HD genes, Essential revisions:oro and sam samples. The authors use these data to define sporophyte and gametophyte genes, an important dataset in itself. Figure 3F shows data for 200 of the gametophyte- and sporophyte-specific genes in the three mutants as well as sporophyte and gametophyte. They show here that the ORO and SAM data are more similar to the WT gametophyte, and state that this supports the conclusion that the two genes are master regulators of the gametophyte-sporophyte transition.1) Regarding the gene expression experiment: RNA-seq data – 13 conditions , although only two biological replicates of each. As described in Supplementary file 9, there are three different wild-type sporophyte conditions, five gametophyte conditions, male and female gametes, and a 'control' mutant sporophyte as well as the ORO and SAM samples are from the gametophyte stage, not parthenotes. If that is the case it doesn't seem surprising that they should resemble WT gametophytes more than WT sporophytes. If the samples in question were partheno-gametophytes, as surely they must have been, that ought to be stated as clearly as possible.How were the WT data in Figure 3F obtained? Are the gametophyte data the average of those five conditions, and the sporophyte data the average of the three sporophyte conditions? Or was one condition of each chosen? Or was the union of several gene lists for the several conditions generated? Please clarify. Furthermore, Supplementary file 9 suggests that the SAM mutants are described as having an effect in the heterozygous state in the sporophyte during the mitotic divisions following meiosis in the unilocular sporangia. While not explicitly stated, this phenotype does not appear to be apparent in oro mutants? If this is the case, do the authors suggest that SAM acts alone at this stage of development, rather than a heterodimer? Or alternatively, the third TALE-HD family member could also act with SAM at this stage – is this hypothesis supported by expression data in Figure 3? Regardless, is the effect non-cell autonomous between the generations? One of the tenets of the model proposed for Chlamydomonas is that the two TALE-HD genes are expressed in the gametes as a mechanism to pre-load the gametes such that upon gamete fusion, a new (diploid) genetic program is initiated. Are ORO and SAM expressed mutually exclusively in the different gametes?2) Physcomitrella are required for induction of the sporophyte program and ectopic expression in the gametophyte can induce the sporophyte program . As written, it suggests that BELL has been shown in Physcomitrella to act in a similar manner as Chlamydomonas, but this is slightly misleading. It is well established that Physcomitrella KNOX is present in the egg , but Horst et al. suggest that BELL too is supplied through the egg, which would make the system surprisingly different from that seen in Chlamydomonas. However, Ortiz-Ramirez et al.  also examine BELL in Physcomitrella and suggest that, in contrast to Horst et al., BELL could be supplied via the sperm , which would be consistent with what is observed in Chlamydomonas. Please add some discussion of the contradictory publications on BELL genes in Physcomitrella.3) In the Introduction, the functions of KNOX and BELL TALE-HD genes in Chlamydomonas are outlined, and then it is stated that BELL genes in  Title: Please consider adding the name of the organism(s) studied; "two eukaryotic supergroups" is not very clear, and for that matter, "life cycle regulators" could probably be stated more precisely (e.g. add TALE-HD).Ectocarpus indicates convergent recruitment of TALE homeodomain life cycle regulators to direct sporophyte development in two eukaryotic supergroups "). We therefore believe that the current title is preferable but we would be happy to use the longer version if the editors think it is necessary.We have modified the title to "Convergent recruitment of TALE homeodomain life cycle regulators to direct sporophyte development in two eukaryotic supergroups ", which adds information about the regulators and the supergroups that are referred to. We have not found a way of adding the name of the study organism without considerably lengthening the title  Regarding the gene expression experiment: RNA-seq data – 13 conditions , although only two biological replicates of each. As described in Supplementary file 9, there are three different wild-type sporophyte conditions, five gametophyte conditions, male and female gametes, and a 'control' mutant sporophyte as well as the oro and sam samples. The authors use these data to define sporophyte and gametophyte genes, an important dataset in itself. Figure 3F shows data for 200 of the gametophyte- and sporophyte-specific genes in the three mutants as well as sporophyte and gametophyte. They show here that the ORO and SAM data are more similar to the WT gametophyte, and state that this supports the conclusion that the two genes are master regulators of the gametophyte-sporophyte transition.How were the WT data in Figure 3F obtained? Are the gametophyte data the average of those five conditions, and the sporophyte data the average of the three sporophyte conditions? Or was one condition of each chosen? Or was the union of several gene lists for the several conditions generated? Please clarify.The gametophyte- and sporophyte-specific genes were identified using duplicate samples for whole gametophytes  and whole sporophytes . We chose to use these samples because the algae had been grown in parallel under identical culture conditions to minimise gene expression differences that may have been induced by differences in the environmental conditions. As pointed out by the reviewer, an alternative approach might have been to compare average expression values for the five gametophyte conditions with average expression values for the three sporophyte conditions. To verify that this alternative approach would not have led to a different result, we tested whether there was a correlation