The main roles of placentas include physical protection, nutrient and oxygen import, export of gasses and fetal waste products, and endocrinological regulation. In addition to physical protection of the fetus, the placentas must provide immune protection throughout gestation. These basic functions are well-conserved; however, placentas are undoubtedly recent evolving organs with structural and cellular diversities. These differences have been explained for the last two decades through co-opting genes and gene control elements derived from transposable elements, including endogenous retroviruses (ERVs). However, the differences in placental structures have not been explained or characterized. This manuscript addresses the sorting of ERVs and their integration into the mammalian genomes and provides new ways to explain why placental structures have diverged. Lerista bougainvillii and Zootoca vivipara, the yellow-bellied three-toed skink, Saiphos equalis, exhibits intraspecific variation in which both oviparity and viviparity exist [It is well accepted that the transition from oviparity to viviparity occurred independently in many animal lineages. Along with two other lizard species, ty exist ,2. Althoty exist . Althougty exist ,5, the c2+-dependent adhesion molecule E-cadherin [The initial development of placental mammals differs from other members of the animal kingdom, in which a blastocyst, consisting of the trophectoderm (TE) and the inner cell mass (ICM), is initially formed; TE becomes the major cell type for the placenta, and ICM forms the body structure, including the amnion, which directly surrounds the fetal body. The blastocyst must implant in the maternal endometrium, after which the three germ layers form from the ICM. TE cell layers are epithelial in nature, each expressing Cacadherin ,7. A typcadherin ,9. For the past two decades, various new technologies to analyze DNA and RNA sequences have become available. In addition, various bioinformatics programs have also been developed to analyze the repetitive and non-repetitive sequences on the genomes, which enable the detection of DNA sequences originating from integrated retroviral sequences. However, mammalian placentas’ evolution and structural diversity have not been fully unraveled. This review endeavors to explain the origin and diversity of mammalian placentas.Following fertilization, the first differentiation of embryonic cells results in the formation of a blastocyst with an inner cell mass (ICM) and an outer trophectoderm. The ICM differentiates into the embryo, amnion, yolk sac, and allantois, while the trophectoderm develops into the chorion, the fetal contribution to the placenta. In most mammals, the yolk sac is displaced with the allantois, forming chorioallantoic placentation . In rodeAn epitheliochorial placenta is found in several orders of animals, including even-toed ungulates, whales, dolphins and lower primates, of which the outer chorioallantoic membrane is in direct contact with the maternal uterine epithelium. The synepitheliochorial placenta is found exclusively in ruminants. As the uterine epithelium is lost, the trophectoderm is directly apposed to the maternal connective tissue, and the placentomal (cotyledonary) chorioallantoic placenta develops at discrete regions of pre-existing, non-glandular caruncular areas on the uterine epithelium . An endoIt should be noted that hyenas belong to the order carnivore, but the hyena undergoes hemochorial placentation . A hemocThe outer layer of trophectodermal cells is basically epithelial cells because they possess apicobasal cell polarity, lateral junctions with neighboring cells, and basal contact with the basement membranes ,41. ThesWhen DNA synthesis takes place in the absence of karyo- and cytokinesis, giant trophoblast cells arise through endoreduplication, as in the murine species. Except for animal species exhibiting epitheliochorial placentation, such as pigs and horses, the outermost layer of trophectodermal cells in many eutherians go through cell fusion, resulting in the formation of bi-nucleated, multi-nucleated or syncytiotrophoblast cells . Like maSNAI2, ZEB1, ZEB2, TWIST1, TWIST2, and KLF8 transcripts [Unlike in murine and primate species, the bovine blastocyst does not invade the uterine endometrium. Instead, it elongates beginning on days 13–14 (day 0 = day of estrus), and as the elongation subsides on days 19–20, the elongated bovine conceptus begins its attachment to the uterine epithelium. Two to three days later, the bovine conceptus starts its adhesion to the uterine epithelium when conceptus trophectoderm expresses mesenchymal markers, such as N-cadherin (CDH2) and vimentin (VIM) . It shounscripts . On day The acquisition of EMT in the non-invasive mode of trophectodermal cells was demonstrated in the following experiments. Using bovine trophoblast CT-1 cells , bovine The presence and importance of syncytiotrophoblast cells are often overlooked, particularly the differences in their cellular structures. These cells are derived from trophectodermal cell fusion and are located next to the maternal cell components. These cells manage efficient nutrient and gas exchange. Because syncytiotrophoblast cells are located next to the maternal cells, they are involved in the immunotolerance of the conceptus by the maternal immune system ,58.The chorionic membrane consists of mono-, di- or tri-layers of trophoblast cells. In hemochorial placentation, the human placenta, discoidal in shape, is classified as a monochorial interface as well as exhibiting villous maternal–fetal interdigitations. In murines, including Muridae and Cricetidae, a labyrinthine placenta is developed in the hemotrichorial organization with three trophoblast layers . The firOn the other hand, beavers, rabbits, and bats have two layers of chorionic membranes, or dichorial placentas, with the first layer consisting of syncytiotrophoblast cells and the second of cytotrophoblast cells. It should be noted that Provaviidae (Hyrax), Tenrec ecaudatus and Dipodidae possess hemochorial placentation, and as such, syncytiotrophoblast cells are not formed in their placentas ,62,63. CDuring the last several decades, virologists have occasionally proposed retrovirus’s role in placental evolution. For example, Haig (2012) argued that the placenta became a mammalian tissue in which retroviral genes were ‘domesticated’ to serve an adaptive function in the host . Such anPEG10 and PEG11/RTL1 are paternally expressed imprinted genes [PEG10 was necessary for the formation of primitive placenta in therians [PEG11/RTL1 [LDOC1/SIRH7/RTL7 [syncytins (see next section). PEG10 and PEG11/RTL1 encode proteins that exhibit homology to GAG and POL proteins of sushi-ichi retrotransposon, while LDOC1/SIRH7/RTL7 encodes only GAG-like proteins of the same retrotransposon. As sushi-ichi retrotransposon is an LTR retrotransposon belonging to the group of gypsy retrotransposons [It has been generally accepted that viviparous therian mammals, the marsupials and eutherians, emerged 166 million years ago (MYA) by diversification from oviparous monotremes. Eutherians with chorioallantoic placenta then diverged from marsupials with choriovitelline (yolk sac placenta) 148 MYA [ed genes . It was therians ,67,68, wG11/RTL1 ,69 and LRH7/RTL7 . The fornsposons ,71, it insposons ,73.PEG10 for placental formation was demonstrated through a gene ablation study in mice, which exhibited embryonic lethality at 10 dpc due to a lack of labyrinth and spongiotrophoblast layers [PEG10 plays an essential role in the differentiation and possibly maintenance of trophoblast cells in these layers. PEG11/RTL1 is essential for maintaining placental fetal capillaries [PEG10 is expressed. Therefore, its gene-ablated mice exhibited middle to late embryonic lethality due to growth retardation, resulting from the destruction of the fetal capillary network. Interestingly, PEG10 protease mutants also exhibited perinatal lethality due to severe impairment of the placental fetal capillary network, demonstrating that together with PEG11/RTL1, PEG10 is also crucial for the maintenance of fetal vasculature [PEG10) and a eutherian-specific gene (PEG11/RTL1), respectively.The necessity of t layers . These oillaries ,74. It iculature . TherefoIt was demonstrated that another GAG-like protein of the sushi-ichi retrotransposon, LDOC1/SIRH7/RTL7, is essential for endocrine regulation of the placenta . It has PEG10 and eutherian PEG11/RTL1 and LDOC1/SIRH7/RTL7 genes alone. Despite the statistical improbability of ERV integration, the phylogenetic record shows multiple independent instances of these genes’ entry into disparate clades over the last 50 million years, which implicate the ERV family genes as prime candidates for the emergence of structural diversification of mammalian placentas. In the human genome alone, remnants of 18 endogenous retroviruses can be found, 16 of which have full coding env genes [env structures possess the fusogenic activity and are highly expressed in the placenta [syncytin-1 (ERVWE1) and syncytin-2 (ERVFRDE1), belonging to the HERV-W family and the HERV-FRD family, respectively [syncytin-1 and -2 transcript expression differ: syncytin-1 mRNA, localized to syncytiotrophoblasts, is highly maintained throughout the gestational period, whereas syncytin-2 transcripts are limited to cytotrophoblasts and decline in late pregnancy [Mammalian placentas are extraordinarily diverse in terms of morphology, beyond the influence of therian nv genes . Only twplacenta : syncytiectively ,82. The regnancy . The synregnancy , whereasregnancy .syncytin-2 gene and its fusogenic function were passed on from Platyrrhini to humans (>87.9%), indicating its integration before the divergence of lineage and placing a lower bound on its age at upwards of 40 million years ago [Syncytin-1, however, exists only in Catarrhini, not in Platyrrhini, which indicates the more recent date of infection of 25 MYA, after the separation from the Old World monkeys [syncytin-2 entered anthropoid lineages and acquired fusogenic as well as immunotolerance activity in cytotrophoblasts, generating the syncytiotrophoblast cell layer. Syncytin-1 then entered Catarrhini, upon which it may have taken over the fusogenic activity in these lineages. Gestation in Platyrrhini and Old World monkeys, which do not possess syncytin-1 or its mutated variant, is considerably shorter than in Catarrhines. In humans, syncytiotrophoblasts form from the fusion between cytotrophoblasts approximately 7 to 11 days into gestation and maintain this activity throughout the pregnancy [Phylogenetic analysis suggests that the ears ago . Syncyti humans >.9%, indiregnancy . It shouERVs characterized, syncytin-Rum1 [BERV-K1 [Syncytin-Rum1, found in cattle, sheep, and goats, entered ruminants’ genomes 20 MYA, whereas BERV-K1, found only in cattle, entered cattle genomes 11 MYA [BERV-K2 and BERV-P envs were also reported to