Cyclin-dependent kinases (CDKs) play critical roles in cell cycle progression and gene expression regulation. In human cancer, transcription-associated CDKs can activate oncogenic gene expression programs, whereas cell cycle-regulatory CDKs mainly induce uncontrolled proliferation. Cyclin-dependent kinase 12 (CDK12) belongs to the CDK family of serine/threonine kinases and has been recently found to have multiple roles in gene expression regulation and tumorigenesis. Originally, CDK12 was thought to be one of the transcription-associated CDKs, acting with its cyclin partner Cyclin K to promote the phosphorylation of the C-terminal domain (CTD) of RNA polymerase II and induce transcription elongation. However, recent studies have demonstrated that CDK12 also controls multiple gene expression processes, including transcription termination, mRNA splicing, and translation. Most importantly, CDK12 mutations are frequently found in human tumors. Loss of CDK12 function causes defective expression of DNA damage response (DDR) genes, which eventually results in genome instability, a hallmark of human cancer. Here, we discuss the diverse roles of CDK12 in gene expression regulation and human cancer, focusing on newly identified CDK12 kinase functions in cellular processes and highlighting CDK12 as a promising therapeutic target for human cancer treatment. Better understanding of the roles played by a protein kinase, an enzyme that adds phosphate groups to other molecules, in healthy and diseased states may help scientists identify novel cancer treatments. Cyclin-dependent kinases (CDKs) are a family of protein kinases crucial to cell cycling and gene expression. CDK12 can activate and modulate cancer-related gene expression, but, according to a review by Seung Hyuk Choi and colleagues at the Salk Institute for Biological Studies in La Jolla, USA, further investigations into its exact functioning and control mechanisms are required. CDK12 mutations are frequently found in aggressive breast and ovarian cancers, while loss of CDK12 function results in abnormal expression of DNA damage response genes and genome instability. CDK12 may also regulate drug resistance in cancer cells. The team suggests that therapies targeting CDK12 are worth exploring. Thus, they can integrate transcription with changes in phosphorylation of RNAPII CTD throughout the transcription cycle2. Phosphorylation of the Ser5 position of RNAPII CTD by CDK7, which is a part of the transcription initiation factor TFIIH, is linked to promoter-proximal transcriptional pausing, which tends to decline during the transition to active transcription elongation, accompanied by an increase in Ser2 phosphorylation mediated by CDK9 and CDK125. Through phosphorylation of RNAPII and associated transcription factors, the nuclear RNAPII CTD kinases coordinate multiple cotranscriptional events important for gene expression, including mRNA splicing and surveillance, termination/cleavage, and the export of nascent mRNAs8. Recent evidence has shown that CDK12 plays a unique role among CTD kinases in regulating genome stability and cancer cell drug resistance9. The expression levels of CDK12 and its partner cyclin, cyclin K (CCNK), are unchanged throughout the normal cell cycle but are frequently elevated in proliferating stem cells and cancer cells. Consistent with its role in the maintenance of genome stability, CDK12 that is lost or mutated is associated with the progression and metastasis of a subset of human cancers, including serous ovarian, breast, and prostate cancers. This review addresses emerging studies to dissect the novel mechanisms of CDK12 in gene expression regulation and its roles in human cancer and consider its potential clinical applications as a biomarker and cancer therapeutic target.The transcriptional cyclin-dependent kinases (CDKs) CDK7, CDK8, CDK9, CDK12, and CDK13 are evolutionally conserved and share a common phosphorylation substrate, which is the RNA polymerase II (RNAPII) C-terminal domain (CTD) heptad repeat  harboring a catalytic domain in the middle of the protein (residues 719–984) and showing 42% identity with the kinase domain of human cdc2 (CDK1). The protein has multiple bipartite nuclear localization sequences (NLS) and is mainly detected in the nucleus and nuclear speckles. A prominent feature of CDK12 includes arginine/serine rich (RS) domains in the N-terminus, which are frequently found in many splicing factors; therefore, it is called a Cdc-related kinase with an arginine/serine-rich (RS) domain (CrkRS). CDK12 contains 21 RS motifs, and the presence of this RS domain is necessary for CDK12 to localize to nuclear speckles, suggesting