between the sporophyte:gametophyte log fold changes for the 200 most differentially expressed genes determined using either the two pairs of samples or the entire wild type data set . This analysis showed that the expression ratios obtained using the two approaches were strongly positively correlated .oro and sam and partheno-sporophytes for imm). We have previously shown that partheno-sporophytes and diploid sporophytes exhibit comparable patterns of gene expression  but to further validate the data presented in the manuscript we carried out a second comparison with a recent dataset that has been obtained using transcriptomic data for haploid gametophyte and diploid sporophyte samples . Again, this analysis showed that the expression ratios obtained using the using duplicate gametophyte and partheno-sporophyte samples (GBP-5/6 vs. GBP-7/8) were strongly positively correlated with those obtained using the haploid gametophyte and diploid sporophyte data .The sporophyte samples used for these comparisons actually corresponded to partheno-sporophytes because we wanted to be able to compare the expression patterns with those of the mutant lines, which were also determined using parthenotes .Furthermore, Supplementary file 9 suggests that the ORO and SAM samples are from the gametophyte stage, not parthenotes. If that is the case it doesn't seem surprising that they should resemble WT gametophytes more than WT sporophytes. If the samples in question were partheno-gametophytes, as surely they must have been, that ought to be stated as clearly as possible.oro and sam samples are indicated as corresponding to partheno-gametophytes. We have also stated in the text  that parthenotes of the three mutant strains were compared for Figure 3.Supplementary file 9 has been corrected. The 2) SAM mutants are described as having an effect in the heterozygous state in the sporophyte during the mitotic divisions following meiosis in the unilocular sporangia. While not explicitly stated, this phenotype does not appear to be apparent in oro mutants? If this is the case, do the authors suggest that SAM acts alone at this stage of development, rather than a heterodimer? Or alternatively, the third TALE-HD family member could also act with SAM at this stage – is this hypothesis supported by expression data in Figure 3? Regardless, is the effect non-cell autonomous between the generations? One of the tenets of the model proposed for Chlamydomonas is that the two TALE-HD genes are expressed in the gametes as a mechanism to pre-load the gametes such that upon gamete fusion, a new (diploid) genetic program is initiated. Are ORO and SAM expressed mutually exclusively in the different gametes?oro mutant does not show a meiotic defect. This has now been stated in the text . And, yes, we agree that these data suggest that SAM acts alone (or at least not as a heterodimer with ORO) to mediate an essential function during meiosis. The exact form of the factor that acts during meiosis is unknown but this could well be a SAM homodimer or a heterodimer with the third TALE-HD protein Ec-04_000450. We do not have expression data for sporophytes bearing unilocular sporangia but Ec-04_000450 transcripts are detected in upright filaments  or gametophytic (after meiosis and therefore non-cell-autonomous between sam and SAM meiotic products). If the effect is sporophytic then, yes, it would have to be non-cell-autonomous between generations because we observe arrested development of the meio-spores, which are the first cells of the haploid phase.The phenotype observed in ORO and SAM are not expressed mutually exclusively in male and female gametes.The current data suggests that 3) In the Introduction, the functions of KNOX and BELL TALE-HD genes in Chlamydomonas are outlined, and then it is stated that BELL genes in Physcomitrella are required for induction of the sporophyte program and ectopic expression in the gametophyte can induce the sporophyte program . As written, it suggests that BELL has been shown in Physcomitrella to act in a similar manner as Chlamydomonas, but this is slightly misleading. It is well established that Physcomitrella KNOX is present in the egg , but Horst et al. suggest that BELL too is supplied through the egg, which would make the system surprisingly different from that seen in Chlamydomonas. However, Ortiz-Ramirez et al.  also examine BELL in Physcomitrella and suggest that, in contrast to Horst et al., BELL could be supplied via the sperm , which would be consistent with what is observed in Chlamydomonas. Please add some discussion of the contradictory publications on BELL genes in Physcomitrella.glr1/2 mutant, 2) that BELL1 is down-regulated in reproductive organs of the glr1/2 mutant isolated 16 days after gametangia induction  and 3) that delayed sporophyte maturation in the glr1/2 mutant can be corrected by expressing BELL1 under control of the GLR2 promoter . We agree that, taken in isolation, these observations could be interpreted to indicate that BELL1 is supplied uniquely through the sperm. However, no direct evidence is presented that the BELL1 is expressed in antheridia whilst, in contrast, the in-fusion GUS expression patterns reported by Horst et al., 2016, provide strong evidence that BELL1 protein accumulates in the egg. We suggest an alternative interpretation of the results presented by Ortiz-Ramirez et al. in which GLR2 is required for sporophytic expression of BELL1 after gamete fusion and the induction of BELL1 expression in the zygote (and probably throughout the developing sporophyte) is essential for sporophyte development. One interesting possibly would be that BELL1 is actually part of the GLR2-regulated signal transduction pathway, so that maternally-supplied BELL1 would auto-activate the BELL1 gene in the zygote.Ortiz-Ramirez et al. (2017) show 1) that sperm (but not archegonium) function is affected in the The expression patterns of KNOX and BEL genes have now been evoked in the Discussion section of the revised manuscript.