be expressed in the bovine placenta, these did not show fusogenic activities [BERV-K3 gag was expressed in the bovine placenta, of which expression is induced by a WNT agonist [ERV-derived genes is still unclear, but they could function for placental development in the bovine species.In ruminants, syncytiotrophoblast cells are not found in their placentas; however, bi-, tri-, or multinucleated trophectodermal cells are instead formed, of which cell fusion starts around the time of conceptus implantation and continues until the end of pregnancy. Among the numerous tin-Rum1  and BERV[BERV-K1  exhibit s 11 MYA . Althougtivities ,37. In a agonist . The molsyncytin-1 and syncytin-2 genes require a transcription factor, glial cell missing factor homolog 1 (GCM1), for their transcriptional regulation. The GCM family genes, regarded as master regulators, are present even in Drosophila [GCM1 and syncytin-1 genes [CD9 mRNA and protein expressions increase in BeWo cells when the cells are treated with forskolin. In addition, CD9 was downregulated by a protein kinase A (PKA) inhibitor, suggesting that GCM1 expression is regulated through the cAMP/PKA signaling system [Both osophila . A multiosophila ,91. CD9 -1 genes . CD9 mRNg system .CRH) and extended flanking regions, including the endogenous retroviral long terminal repeat transposon-like human element 1B (THE1B), Dunn-Fletcher and colleagues provided strong evidence that anthropoid primate-specific retroviral element THE1B controls placental CRH expression [CRH by THE1B is through a transcription factor, distal-less homeobox 3 (DLX3).Through the analysis of transgenic mice carrying a 180-kb human bacterial artificial chromosome DNA that contains the full length of corticotropin-releasing hormone , was originally found in syncytin-1 and syncytin-2 [syncytin family genes, such as mac-syncytin-3 of macaque, syncytin-Ten 1 of tenrec, and syncytin-Car1 of Carnivora. Still, this motif is not found in any exogenous viral sequences [ERV genes in host genomes [In addition to ncytin-2 . The SPRequences . Through genomes .ERVs are involved in mammalian placentation. It is probable that those integrated into the mammalian genome may work in biological systems other than placentation. For example, one study showed that the transcriptional network of proinflammatory cytokine interferon-gamma (IFNG) could have been shaped by ERVs [ERVs, which had dispersed their numerous IFN-inducible enhancers in mammalian genomes. For example, a gammaretrovirus, MER41, was endogenized in the genome of an anthropoid primate ancestor 45-60 MYA with 7190 LTR elements, from which six subfamilies  are found in the human genome. MER41A is located 220 bp upstream of the gene Absent in Melanoma 2 (AIM2), one of the interferon-stimulated genes that encodes a sensor for foreign cytosolic DNA and activates an inflammatory response. This ERV was found to regulate AIM2 through IFN signaling in anthropoid primates [MER41A/B insertions regulate dozens of lineage-specific serum response factor-binding loci, including one adjacent to FBN2. In humans, an increase in FBN2 expression leads to the production of the peptide hormone placesin, which stimulates glucose secretion and trophoblast invasion [Accumulated evidence indicates that  by ERVs . This inprimates . Furtherinvasion . These oERV is not a one-time event but can be just a temporary link in a chain of successive invasions by new ERVs. The new interloper retroviral genes may subsume their role, often with beneficial enhancements such as increased efficacy in trophoblast cell fusion, as observed with Fematrin-1 versus the older syncytin-Rum1 [ERV genes could be lost, but in other cases, they may remain integrated within the genome, repurposed for uses such as immunosuppression as in the case of syncytin-2 and syncytin-Rum1 [In primate evolutions, including humans, syncytin-2 entered the lineages approximately 40 MYA, followed by syncytin-1 integration 25 MYA. Similarly, in Bovidae, syncytin-Rum1 integrated into the bovine genomes 20 MYA, followed by BERV-K1 (Fematrin-1) 11 MYA. In fact, syncytin-Rum1 is found not only in the bovine subfamily but also in sheep and goats, whereas Fematrin-1 is found only in the bovine subfamily and not in sheep or goats. It appears that during evolution, the exaptation of one tin-Rum1 ,19. Meantin-Rum1 ,98,99. Ttin-Rum1 . For exatin-Rum1 . The batERV-env (syncytin) genes are the main reasons for placental diversity in mammals  or non-invasive (ruminants). Although much research on elucidating placental diversity has been done, molecular mechanisms associated with the construction of structural differences in mammalian placentas have not been definitively characterized. It has been generally accepted that successive integrations of  mammals . The facTHE1B sequence serves as a cis-element for regulating corticotropin-releasing hormone (CRH) gene expression. Further support is expected as genomic and transcriptomic data analytical tools are developed and refined. Recently, progress has been made in research into ERV sequences serving as transcriptional and translational regulators [Dunn-Fletcher and colleagues demonstrated that the retroviral gulators ,101,102.ERVs is also contingent on the perfect confluence of criteria as follows:(a)Fematirn-1 integration into the intron 18 of pregnancy-specific FAT2 gene;The insertion of ERVs can make functional genes of the host placenta-specific. i.e., (b)IFNG, THE1B on CRH;Its own LTR is sufficient to transcribe its own gene segments, which serve as the cis-acting element(s), resulting in the activation of a host gene. i.e., (c)syncytin-1 and syncytin-2.It can make use of transcription factors utilized by the pre-existing gene, as per the baton-pass hypothesis. i.e., A transcription factor GCM1 for (d)The ERV is co-opted along with its promoter/enhancer in the integrated genome; i.e., SPRE ;(e)ERVs.There is cooperation with miRNAs and/or lncRNAs, yet not definitely characterized under placental/trophectodermal conditions, either alone or together with Nevertheless, solid confirmation of a retrovirus integration into sperm or egg has not been obtained, and the mechanism of integration remains unclear. The rarity of such events owes in no small part to the narrow windows of possibility for infection, but conversion to active ERVs and transposons during gestation, thereby facilitating the selection of advantageous genes from a broader market. Such extraembryonic circumstances might have allowed for not only the domestication of ERVs to establish novel endogenous genes via multiple selections but also the dissemination of ERVs and transposons throughout genomes as transcriptional regulators. Moreover, ERVs could serve as cis- and/or trans-acting factors for functional genes of the host. Similarly, various degrees of maternal-fetal cell interactions in the uterine compartment may [In general, the placentas have lower DNA methylation levels than embryos, allowing free expression of ment may  have ledment may ,103.ERVs linked to such variations; however, the mechanisms associated with placental diversity have not been characterized.Exhaustive research on the elucidation of cellular mechanisms associated with structural diversity of the placenta has continued for several decades. Still, in the last twenty years, new methods such as RNA-seq, iTRAQ, and ChiP-seq analyses have become available to analyze whole transcripts, proteins, and their upstream events to regulate such expression. These advancements have made it possible to isolate molecular events regulating placental diversity and the PEG10 and eutherian PEG11/RTL1 and LDOC1/SIRH7/RTL7 genes, followed by independent yet successive integrations of syncytin-type genes for structural variations. A question remains whether the placental structures we know now are the ultimate forms or are still evolving. If it is the latter, placental structures may still be diversifying, and new variations await discovery.It is now clear that the emergence of mammalian placentas was made possible with the acquisition of therian


Cardiovascular disease (CVD) remains the number one cause of death worldwide. Ischemic heart disease contributes to heart failure and has considerable morbidity and mortality. Therefore, alternative therapeutic strategies are urgently needed. One class of epigenetic regulators known as pioneer factors has emerged as an important tool for the development of regenerative therapies for the treatment of CVD. Pioneer factors bind closed chromatin and remodel it to drive lineage specification. Here, we review pioneer factors within the cardiovascular lineage, particularly during development and reprogramming and highlight the implications this field of research has for the future development of cardiac specific regenerative therapies. Cardiovascular disease is the number one cause of death in the U.S and worldwide. Vascular and cardiac disease results in considerable morbidity and mortality , 2. The Reprogramming of lineages has received intense interest and several outstanding reviews are available and provide a comprehensive overview of the field , 6. ThisWhile the role(s) of master regulators focused primarily on their ability as transcription factors to govern lineage specific gene expression, pioneer factors function to bind nucleosomal DNA and relax the chromatin landscape upstream of lineage specific genes , 8, 9. ICardiovascular development is a well-coordinated and complex process that requires the specification, proliferation, migration and differentiation of progenitor cells that become coupled to form a functional syncytium within the heart –14. ProgPdx1 in the mouse results in the absence of the pancreas and similar to MYOD, ectopic overexpression of PDX1 converts somatic cells to pancreatic acinar cells  to specific lineages , 16. In ar cells , 19. Othar cells , 20–24. ar cells , 26  shares a similar structure to that of linker histones, which enables this pioneer transcription factor to displace linker histones from nucleosomes to remodel chromatin and promote liver development  on pioneer factor binding. A mechanism that has been observed is the binding of pioneer factors to nucleosomal DNA sites without any apparent effect. These regions are termed “pioneer factor resistant sites” and are being examined to define the co-regulatory mechanisms that are present in the chromatin environment to control cell fate by promoting or preventing chromatin remodeling and lineage specification by a pioneer factor following binding . For exaA majority of the pioneer factor studies have focused on the characterization of the activation of lineage transcriptional programs and the initial functions of pioneer factors. However, the mechanisms driving lineage repression and the later stages of lineage specification are not well understood and warrant further investigation. ASCL1 is one of the few pioneer factors whose repressive role has been explored and found to recruit cooperating factors such as the myelin transcription factor 1- like protein (MYT1L), which represses the myogenic lineage program during the reprogramming of fibroblasts to neuronal cells . StudiesBrg1 null embryos are lethal prior to implantation  is necessary in order to drive lineage development and reprogramming , 25, 39.antation . Using iantation , 43. BRGantation   , and was recently identified as a pioneer factor . Isl1 KOs (CPCs) –56. In rs (CPCs) . They alGata4 has been shown to lead to early cardiac defects and results in bifed (non-fused) heart fields and embryonic lethality  to remodel chromatin . ET. ET37). utations , 51, 111 in vivo . Our rec in vivo . We charectively . Understectively . Forkheahromatin . Furtherhromatin , 114.forkhead factors have demonstrated that the winged helix DBD of the pioneer factor FOXA resembles that of the structure of linker histones, while Ets factors have been shown to use their short α-helix structure to bind the major groove of DNA to target nucleosomes (ETV2 possesses an Ets DNA binding domain (DBD) characterized by a winged helix-turn-helix structure which needs to be studied in terms of how it interacts with nucleosomal DNA to allow for chromatin binding and remodeling to occur . Previouleosomes . We hypoUnlike ISL1 and GATA4, the pioneer function of ETV2 in the cardiovascular lineage is independent of its cellular context, whether it is cellular differentiation or reprogramming, it functions in a similar fashion in both model systems. Future studies will need to focus on further characterization of the molecular mechanisms driving endothelial cell development/reprogramming by ETV2 to enhance therapeutic approaches to develop mature vasculature for ischemic diseases. While ETV2 is an essential regulator of hematopoietic development, we did not define ETV2 as a pioneer factor for hematopoietic lineages and therefore we hypothesize that other co-factors and pioneer factors might facilitate this developmental process. For example, EBF1, PU.1 and C/EBPα regulate hematopoietic development and act as pioneer factors for the B cells, DN3 t cells and macrophages –122 Tab. Whetherin vivo (sequencing) and in vitro , (2) pioneer factors need to promote chromatin remodeling around DNA binding sites by themselves or by interacting with chromatin remodelers and (3) pioneer factors need to enact global epigenetic changes  and recruit other co-factors that further promote the development or reprogramming of a cellular lineage  that comprise the four chambered organ. For example, while ETV2 sits at the top of the endothelial lineage developmental hierarchy, ISL1 and GATA4 are two of many regulators of the cardiac muscle lineage with very specific functions. We predict that multiple pioneer factors will be required to regulate cardiac muscle development and reprogramming. Other cellular lineages that were not discussed include: smooth muscle and cardiac fibroblasts as no pioneer factors have been identified for these lineages. Pioneer factors can be powerful tools for the development of regenerative therapies whose goal is to generate mature and functional cell lineages. Understanding the molecular mechanisms that drive lineage development by these and other pioneer factors within the cardiovascular lineage will be instrumental because coupling these pioneer factors along with chromatin remodelers and downstream targets genes can amplify the molecular effect needed to better develop regenerative therapies for cardiovascular disease.JS-P and DG: conceived the review and wrote the manuscript. All authors contributed to the article and approved the submitted version.We are grateful for the support of the Department of Defense (Grant # 3002-11765-00089113).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The mature SP cells then emigrate out of the thymus and further differentiate into distinct subsets under different environment signals to perform specific functions. Each step is regulated by various transcriptional regulators downstream of T cell receptors (TCRs) that have been extensively studied both in vivo and vitro via multiple mouse models and advanced techniques, such as single cell RNA sequencing (scRNA-seq) and Chromatin Immunoprecipitation sequencing (ChIP-seq). This review will summarize the transcriptional regulators participating in the early stage of T cell development reported in the past decade, trying to figure out cascade networks in each process and provide possible research directions in the future.T-lymphocytes play crucial roles for maintaining immune homeostasis by fighting against various pathogenic microorganisms and establishing self-antigen tolerance. They will go through several stages and checkpoints in the thymus from progenitors to mature T cells, from CD4 T cells widely participate in the innate and adaptive immune responses throughout the lifetime. T cell development is tightly regulated by numerous factors including transcriptional and epigenetic regulators. The proper development and differentiation of thymocytes is the foundation for the function of the immune system.There is no doubt that the thymus is the fundamental place of thymocytes development that is highly organized, where thymocytes go through several stages and checkpoints before maturation and under-control of a network of multiple players , 2. Thym-CD8- double negative (DN), CD4+CD8+ double positive (DP) and CD4+CD8- or CD4-CD8+ single positive (SP). Then, mature SP cells will migrate to the periphery. Particularly, the DN population can be divided into four stages according to the expression of CD25 and CD44, starting from DN1 (CD44+CD25-), followed by DN2 (CD44+CD25+), DN3 (CD44-CD25+) and DN4 (CD44-CD25-) (The early thymic precursor (ETPs) that come from bone marrow will go through different thymocyte developmental stages including CD4At the DP stage, TCRα gene rearrangements initiate and mature αβ-TCR will be produced. Subsequently, thymocytes must pass through both positive and negative selections to become mature T cells. Thymocytes with functional TCRs interact with the major histocompatibility complex (MHC) on cortical epithelial cells (cTECs) presenting foreign antigens will survive . Thus, pEach developmental step requires the participants of transcriptional regulators that have been elucidated through advanced genomic techniques to identify the binding sites –9. The tTcf7 gene) expression at the ETP stage. Then lead to the activation of two major target genes, Gata3 and Bcl11b (B-cell lymphoma/leukemia 11B)  , 13. TCFigenesis . Moreoveigenesis . Most reigenesis . Collectvia Notch1  of Cd4 gene cooperates with RUNX which is involved in T-helper inducing POZ-Kruppel factor (ThPOK) pathway (The function of each RUNT-related transcription factors (Runx) family member is still poorly understood based on current studies. Nevertheless, it is well known that Runx family members, including Runx1, Runx2 and Runx3 are another crucial transcription factors facilitating early T cell development. The activity of Runx1 has been highlighted in the relationship with other key transcription factors such as Bcl11b and Pu.1, which regulate the dynamic changes of transcriptional signatures before and after T cell commitment respectively. In addition, enforced expression of Runx2 affects β-selection resulting in an expansion of DN cells . The int pathway . Herein, pathway . The hyp pathway , 31.Tcra, Tcrb, Tcrg and Tcrd). Murine Tcra and Tcrd are organized into a single genetic locus (Tcra/Tcrd locus) that undergoes V(D)J recombination in DN thymocytes to assemble the Tcrd gene and in DP thymocytes to assemble Tcra gene, to generate diverse TCR repertoires  and turns off-target gene expression in the absence of Wnt signals. In the absence of TCF-1, ISP thymocyte development is blocked which contributes to DP thymocytes reduction  , 42. Anoeduction .CD4 silencer and represses transcription factors in immature DN thymocytes followed by CD8 expression to promote DN-to-DP transition, then down regulate in DP stage  and Nur77 are TCR-induced proteins with pro-apoptotic function. Bim is important for clonal deletion whereas Nur77 is often dispensable but able to influence late DP thymocytes apoptosis , 62.Interestingly, nuclear speckle-related protein 70 (NSrp70) is selectively expressed on developing thymocytes and highest at DP stage. NSrp70 could regulate cell cycle and survival of thymocytes by governing the alternative processing of various RNA splicing factors, such as oncogenic serine/arginine-rich splicing factor SRSF1 . This fi+ or CD8+ expressing SP cells. Calcium flux signaling is required for positive selection of T cells. Our results demonstrate a newly discovered adaptor named Tespa1  is essential for positive selection by modulating the interaction with a Ca2+ release channel - inositol 1,4,5-trisphosphate receptor (IP3R) which express on ER membranes  family. In a way, the pioneer transcription factors Foxa1 and Foxa2 (forkhead box protein A) regulate alternative RNA splicing during thymocyte positive selection. Another Fox protein Foxo1 may induce the selection and maturation of DP thymocytes that can be accelerated in the deficiency of transcription repressor Gfi1(Growth factor independent 1). Thus, the Gfi1-Foxo1 axis shapes the proper generation of SP cells , 68. AddLastly, the achievement of positive selection is inseparable from epigenetic regulation which cross-work with transcriptional signals. HDAC7 (Histone deacetylase 7) has been reported exporting from the cell nucleus during positive selection in mouse thymocytes and modifying genes to mediate the coupling between TCR engagement and downstream events that determine cell survival including MAPK activity .Negative selection is critical to delete highly self-reactive thymocytes to prevent autoimmunity. The thymocytes who pass the negative selection will become mature T cells with low self-reactivity and export to periphery immune organs.The proceed of negative selection depends on functional mTECs, whose development is powerfully promoted by transcription factors Foxn1 (forkhead box family N1) and Aire (autoimmune regulator), which control the differentiation and maturation repectively . ConditiOn the other hand, the function of mTECs is highly dependent on their characteristic features such as ectopic expression of tissue-restricted antigens (TRAs) and their master regulator Aire, whose expression is restricted to a mature subset of mTECs. Aire induces tissue-specific antigens to ensure negative selection by directly binding the promoter of the target gene within the medulla , 76. TheIn addition to the promoters, the transcriptional repressors of negative selection are indispensable. NCoR1 is a nuclear receptor co-repressor to connect repressive chromatin-modifying enzymes to gene-specific transcription factors. NCoR1 restrains negative selection by repressing pro-apoptotic factor Bim expression, which is expressed elevated in the absence of NCoR1. NCoR1-null thymocytes show excessive negative selection and reduced mature SP thymocytes –81. NCoRin vivo and vitro using ingenious animal models and molecular biological approaches.We conclude the map of T-lymphocyte development in the thymus and related transcriptional regulators that have been reported in the past decade .