Saccharomyces cerevisiae and Schizosaccharomyces pombe. Also, screenings for lncRNAs in ascomycetes were performed and, just recently, the first full characterization of a lncRNA was performed in the filamentous fungus Trichoderma reesei. In this review, we provide a broad overview about currently known fugal lncRNAs. We make an attempt to categorize them according to their functional context, regulatory strategies or special properties. Moreover, the potential of lncRNAs as a biotechnological tool is discussed.Long noncoding RNAs (lncRNAs) are crucial players in epigenetic regulation. They were initially discovered in human, yet they emerged as common factors involved in a number of central cellular processes in several eukaryotes. For example, in the past decade, research on lncRNAs in yeast has steadily increased. Several examples of lncRNAs were described in  However, also yeast lncRNAs with a repressing function in trans are known. For example, members of the class of the Xrn1 unstable transcript (XUTs), such as TY1, frequently mediate silencing of trans-located target genes  inhibits the expression of the downstream located and partially overlapping sense-oriented gene IME1 (inducer of meiosis 1), thereby preventing erroneous germ cell differentiation and sporulation in haploids  induces the production of the lncRNA IRT1. IRT1 then mediates depositioning of repressive chromatin marks by recruiting the histone methyltransferase Set2 and the histone deacetylase Set3, which leads to a block of transcription initiation of IME1. Moreover, IRT1 hinders binding of the transcription activator Pog1. Upon the onset of meiosis as a response to carbon source and nitrogen starvation in MATa/MATα heterozygous diploid cells, the expression of the Rme1 encoding gene is inhibited by binding of the diploid-specific a1-α1 repressor complex  and TORC1 (target of rapamycin complex I) are produced in both haploid and diploid cells. Those factors are required for binding of the Tup1–Cyc8 complex to the IME1 locus and hence direct inhibition of both IME1 and IRT1 when nutrients are available  prevents germ cell differentiation in haploids and interferes with the transcription of its target gene IME4 depending on the presence or absence of Rme1  represses the expression of its adjacent gene ZIP2, thereby preventing formation of the synaptonemal complex, which is required for chromosome pairing during meiosis  and the telomerase RNA TCL1. TERRA acts as a scaffold for telomeric DNAs and chromatin-modifying enzymes during telomere synthesis and regulates telomerase activity  . Also in this case, pho1 is repressed in response to prt1 transcription under phosphate-rich conditions and expressed upon phosphate starvation, strictly depending on activation by Pho7 , depending on its phosphorylation status  site of prt, thus resulting in the loss of pho1 initiation  motives which are recognized and bound by the central silencing factor Mmi1. Yet, this mechanism is independent from the regulatory impact on their adjacent genes  and causes nucleosome rearrangement and displacement of the factor SFB from the HO promoter. The loss of the activating signal from SFB results in a shut-off of HO expression and thus prevention of mating-type interconversion during re-entering into the cell cycle.The quite newly identified pHO-lncRNA forces nucleosome repositioning at the locus of the downstream located SUT169  is switched on in the presence of galactose and repressed in the presence of glucose to allow regulation of galactose metabolism. Under repressing conditions, GAL10 ncRNA is produced and mediates di- and trimethylation of K4 and dimethylation of K36 on histone 3 by Set2  is expressed in the absence of glucose. The production of mlonRNAs in S. pombe is anticorrelated with antisense transcripts, which are produced from the fbp1 locus under glucose-rich conditions  and encodes asparaginase II, an enzyme performing hydrolysis of asparagine to aspartate and ammonium cations in response to nitrogen starvation   or ribonucleo-proteins (e.g. telomerase RNAs). In human, attempts for the prediction of lncRNAs based on structural mappings were presented , telomere maintenance or gene silencing. Their regulatory mechanisms range from transcription interference and local nucleosome rearrangement, over depositioning of histone modifications and chromatin remodelling, to physical interactions with several factors, acting as a scaffold or bait. Some lncRNAs might be distinguished upon certain characteristics , and others acting on the same cellular process share a similar mode of action. However, in general, classification of lncRNAs is a challenging task. One possible property for categorizing lncRNAs in the future might be their higher ordered structure. Unfortunately, at the current state of knowledge, not enough data are available on this issue.T. reesei. It was presented as a potential tool for strain improvement and the industrial exploitation of T. reesei. Similarly, a specific use of yeast lncRNAs for industrial purposes might be considered. Both S. cerevisiae and S. pombe are commonly applied for biotechnological production processes. Hence, targeted intervention in processes influenced by lncRNAs represents a promising strategy for process optimisation. Ongoing research and steadily improving technologies provide the basis for uncovering yet unknown mechanisms of lncRNAs, their biotechnological applications and the identification of new candidates in yeasts and higher fungi.In addition to yeast lncRNAs, recently, the first functional characterization of a lncRNA was reported for a filamentous fungus, namely