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The DSIF complex comprising the Supt4h and Supt5h transcription elongation proteins clamps RNA polymerase II (RNAPII) onto DNA templates, facilitating polymerase processivity. Lowering DSIF components can differentially decrease expression of alleles containing nucleotide repeat expansions, suggesting that RNAPII transit through repeat expansions is dependent on DSIF functions. To globally identify sequence features that affect dependence of the polymerase on DSIF in human cells, we used ultra-deep ChIP-seq analysis and RNA-seq to investigate and quantify the genome-wide effects of Supt4h loss on template occupancy and transcript production. Our results indicate that RNAPII dependence on Supt4h varies according to G + C content. Effects of DSIF knockdown were prominent during transcription of sequences high in G + C but minimal for sequences low in G + C and were particularly evident for G + C-rich segments of long genes. Reanalysis of previously published ChIP-seq data obtained from mouse cells showed similar effects of template G + C composition on Supt5h actions. Our evidence that DSIF dependency varies globally in different template regions according to template sequence composition suggests that G + C content may have a role in the selectivity of Supt4h knockdown and Supt5h knockdown during transcription of gene alleles containing expansions of G + C-rich repeats. Elongatg factor ,3), whicg factor . The DSIg factor  to closeg factor ,13.HTT gene  and  and HTT P56 gene —which arA lentivirus construct carrying human Supt4h shRNA (clone ID: TRCN0000019645 from Dharmacon having the antisense sequence TTAAAGTTACTGACTCGCTGC) and a gene encoding resistance to the antibiotic puromycin was introduced into the iPSC cell line GM23225 (Coriell Institute). Puromycin was used to select cells that acquired the construct. Knock-down of Supt4h in iPSCs and NPCs was confirmed by western blotting . Knock-dAs described , iPSCs wTotal RNA was extracted using RNeasy kit (Qiagen) according to the manufacturer's instructions. ScriptSeq v2 RNA-seq kit (Epicenter) was used following the manufacturer's instructions. The workflow included rRNA removal (RiboZero technology) followed by RNA fragmentation and ligation-free cDNA synthesis for preparing directional RNA-seq libraries. The amplified libraries were further purified by gel excision and extraction and were analyzed using an Illumina HiSeq 2000 sequencer.DNA occupancy by RNAPII-S2 was evaluated by ChIP-seq analysis. Human ChIP-seq samples  produced7 cells were collected and incubated in 1% formaldehyde for 10 min at room temperature and then quenched by addition of 125mM Glycine. Nuclear lysates were prepared by sonication using a Branson 250 Sonifier. Clarified lysates were treated overnight at 4°C with 5 μg anti-pol II CTD repeat YSPTSPS (phosphor S2) (Abcam ab5095) antibody. Protein–DNA complexes were captured on Protein A/G agarose beads (EMD Millipore) and eluted in 1% SDS TE buffer at 65°C. Following cross-link reversal and purification, ChIP DNA sequencing libraries were prepared according to Illumina DNA sample kit instructions (Illumina). Libraries were sequenced on an Illumina HiSeq 4000 instrument. The experiments were done in two biological replicates  was calculated by PEARSON function of Microsoft Excel to reflect the extent of a linear relationship between data sets.Mouse RNA-seq (GEO accession number: GSE33497) samples  had an aGene lengths were defined as the number of nucleotides from the TSS to TTS, including introns. To increase precision in the identification of short template segments occupied by RNAPII-S2, >300 million reads, each ∼100 bp in length, were sequenced for each sample , produciFor each gene, we determined the G + C content in the most abundant isoform as defined using a corresponding RNA-seq control sample. A 100 bp sliding window was used to scan the gene body, including exon and intron, from TSS to TTS. We calculated the average G + C% for each sliding window along the gene for heatmap visualization. We also calculated the average G + C% for the whole gene body, excluding the first 1kb after TSS, which for virtually all genes contained a promoter region high in G + C, to represent overall G + C content of the gene.2(ChIP-seq_FPKM_Supt4h KD/ChIP-seq_FPKM_UNT). ESKOR value can be either positive or negative. Zero ESKOR represents no difference. Positive ESKOR means more RNAPII occupancy in Supt4_KD than untreated, and vice versa.RNAPII-S2 ChIP-seq fragments per kilobase of transcript per million fragments mapped (FPKM) was calculated from the ChIP-seq read count and normalized according to whole gene body length, using procedures employed for RNA-seq FPKM calculations. Genes were chosen for further analysis based on the following criteria: (i) a gene body length (from TSS to TTS including introns) no longer than 5 kb; (ii) RNAPII-S2 ChIP-seq FPKM measurements >0.5 and (iii) transcription of these genes to an extent that resulted in their inclusion also in the RNA-seq list. ESKOR (Effect of Supt4h Knockdown on Occupancy by RNA polymerase) was calculated as logEarlier work indicates that knockdown of components of the DSIF complex differentially reduces expression in neural cells of mutant genes containing expanded nucleotide repeats ,21,22. IDrosophila polytene chromosomes indicates that Spt5 co-localizes with the elongating Ser2-phosphorylated form of RNAPII  and confirmed by Western blotting . PreviouNAPII-S2 ,8). We tNAPII-S2  during CNAPII-S2 . Read coNAPII-S2 . 8779 geAs shown in Figure 2(RNAPII-S2 ChIP-seq_FPKM_KD/RNAPII-S2 ChIP-seq_FPKM_UNT). Altered template occupancy resulted in ESKOR values deviating from zero  . ESKOR values were defined as the log ratio of FPKMs obtained from Supt4h knockdown (KD) versus untreated (UNT) NPC cells: logo Figure . Overallo Figure . RNA-seqo Figure  resultedr = –0.78). Consistent with this correlation were the results of heatmap analysis of G + C content in 100bp sliding windows  and template sequence were examined. When chromosomal genes were ranked from high to low according to ESKOR Figure  for eachs Figure  that extTo further understand genome sequence characteristics that affect ESKOR, we investigated additional correlations among the 8779 genes studied, . Our anaP = 1.68 × 10−23), while read counts from the lowest G + C segments were barely affected by Supt4h knockdown (P = 0.48)  (C9orf72) (NOP56) (Transcription through genes containing repeat expansions in Huntington's Disease (the TT gene) , AmyotroC9orf72) ,21,56, a (NOP56)  has been (NOP56) ,58. Such (NOP56) . We suggIn addition to performing ChIP-seq in NPC samples, we re-analyzed previously published ChIP-seq data obtained from mouse embryonic fibroblasts in which Supt5h was absent . Notwithhttps://www.ncbi.nlm.nih.gov/geo/) under accession number GSE169466.The sequencing data generated in this study have been submitted to the Gene Expression Omnibus (GEO; lqac054_Supplemental_FileClick here for additional data file.


Epigenomics encompasses a broad field of study, including the investigation of chromatin states, chromatin modifications and their impact on gene regulation; as well as the phenomena of epigenetic inheritance. The epigenome is a multi-modal layer of information superimposed on DNA sequences, instructing their usage in gene expression. As such, it is an emerging focus of efforts to improve crop performance. Broadly, this might be divided into avenues that leverage chromatin information to better annotate and decode plant genomes, and into complementary strategies that aim to identify and select for heritable epialleles that control crop traits independent of underlying genotype. In this review, we focus on the first approach, which we term ‘epigenome guided’ improvement. This encompasses the use of chromatin profiles to enhance our understanding of the composition and structure of complex crop genomes. We discuss the current progress and future prospects towards integrating this epigenomic information into crop improvement strategies; in particular for CRISPR/Cas9 gene editing and precision genome engineering. We also highlight some specific opportunities and challenges for grain and horticultural crops. Epigenomics, through its exploration of the combinatorial code of chromatin modifications, can play a role in crop improvement strategies by enriching our understanding of crop genomes and as the molecular basis of traits. The investigation of heritable epigenetic variation that underpins traits in crops is an accelerating area of research with many exciting challenges on the horizon, including better understanding the epigenetic basis of traits, the stability and heritability of epialleles, and the sources of epigenetic variation in crops. These aspects have been reviewed extensively in recent years . In thisteosinte branched 1 (tb1) [VEGETATIVE TO GENERATIVE (VGT1) enhancer of ZmRap2.7 [Crop genomes vary dramatically in size , ranging 1 (tb1)  and the ZmRap2.7 , where gde novo annotate or refine existing genome annotations [Different parts of the genome, such as expressed genes, silent genes, TEs and CREs are marked by different chromatin states. These are defined by the presence or absence of specific covalent histone or DNA modifications. Studying the chromatin states of different genomic elements has greatly advanced our understanding of the roles of various chromatin modifications. However, researchers have noted the power of reversing this approach and using chromatin states to otations ,20,21 orotations . There aThe specificity and quantity of transcription are regulated via the interactions between sequence-specific DNA-binding transcription factors (TFs) and the CREs in promoters and distal enhancers . TranscrMedicago truncatula, rice, tomato [ATAC-Seq (the assay for transposase-accessible chromatin with high-throughput sequencing) is a powerful approach for the identification of CREs. It takes advantage of an engineered Tn5 transposase's preference to insert into accessible chromatin regions . This me, tomato , sorghum, tomato . Signifi, tomato . Additio, tomato , includi, tomato ,28 and M, tomato . These cHistone modifications are an important chromatin modification that can be used to guide genome annotation. Histones are primarily defined by their role in the packing of DNA into chromatin fibers. Nucleosomes represent a barrier to transcription, accordingly, their placement and modifications affect transcription or silencing of particular DNA regions; some modifications are more favorable to transcription. To facilitate these processes, histones are subject to a wide variety of post-translational modifications including methylation, acetylation and phosphorylation . The golThese patterns of chromatin modifications, especially histone modifications, can give insight into the regions of the genome that may be fruitful targets for gene editing. In particular, this information is useful not only to identify functional genes but also to refine gene boundaries and resolve inconsistencies in gene annotations. Mendieta et al. , appliedaccessible gene distal plant CREs are likely characterized by three types of chromatin signatures /CRISPR-associated protein 9 (Cas9) have paved the way for a new era of genome engineering . These tWUSCHEL (WUS) and CLAVATA3 (CLV3) in meristems is to maintain the balance between cell differentiation and self-renewal. In tomato, natural and induced mutations that affect the expression of WUS and CLV3 can positively affect the size of tomato floral meristems that leads to enlarged fruits compared with the wild progenitors [CLV3 has generated a range of new alleles that in turn produce a continuum of phenotypic variation for tomato fruit size [CLE genes [CLE knockout alleles had a deleterious effect on yield, while cis-regulatory editing to change gene expression boosted yield. Other recent work in both rice [In contrast with targeting a coding sequence, there is great potential in using CRISPR/Cas to generate expression variation by targeting gene-regulatory sequences ,57. For genitors . In factuit size  and indeuit size . EngineeLE genes . In thisoth rice  and tomaoth rice  has usedoth rice ; thus, wFWA leading to highly heritable changes in gene expression and flowering time [Epigenome editing might also help address some of the shortcomings of gene knockout. Promoter DNA methylation is strongly associated with decreased gene expression ,65. Receing time . TherefoVERNALIZATION1 (VRN1) affect both flowering, shoot architecture and root architecture in wheat and barley [TEOSINTE BRANCHED1 (TB1) affects both lateral branching and inflorescence architecture in maize and wheat [CLV3–WUS stem cell homeostasis network [Editing gene promoters and CREs may offer the possibility to introduce novel alleles in ways that cannot be achieved by knockout mutations or conventional breeding. This may be extremely useful in some specific situations as outlined in d barley ; likewisnd wheat ,74. Targnd wheat  and the  network , or to gDNA methylation is highly stable throughout most of the vegetative development of a plant, this means that the profile of a single tissue, for example, a leaf could potentially be extrapolated to predict genes and CREs relevant in other tissues or organs ,77–79. IDEMETER-like DNA demethylases mutant, SlDML2, where the resulting phenotype was a non-ripening tomato [In several species, DNA methylation patterns have been observed to change during fruit ripening, including tomato , pepper g tomato . The mecg tomato . In orang tomato , presentDNA methylation patterns in grains also need careful consideration. In developing rice grains DNA methylation profiles fluctuate through grain development as part of the regulatory mechanism for seed maturation . DNA metInstances in which DNA methylation is more dynamic could present issues when using UMRs from vegetative tissues as predictions for CREs in reproductive tissues. One possibility is that predicted CREs might become methylated and no longer function in these tissues. Yet, these regions could still be edited to alter expression patterns in non-reproductive tissues, for example, by removing a repressor element. More likely, the more dynamic nature of DNA methylation in fruits and grains raises the possibility that cryptic CREs could be uncovered in these tissues due to the removal of DNA methylation. Indeed, this is the case in tomatoes, where cryptic binding sites for the transcription factor RIN are uncovered during ripening . The samEpigenome guided improvement is already being deployed for effective modification of traits in crops.The presence, absence and combination of chromatin modifications can be used to locate genes and gene-regulatory regions in crop genomes.The fruits and grains of crop species may represent special cases that require further investigation because patterns of DNA methylation can change in the cells that comprise these reproductive organs.We foresee great potential for epigenome guided improvement in the future, most notably to pinpoint targets for genome engineering to fine-tune gene expression.


Drosophila and humans, the molecular mechanisms by which euchromatin is preserved from heterochromatin spreading have been extensively studied, leading to the identification of insulator DNA elements and associated chromatin factors (insulator proteins), which form boundaries between chromatin domains with antagonistic features. In contrast, the identity of factors assuring such a barrier function remains largely elusive in plants. Nevertheless, several genomic elements and associated protein factors have recently been shown to regulate the spreading of chromatin marks across their natural boundaries in plants. In this minireview, we focus on recent findings that describe the spreading of chromatin and propose avenues to improve the understanding of how plant chromatin architecture and transitions between different chromatin domains are defined.As in other eukaryotes, the plant genome is functionally organized in two mutually exclusive chromatin fractions, a gene-rich and transcriptionally active euchromatin, and a gene-poor, repeat-rich, and transcriptionally silent heterochromatin. In  By using X-ray mutagenesis, Muller identified partial suppressors of white in which some facets of the eye remained white while others developed the red pigmentation, suggesting that the white allele itself was not mutated , which limit the spreading of the Silent Information Regulatory proteins (Sir) promoting the establishment of silenced chromatin in revisiae . Similarrevisiae . Anotherrevisiae . Howeverrevisiae . In generevisiae . This raDe novo methylation of DNA  involves the RNA-directed DNA methylation pathway (RdDM) as well as the DOMAINS REARRANGED METHYLASE 1 and 2 (DRM1/2) proteins  histone methyltransferases . CHH andproteins ,b.Arabidopsis thaliana, the RDR6 pathway starts with the processing of TE mRNAs by RDR6 in order to produce a double-stranded RNA, which is cleaved by the endonucleases DCL2 and DCL4 (in situ by Pol V. This complex recruits the DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which catalyzes de novo cytosine methylation  were initially defined in humans as small euchromatin regions within large heterochromatin regions, which contain expressed genes, are depleted for H3K9me2, and are enriched for CTCF binding sites . Despiteposition . Hence, RDR2 ortholog MOP1 decreased the production of 24-nt siRNAs by 100-fold and altered the formation of mCHH islands  binding motif . FV is a recipient of a PhD fellowship from the French Ministry of Higher Education, Research and Innovation. Work by LVM was supported by the French National Research Agency (ANR-20-CE43-0010).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Salmonella typhimurium were shown to exhibit increased MNase sensitivity specifically at genes implicated in immune responses. Increased sensitivity at the −1-nucleosome permitted transcription factor and RNA Pol II binding events. This system illustrates how cytoplasmic signals induce altered chromatin states to produce a specific cellular response to a stimulus. Innate immune activation is a longstanding model for inducible promoters, transcriptional activation, and differential nucleosomal sensitivity in response to immune activation and offers a model that may be largely applicable to other specific cellular responses including viral infection and cancer. Previous work has shown that early transformation events are associated with prolonged nucleosome occupancy changes that are not observed later in cancer progression. Herein, we propose a model in which we suggest that detailed studies of nucleosomal occupancy and sensitivity in response to specific stimuli will provide insight into the regulation of nuclear events in cancer and other biological processes.The nucleosome, consisting of ~150bp of DNA wrapped around a core histone octamer, is a regulator of nuclear events that contributes to gene expression and cell fate. Nucleosome organization at promoters and their associated remodeling events are important regulators of access to the genome. Occupancy alone, however, is not the only nucleosomal characteristic that plays a role in genome regulation. Nucleosomes at the transcription start sites (TSSs) of genes show differential sensitivity to micrococcal nuclease (MNase) and this differential sensitivity is linked to transcription and regulatory factor binding events. Recently, lymphoblastoid cells treated with heat-killed  The nucleosome has long been considered a regulator of nuclear events. Consisting of ~150bp of DNA wrapped 1.65 times around a histone octamer, the nucleosome serves as the functional subunit of chromatin –4. SimilThe availability of massively parallel sequencing technologies in the early 2000s allowed for more detailed studies that mapped nucleosome occupancy in a highly time-resolved manner and provided insights into the dynamics of nucleosome remodeling. Time-resolved studies demonstrated widespread transient changes in nucleosome occupancy in response to reactivation of Kaposi’s sarcoma associated herpesvirus (KSHV) to produce a transient intermediate chromatin conformation favorable to an immune response followed by a return to the basal state . SimilarThis type of transient remodeling may also play a role in response to other stimuli including viral infections and resulting immune-mediated pathologies like those seen in acute SARS-CoV-2 infections and persistent systemic symptoms classified as Post-Acute Sequelae of COVID-19 (PACS). Chromatin remodeling complexes are essential for viral infection and replication indicating a direct role for chromatin dynamics during viral infection . InitialSalmonella typhimurium. However, increased sensitivity at the −1 nucleosome in highly expressed genes is observed 20 minutes after stimulation which is consistent with the timeframe for the innate immune response to bacterial stimulation [Nucleosome mapping is often achieved by digesting crosslinked chromatin in micrococcal nuclease (MNase) to produce ~150bp fragments which correspond to nucleosome-protected immune-specific regions  Figure . Biochemmulation ,26. MNasmulation . The posGenomic instability and altered chromatin organization are the hallmark of the transformed phenotype and are associated with cancer progression. Features of chromatin organization  are regulated by ATP-dependent chromatin remodeling complexes . MutatioSalmonella example above. The longer-lived nucleosome sensitivity changes explain differential gene activation through altered regulatory factor accessibility in conditions, such as high-grade cancers, where nucleosome occupancy appears largely static.Early grade adenocarcinomas show widespread nucleosome depletion at transcription start sites particularly in genes associated with chromatin structure and cancer progression. While it is unknown exactly how long this remodeled state persists, an appealing explanation is that this remodeled state persists longer than physiologically expected due to dysregulation of ATP-dependent chromatin remodeling complexes. Consistent with this explanation, differential occupancy is reduced in higher grade tumors , hintingThe above observations suggest that measurements of nucleosome occupancy and sensitivity will provide valuable insights into gene expression and cell fate outcomes in cancer progression. We propose a model to explain these relationships . In this


Histone H2B mono-ubiquitination at lysine 120 (ubH2B) has been found to regulate transcriptional elongation by collaborating with the histone chaperone FACT (Facilitates Chromatin Transcription) and plays essential roles in chromatin-based transcriptional processes. However, the mechanism of how ubH2B directly collaborates with FACT at the nucleosome level still remains elusive. In this study, we demonstrate that ubH2B impairs the mechanical stability of the nucleosome and helps to recruit FACT by enhancing the binding of FACT on the nucleosome. FACT prefers to bind and deposit H2A-ubH2B dimers to form an intact nucleosome. Strikingly, the preferable binding of FACT on ubH2B-nucleosome greatly enhances nucleosome stability and maintains its integrity. The stable altered nucleosome state obtained by ubH2B and FACT provides a key platform for gene transcription, as revealed by genome-wide and time-course ChIP-qPCR analyses. Our findings provide mechanistic insights of how ubH2B directly collaborates with FACT to regulate nucleosome dynamics for gene transcription. Here the authors investigated the direct collaboration between ubiquitinated histone H2B (ubH2B) with FACT at the nucleosome level. They found ubH2B enhances FACT’s chaperone property, recruits FACT to form a stable altered nucleosome state, and provides a key platform for transcription. Generally, the nucleosome acts as a repressor to limit the access of DNA and provides a structural platform for DNA-related processes including DNA transcription and replication, in which the nucleosome barrier needs be temporarily displaced to expose DNA, and then rapidly restored afterwards to preserve the original epigenetic identity4. During the transcriptional elongation process, the nucleosome structure has been found to be dynamically regulated, with the nucleosome disassembled in front of transcribing RNA polymerase II (Pol II) and rapidly reassembled in its wake. Recent investigations have implicated that post-translational modifications (PTMs) of core histones play a critical role in the process. One such typical modification is the mono-ubiquitination of H2B C terminus at lysine 120 in human , in which the bulky 76 amino-acid protein ubiquitin of approximately 8.5 kDa is covalently attached through its C-terminus to the ε-amino group of lysine residue5. Histone H2B is transiently ubiquitinated and deubiquitinated at coding region of actively transcribed genes5. In mammalian, H2B mono-ubiquitination is catalyzed primarily by heteromeric RNF20/40 complex7 and deubiquitinated by deubiquitinating enzyme (DUB) including several ubiquitin-specific proteases (USPs) as USP12, USP22, USP44 and USP46 etc10. Aberrant ubH2B levels have been shown to affect cell differentiation and development, and linked with a variety of cancers12.Eukaryotic DNA is packaged into chromatin by histones. Nucleosome, the basic structural unit of chromatin, is composed of a histone octamer  wrapped by 147 base pairs (bp) of DNA about 1.7 turns in a left-handed manner13. With a bulky moiety addition onto histones, ubH2B has been proposed to disrupt the folding of higher-order chromatin structure and lead to an open and biochemically accessible fiber conformation by in vitro analysis of nucleosome arrays containing semisynthetic ubH2B14. Although a mild nucleosome destabilizing effect of ubH2B was measured by in vitro Nap1-assited FRET (Fluorescence Resonance Energy Transfer)-based assay15, Chandrasekharan et al. showed that the nucleosome stability is enhanced when the level of ubH2B increases by using a repertoire of biochemical and genetic analyses in yeast cells16. In addition, single-molecule DNA unzipping invesigations by optical tweezers showed that ubH2B strengthened the proximal dimer region of nucleosome and increased the overall barrier strength of Pol II transcription through a nucleosome17. ubH2B has also been found to be required for efficient reassembly of nucleosomes at gene bodies in the wake of Pol II-mediated transcription elongation18. Nevertheless, how ubH2B directly affects the nucleosome dynamics still remains to be answered.H2B mono-ubiquitination has long been proposed to play an important role in gene transcription. ubH2B is broadly associated with transcribed region of highly expressed genes in human cells19. The highly conserved FACT complex is initially identified as a chaperone of histone H2A/H2B dimer20, and essential for cell viability22. FACT has been found to not only facilitate the progression of DNA and RNA polymerases on chromatin templates20, but also maintain the genome-wide integrity of chromatin25. Recent in vitro structural and single-molecular investigations have revealed the dual functions of FACT on nucleosome by facilitating both the nucleosome disassembly and re-assembly27. FACT has been found to tether both the (H3/H4)2 tetramer and H2A/H2B dimer on DNA by its two subunits SPT16 and SSRP1, which function distinctly but coordinately to fulfill these apparently opposite functions of FACT on nucleosome27. It is of great interest to investigate how the FACT’s functions are regulated by different epigenetic factors, as for histone mono-ubiquitination. The H2A mono-ubiquitination at lysine 119 (ubH2A) does not affect the FACT’s chaperone function in nucleosome assembly, but greatly blocks the FACT’s function in nucleosome disassembly28. Different from ubH2A, ubH2B presents unique interactions with FACT for gene transcription. Previous analysis by in vitro transcription assays has showed that ubH2B can cooperate with the FACT complex to stimulate the efficiency of elongation by RNA Pol II, probably by the efficient displacement of H2A/H2B dimer from nucleosome29. But to date, how ubH2B directly regulates the function of FACT on nucleosome dynamics are still poorly understood.The FACT complex, who earns the name “Facilitates Chromatin Transcription” from its ability to allow RNA Pol II to transcribe DNA templates at the level of chromatin, has been characterized to play an essential role in the chromatin-related processesIn this work, we focus on the regulation of ubH2B on nucleosome dynamics and its effect on the function of FACT complex. We reveal that the mono-ubiquitination on H2B impairs the stability of nucleosome. ubH2B facilitates the recruitment of FACT by enhancing the binding of FACT on nucleosome, and FACT greatly enhances the stability of ubH2B-nucleosome and helps to maintain nucleosome integrity. In addition, ubH2B promotes the binding of FACT on H2A/H2B dimer, maintains and enhances the FACT’s chaperone function to deposit H2A/H2B dimer to form intact nucleosome. The genome-wide study and time-course ChIP-qPCR analyses show that ubH2B helps to recruit FACT to maintain nucleosome integrity and facilitate gene transcription. Our findings provide mechanistic insights of how H2B ubiquitination directly collaborates with FACT to regulate nucleosome dynamics during the chromatin-related transcription process.z direction at 10 μm/s , still two-step unfolding dynamics was observed, with force lower to around 2pN for the outer wrap and around 15pN for the inner wrap, as compared to that of H2B-nucleosome . Based on the distribution of rupture force, the free energy cost to unfold the outer wrap is evaluated to be ~40 kJ/mol and ~20 kJ/mol for H2B- and ubH2B-nucleosome respectively. Compared to H2B-nucleosome, a less energy cost to unfold the outer DNA wrap is caused by the mono-ubiquitination of H2B in nucleosome.The effect of ubH2B on the stability and folding kinetics of nucleosome was directly investigated by single-molecule magnetic tweezers. We traced the dynamics of force-dependent conformational transition of mono-nucleosome reconstituted in vitro based on a 409 bp DNA containing one Widom 601 nucleosome positioning sequence in the middle Fig. . To invem/s Fig. . The strm/s Fig. . For the27. Intriguingly, for ubH2B-nucleosome, FACT does not impair, but greatly enhances the stability of ubH2B-nucleosome, and still maintains its integrity. As shown in Fig. d for FACT binding to the 409-bp nucleosome were measured by single-molecule magnetic tweezers27. The resulting Kd for FACT binding is 16.3 ± 3.9 nM (mean ± SE) for H2B-nucleosome and 8.0±4.2 nM (mean ± SE) for ubH2B-nucleosome with significant difference p < 0.01 . The results showed that the purified FACT has a higher affinity for the ubH2B-nucleosome, which agree with the mono-nucleosomes pull-down assays. In addition, previously investigations have shown that FACT prefers to bind the altered nucleosome structures, including partially unwrapped nucleosome, the sub-nucleosome, or the nucleosome with double-strand break site36. As we revealed that ubH2B can impair the stability of nucleosome, the allosteric effect of ubH2B on the nucleosome state may also function to recruit and correlate with FACT. Taken together, our results indicated that ubH2B recruits FACT to maintain a stabilized structural state at nucleosome level.To investigate how ubH2B affects the binding of FACT on nucleosomes, the recombinant FACT complex with the two subunits SPT16 and SSRP1 were purified and incubated with biotin-labelled unmodified H2B or ubH2B mono-nucleosomes  in vitro with high efficiency, but not by its catalytically inactive DUB mutant (DUB-mut), as examined by western blot experiments Fig. . In addi2 tetrasome (the particles made of DNA wrapped around an H3/H4 tetramer) to form intact nucleosome27. We have found that ubH2B recruits FACT to stabilize and maintain the nucleosome state, then how does ubH2B regulate the chaperone property of FACT? We first examined the effect of ubH2B on the binding property of FACT with H2A-H2B dimer. The recombinant H2A-H2B dimer or H2A-ubH2B dimers were incubated with the purified recombinant FACT complex, composed of Flag-SPT16 and SSRP1. As shown by the immunoprecipitation assays 2 tetrasome, and ubH2B does not have a pronounced effect on the binding of FACT-H2A-H2B on (H3-H4)2 tetrasome ays Fig. , FACT prome Fig. .Fig. 3FA2 tetrasomes were reconstituted in vitro onto the 409 bp DNA fragment with one Widom 601 nucleosome positioning sequence, as characterized by AFM imaging rap Fig. , which iity Fig. , with thity Fig. , which iity Fig. . Obviousity Fig. , similarity Fig. . The staity Fig.  and the ity Fig.  further ity Fig. . As a coOur in vitro investigation showed that the ubH2B helps to recruit FACT to form a stable and robust structural state at the nucleosome level. We then explored how the ubH2B cooperates with FACT to regulate gene expression in mouse embryonic stem cells (mESCs). The genome-wide correlation between the levels of ubH2B and SSRP1 (one subunit of FACT complex) by chromatin immunoprecipitation (ChIP)-seq analysis were examined as shown in Fig. Phrf1, Sipa1, Erf and Sxrn1 were shown in Fig. To further explore the dynamic interplay of ubH2B and FACT on the transcriptional process, time-course ChIP-qPCR analysis was employed on the gene body regions of RAR-RXR regulated genes during transcriptional activation by all-trans retinoic acid (tRA) induction. The results for the typical examples of genes 7, while how ubH2B directly regulates the dynamics of nucleosome and functions in transcriptional process remain largely unknown. In this study, we intensively discovered the direct effect of ubH2B on nucleosome dynamic and its correlation with FACT complex at nucleosome level  has long been revealed to play an important role in regulating genome functions, especially in transcriptional process. Previous investigations on the roles of ubH2B mainly rely on the manipulation of the major H2B ubiquitination ligase BRE1 in yeast or RNF 20/40 in mammalian cellsvel Fig. . We foun18. How can ubH2B fulfill these functions in cells? Our research suggests that although the ubH2B itself cannot enhance the nucleosome stability and help nucleosome reassembly, but it can act as a structural platform to recruit FACT complex to fulfill these functions. We showed that ubH2B can help to recruit FACT by enhancing the binding of FACT on nucleosome, and the binding of FACT on ubH2B-nucleosome greatly enhances the nucleosome stability. The free energy cost for the outer wrap of ubH2B-nucleosome in the presence of FACT is increased up to ~100 kJ/mol. At the same time, FACT helps to maintain the ubH2B-nucleosome integrity. In addition, ubH2B promotes the binding of FACT on H2A/H2B dimer, maintains and enhances the FACT’s chaperone function to deposit H2A/H2B dimer to form intact nucleosome. Our genome-wide analysis and time-course ChIP-qPCR analysis on specific genes also indicated that ubH2B helps to recruit more FACT to facilitate gene transcription. As a structural platform, ubH2B recruits FACT complex to help to maintain the integrity of ubH2B-nucleosome and form a stable and altered nucleosome state, which is ready for transcriptional activation  and on lysine 79 (H3K79) by recruiting histone methyltransferase Set1/COMPASS and Dot1 respectively, and has been found to associate with the SWI/SNF chromatin remodeling complex for transcriptional activation39. Structural analyses on the ubH2B-nucleosome with COMPASS or Dot1L revealed that the ubiquitin modified on H2B serves as a structural platform to recruit and contact with the proteins for functional stimulation47. Other than the direct effect of ubH2B on the property of chromatin, the indirect effect of ubH2B to act as a structural platform by recruiting or preventing the binding of some specific proteins, might function more importantly to regulate the transcriptional process. In addition to its role in gene transcription, ubH2B has also been found to play roles in the processes of DNA replication and DNA damage response49. Our investigation on the directly mechanism of ubH2B’s functions in the nucleosome dynamic and in its correlation with FACT may help to understand these related processes.Our and previous in vitro investigations on the direct effect of ubH2B on nucleosome dynamic revealed that ubH2B impairs the stability of nucleosome, especially the strength of interactions between the DNA and histones. However, previously investigations in yeast cells have showed that ubH2B is required for efficient reassembly of nucleosomes during Pol II-mediated transcription elongation, enhance the nucleosome stability, and mediate both activation and repression of transcription depending the localization and context of the modificationion Fig. . Previou4. Previous studies have found that FACT appears to function not only in destabilizing nucleosome to facilitate the progression of DNA and RNA polymerases on chromatins, but also in establishing and maintaining the genome-wide integrity of chromatin structure25. Most recently, we and others have revealed that FACT can tether both the (H3-H4)2 tetramer and H2A-H2B dimer on DNA by its two subunits SPT16 and SSRP1, which function distinctly but coordinately to fulfill the apparently opposite functions of FACT on nucleosome by in vitro structural and single-molecular investigations27. It is of great interest to investigate how the FACT’s functions are regulated by different epigenetic factors, as for histone mono-ubiquitination. Our previous study has revealed that the direct regulation of H2A ubiquitination on the two-face functions of FACT complex at nucleosome level. ubH2A has been found to inhibit the binding of FACT on nucleosome to block the FACT’s function at nucleosome level, but not affect the FACT’s chaperone function in nucleosome assembly. At this point, FACT functions as a “salvager” to maintain ubH2A-nucleosome as a repressed state of genes, by helping to reassemble the ubH2A-H2B to form intact ubH2A-nucleosome, but cannot disassemble the ubH2A-nucleosome. Previous analysis by in vitro transcription assays showed that ubH2B can cooperate with the FACT complex to stimulate the efficiency of RNA Pol II elongation, and suggested that it may caused by the efficient displacement of H2A/H2B dimer from the nucleosome barrier29. However, our purified system in this work clearly showed that although ubH2B does not affect the FACT’s chaperone function in nucleosome assembly, it helps to recruit FACT and greatly impairs the destabilized effect of FACT on nucleosome state. The preferable binding of FACT on ubH2B-nulceosome helps to maintain a stable and altered nucleosome state for transcriptional activation.The histone chaperone FACT has been found to play essential roles in nucleosome remodeling during the chromatin-related processes, including DNA transcription, replication and repair. In these processes, the nucleosome barrier needs be temporarily displaced to expose DNA for processing, and then be rapidly reassembled to protect the DNA and preserve the original epigenetic identity26. The ubiquitin modified at H2A-K119 site around the dyad of nucleosome may clash and exclude the binding of FACT on nucleosome; while the ubiquitin modified at H2B-K120 site on the face of nucleosome may contact with the middle domains of SPT16 and SSRP1 to recruit and stabilize the binding of FACT on nucleosome. In addition, FACT has been found to prefer to bind the partially unwrapped nucleosome36, the sub-nucleosome26, or the nucleosome with double-strand break site35, rather than the intact nucleosome. Our maganeitc tweezer investigations have revealed that ubH2A greatly enhances, but ubH2B impairs the stability of nucleosome, which indicate that the allosteric effect of ubH2A and ubH2B on the nucleosome state may also function to correlate with FACT. It will be of great interest to resolve the structure of FACT with ubH2B-nucleosome to give more details on the regulation. The FACT complex, functioned as an essential and conserved factor in almost all chromatin-based processes, including DNA transcription, replication and repair in eukaryotes. It is critically important to understand how the core functions of FACT are regulated in different chromatin circumstances. Our research on the distinct regulation of ubH2B on FACT’s functions provides a mechanistic insight to understand the FACT’s function. It will be of great interest to investigate the regulation of FACT’s function by other related epigenetic factors, which may help to propose a unifying model for FACT’s activity at the chromatin state.The same “ubiquitin” modified at different sites on nucleosome has totally different effect on FACT’s function. Recent cryo-EM structural analysis on the FACT complex with partially assembled sub-nucleosome has revealed that the dimerization domains of SPT16 and SSRP1 straddle on the dyad of nucleosome with the middle domains of SPT16 and SSRP1 projecting down on the either side of nucleosome’s face, as like a unicycleE. coli, and purified from inclusion bodies50. The purified histone ubH2B was purchased from KS-V Peptide Biological Technology Co., Ltd , by total chemical synthesis utilizing standard solid phase peptide synthesis and peptide fragments ligation reactions to generate the desired protein51. For the purification of recombinant FACT complex27, Sf9 cells (1.5–2 × 106/ml) were infected with baculovirus containing Flag-SPT16 and His6-SSRP1, and incubated at 27 °C for 72 h. The infected cells were collected and washed with ice-cold PBS, and then lysed in lysis buffer . 10 mg (60 mL) of the cell extracts were incubated with 500 μL anti-Flag M2-agarose  for 4 h at 4 °C, with the resin washed by lysis buffer. Bound proteins were eluted in the presence of 0.5 mg/ml Flag peptide  and further purified by a Heparin HP column . The fractions containing FACT complex were dialyzed against BC-100 buffer  and stored at −80 °C.Histones H2A, H2B, H3 and H4 were cloned into pET3a histone expression plasmid, overexpressed in 27, equimolar amounts of individual histones in unfolding buffer  were dialyzed into refolding buffer , and purified by a Superdex S200 column. For magnetic tweezers’ investigation, a 409 bp DNA template with a single 601 sequence in the middle was prepared by PCR from plasmid using a biotin(bio)-labeled forward primer and a 3×digoxigenin (3×dig)-labeled reverse primer. Chromatin samples were assembled using the salt-dialysis method50. The reconstitution reaction mixture with octamers and 601 based DNA templates in TEN buffers  were dialyzed over 16 h at 4 °C in TEN buffer, which was continuously diluted by slowly pumping in TE buffer  to a lower concentration of NaCl from 2 M to 0.6 M, followed by a final dialysis step in TE buffer for 4 h. The stoichiometry of octamer/tetramer to DNA template was determined by gel electrophoresis analysis and AFM50.To reconstitute the respective histone octamers, H3/H4 tetramers and H2A/H2B dimers1 μg ubH2B-nucleosomes were incubated with increasing concentrations  of wild-type DUB  and its catalytically inactive mutant  in deubiquitination reaction buffer  at 30 °C for 1 hr. The reaction was terminated by the addition of SDS–PAGE sample loading buffer. The proteins were then resolved on SDS–PAGE and blotted with the anti-H2B antibody  and anti-His antibody  for his-tagged DUB, with the uncropped and unprocessed scans of all of the blots shown in the Source Data file.28. The beads were washed by wash buffer  for 3 times, 10 min each time. The proteins were eluted with SDS-loading buffer  and analyzed by western blot, with the uncropped and unprocessed scans of all of the blots shown in the Source Data file.Biotin-labeled DNA was prepared by PCR with biotin-labeled primer. 1 μg Biotin-labeled mono-nucleosomes and streptavidin-agarose-beads (10 μL) were mixed in BC300 buffer , and incubated with increasing concentrations (4 nM and 8 nM) of FACT complexes overnight at 4 °C28. The beads were washed 3 times with the wash buffer , 10 min each time. The proteins were eluted by SDS-loading buffer and analyzed by western blot, with the uncropped and unprocessed scans of all of the blots shown in the Source Data file.10 μL anti-Flag M2-agarose  beads for each reaction was washed by PBS twice and pre-cleaned in BC300 buffer for 1 hr at 4 °C. Then Flag-FACT(8 nM) and increasing concentrations (1.5 nM and 3 nM) of H2A-H2B dimer or H2A-ubH2B dimer were added and incubated overnight at 4 °CTetrasomes for histone deposition assays were reconstituted on 87 bp DNA fragments using the salt-dialysis method. Different concentrations (25 nM and 50 nM) of FACT complexes were first incubated with H2A/H2B dimer (100 nM) at 4 °C for 30 min, and then mixed with the constant reconstituted tetrasomes (100 nM) in reaction buffer . The samples were incubated at 30 °C for 1 hr prior to electrophoresis on 5% native PAGE electrophoresis in 0.25 × TBE buffer  for 1.5 hr at 80 V. The gels stained with ethidium bromide were scanned by a Gel DocTM EZ Imager system (Bio-Rad), with the uncropped and unprocessed scans of all of the blots shown in the Source Data file.2O for 4 times and blow to dry mica surface briefly with nitrogen gas. 10 μL sample solution was added onto the mica surface, incubated for 10–15 min. Finally, wash the mica with 200 μL ddH2O for 3 times and blow to dry gently. The prepared AFM samples were examined using ScanAsyst Mode of AFM .The reconstituted nucleosome samples (20 ng/μL) were prepared in HE buffer and fixed with 0.1%glutaraldehyde (Fluka) on ice for 30 min. Rinse the mixture with 500 μL HE buffer for 2–3 times in a vivaspin500 column. The column was spined at 15 000 g for 2–5 min at 4 °C to get rid of the liquids and diluted to 0.5–1 ng/μL in HE buffer. 20 μL spermidine (1 mM) was dropped onto newly cleaved mica surface and incubated for 10 min. Then rinse the mica with 200 μL ddH31. Briefly, the two DNA ends of reconstituted nucleosome were tethered by digoxigenin and anti-digoxigenin ligation to a glass coverslip and by biotin-streptavidin ligation to a 2.8-μm diameter Dynabeads , respectively. The applied tensions on the super paramagnetic beads arised from the strong magnetic field gradient of the two small NdFeB magnets, which were controlled to pull on the Dynabeads and thus stretch the DNA molecule on nucleosome. The bead image was projected onto the CCD camera  through an inverted microscope objective . The real-time position  of the bead at various forces was recorded by comparing the diffraction pattern of the bead with calibration images at various distances from the focal point of the objective. The quadrant-interpolation (QI) algorithm was applied by LabView software to trace the three-dimensional position of the beads in the flow cell52. About 80 samples were traced simultaneously in each measurement. In the repeated stretching cycle, magnets were moved near to the samples at a rate of 10 μm/s, during which the dynamic unfolding of the nucleosome was recorded. After that, force was reduced to 0.1pN rapidly and waited for 5 min, and then repeated the stretching cycle on the same nucleosome. All measurements were carried out at 25 °C. To calibrate the force in the force-extension measurement, the force calculation for a 10,000 bp DNA tethered between the bead (M280) and the coverslip was carried out. At each magnet position, the y-position of the bead was recorded at 500 Hz for 5 mins and the corresponding force was calibrated by power-spectral-density (PSD) analysis53. The force measurements were repeated for 10 independent DNA tethers. The relationship between force and magnet position was fitted well with a double exponential function. Forces in force-extension measurements of nucleosomes were derived from the fitted double exponential function with magnets positions.The single-molecule stretching experiments were performed by magnetic tweezersForce-dependent folding free energy The kinetic rate constants f54. 55 and contour length of When entclass1pt{minimaL is 119 nm for Feq=3.5 pN from Supplementary Fig. For a molecule of H2B-nucleosome as shown in Supplementary Fig. Based on the kinetic rate analysis under tensions, the equilibrium force Kd measurement for FACT binding to the 409 bp nucleosome by single-molecue magnetic tweezers27.We performed the Kd is defined as:In the case: Kd value could be obtained by calculating the ratio of the number of nucleosome alone to the number of complex at a certain concentration of FACT. The force-extension measurement has revealed that canonical nucleosome are totally disrupted at the tension around 25pN in the absence of FACT, and at the tension less than 10pN in the presence of FACT. When we exerted 11pN on the nucleosome sample, the inner wrap of nucleosome without FACT will not be disrupted while the inner wrap of the nucleome bound with FACT will be disrupted  and of nucleosome combined with FACT (Ncomplex) counted at 83 nM FACT concentration. The similar measurements of the Kd between FACT and ubH2B-nucleosome were performed. The outer wrap of the ubH2B-nucleosome disrupted at ~3pN in the abence of FACT, and disrupted at the tension around 10pN in the presence of FACT. When the force of 6pN were exterted on the ubH2B-nucleosome sample, the outer wrap of ubH2B-nucleosome without FACT will be disrupted while the nucleosome bound with FACT will not  for the binding of FACT with unmodified nucleosome, and 8.0 ± 4.2 nM  for the binding of FACT with ubH2B nucleosome with significant difference p < 0.01  into the flow cell, and traced the deposition process immediately at 1.5pN. Two sequential 10-nm deposition processes occurred, corresponding to the formation of the two halves of the outer DNA wrap, respectively. We then carried out the force-extension measurement for the same reassembled sample.To confirm the chaperone property of FACT on H2B- or ubH2B-nucleosome, we traced the real-time deposition of H2A-H2B or H2A-ubH2B dimer on tetrasome in the presence of FACT, respectively. We anchored the mono-(H3/H4)2 at 37 °C.The mouse embryonic stem cell (mESC), R1 cell line (ATCC® SCRC1011™) was obtained from American Type Culture Collection (ATCC) and used for biochemical, cellular and genomic analyses. R1 cells were cultured in the medium with 80% DMEM, 15% FBS, 1% Lglutamine, 1% nucleosides, 1% nonessential amino acids, 1% 2-mercaptoethanol, 1% Pen/Strep, and 1000U/ml leukemia inhibitory factor (LIF) in standard incubator with 5% COmESCs were treated with tRA for 15 min, 30 min, 1 hr, 2 h, 4 h, 8 h, and then fixed with 1% formaldehyde. The fixed mESCs were resuspended in nuclei lysis buffer  and sonicated to 300–500-bp-sized fragments. 10 μg of the sonicated chromatin sample were used as input to calculated the relative enrichment of histone modifications or proteins on genome. Protein A  and protein G Dynabeads  were pre-incubated with H2B antibody , ubH2B antibody  and SSRP1 antibody  for 6 h at 4 °C and then incubated with 100 μg of the sonicated chromatin sample overninght at 4 °C. The beads were then extensively washed with RIPA150  once, RIPA500  twice, RIPA-LiCl  twice and 1×TE twice, and resuspended in freshly made elution buffer . The mix was vortexed at 65 °C for 30 min and placed on a magnetic stand for 2 min. The supernatant was collected and de-crosslinked by incubation in 1% SDS at 65 °C for 6 h, followed by treatment with RNase A and proteinase K. The ChIPed and input DNA was extracted with phenol: chloroform: isoamyl (25:24:1) and precipitated with 75% ethanol and 300 mM NaAc followed by qPCR analysis. All the qPCR experiments were performed on the real-time PCR  using FastStart Universal SYBR Green Master (ROX). The primer pairs used for the qPCR experiments were the following: PHRF1: sense 5’ -GGATGGGGAGTCGGACTGTA- 3’, antisense 5’ -AGTGCACCCATACTCTTCCG- 3’; SIPA1: sense 5’ -TCCACTACGTACCGCCTCCATCCTGG- 3’, antisense 5’ -AGTACCTCTCGGGCTCAAGC- 3’; ERF: sense 5’ -GATATTAACCCGGGAGGCGG- 3’, antisense 5’ -CTGTGTCCGCCGGGGTCTTC- 3’; SXRN1: sense 5’ -CCAAGGAAGAGGTATGGGGC- 3’, antisense 5’ -GGCGATTGGTACGTTGTGC- 3’. The ChIP enrichment were normalized to the input, caculated with the formula [ChIPed DNA/(input DNAx10)]x100%. The results were calculated by three independent replications.mESCs were harvested after treated with tRA for 15 min, 30 min, 1 hr, 2 h, 4 h, 8 h, and then the total RNA was extracted using TRIzol reagent (Invitrogen), and the first strand of cDNA was reverse-transcribed using 2 μg of RNA. cDNA products were used for quantitative real-time PCR using the SYBR Premix Ex Taq (Takara) to validate the efficiency of tRA induction. The primer sequences used for real-time PCR were as follows: ERF: sense 5’ -CATGAAGACCCCGGCGGACACAG- 3’, antisense 5’ -CTTGATGACAAACTCCCCGTA-3’; PHRF1: sense 5’ -GACAGAGGCGGGATCGTCTAG- 3’, antisense 5’ -ATCACTGGCCAGTTCTGAG- 3’; SIPA1: sense 5’ -GCGGTGGCCAGCTTGAGCCCG- 3’, antisense 5’- TGCTCTGTGGGCCTGACTCTC- 3’; SRXN1: sense 5’ -CTGCATCGCCACGGTGCACAACGTAC- 3’, antisense 5’ -TGCAGCTGCTGGTAGGCTGCAT- 3’. The results were calculated by three independent replications.GSE90906, PRJNA604675, GSE153584 and PRJNA643279). All the ChIP-seq data were mapped to the mouse genome (mm9) using bowtie256 with the default parameters, and the input controls were used in the peak-calling ananlysis for the ChIP-seq data. The single-end RNA-seq reads were mapped to the Mus musculus mm9 gene annotation model using hisat2, with the default parameters, and converted to bam files in samtools. Fragments overlapping representative transcripts from annotated genes (genecode vM1) were counted in the featureCounts software. The normalization was performed with R script. Low quality reads and PCR replicates were removed by samtools57 and only uniquely mapped reads which mapping to a unique genomic location and strand were kept. Enriched peaks were called using MACS58. The p-value cutoff for peak detection was 1e-5 with MACS2.The SSRP1 ChIP-seq data, RNA-seq expression data, and ubH2B ChIP-seq data were downloaded from Gene Expression Omnibus (GEO: Further information on research design is available in the Supplementary informationnPeer Review FileReporting Summary