its active role in transcription regulation. CDK12 and its most homologous kinase, CDK13 , are uniquely long among CDKs with distinct structural characteristics, including an RS domain and a proline-rich (P) domain, which might mediate RNA–protein or protein–protein interactions. Schematic diagrams of the structural features of CDK12, Cyclin K, and related proteins are illustrated in Fig. Drosophila melanogaster) CDK12 has structural features similar to those of human CDK12, but the yeast (Saccharomyces cerevisiae) ortholog CTK1 shows low structural similarity, with its core kinase domain mostly conserved. Another name for CDK12, CrkRS, is no longer used because several studies confirmed that CDK12 is associated with a unique cyclin, Cyclin K, a characteristic feature of a CDK complex; it is now called CDK125. CDK13 also binds to Cyclin K and forms a separate complex4. The CDK12–Cyclin K and CDK13–Cyclin K complexes may share similar functions because of the similarity of their structures and associated cyclin. Cyclin K is a 70 kDa protein with a conserved cyclin box in the N-terminus and a proline-rich region in the C-terminus. Knocking down CDK12 expression results in decreased stability of Cyclin K and vice versa11, indicating that the interaction between CDK12 and Cyclin K is important for complex integrity and stability. The yeast ortholog of human CDK12/CDK13 is CTK1. The yeast kinase protein complex (CTDK-I) consists of CTK1 (kinase), CTK2 (a cyclin partner), and CTK3 (a yeast-specific subunit). In comparison, the yeast ortholog of human CDK9/Cyclin T (P-TEFb) is the Bur1/Bur2 complex.CDK12 was first cloned from a human cDNA library to screen for Cdc2-related genes12. The CTD kinase activity of CDK12 was first reported based on an in vitro kinase assay. In this experiment, anti-CDK12 obtained from mammalian cells via immunoprecipitation were able to phosphorylate substrates, including SR-type splicing factor ASF, myelin basic protein (MBP), and a recombinant yeast CTD protein, which led to the speculation that CDK12 is a novel CTD kinase10. Notably, the initial purification of the CDK12 protein expressed in baculovirus-infected cells was not successful due to its insolubility. Additional in vitro kinase experiments using fly CDK125 and human CDK12 from other groups4 also showed that CDK12 effectively phosphorylated the CTD, confirming that CDK12 was a bona fide CTD kinase with potential roles in transcription regulation. The in vivo kinase activity of CDK12 as a CTD kinase was also demonstrated using RNAi in both fly cells and human cells5. Knocking down CDK12 or Cyclin K resulted in a significant reduction in CTD phosphorylation, with particularly remarkable effects on Ser2 phosphorylation. Interestingly, this effect was not as profound after CDK13 knockdown, indicating that CDK12–Cyclin K is a dominant CTD kinase complex with global control of CTD phosphorylation. A more detailed study using a phosphorylated CTD peptide substrate revealed that CDK12 prefers a CTD substrate that is prephosphorylated at the Ser7 position but has low activity toward an unmodified CTD peptide. However, P-TEFb actively phosphorylated the unmodified CTD13, indicating that Ser7 phosphorylation is a necessary element of the kinase motif recognized by CDK12 and that other kinases must be in place to prime this phosphorylation before CDK12 properly recognizes the CTD as the preferred substrate. Usually, higher levels of Ser7 phosphorylation in CTD are found among actively transcribed genes15. For example, in yeast, Ser7 is phosphorylated early in transcription and persists at robust levels throughout the whole transcription process16. Because of the CDK12 preference for this substrate, it is possible that higher CDK12 activity is associated with actively transcribed genes marked by high levels of Ser7 phosphorylation. Furthermore, using an affinity purified CDK12 complex in an in vitro kinase assay, this study also demonstrated that CDK12 can phosphorylate both the Ser2 and Ser5 positions, a finding consistent with the property of the CDK9 kinase17. Therefore, the substrate specificity of CDK12, according to the phosphorylation pattern in the CTD, has largely remained elusive due to the different combinations of phosphorylation at multiple sites in the CTD heptad (CTD code) and the limited number of available phospho-specific antibodies that can distinguish among them. To replace the use of these CTD-phospho-specific antibodies, a new mass-spectrometry (MS)-based approach can be used to determine and map diverse phospho-sites along the entire CTD18. This technique is expected to reveal the exact positions and provide quantitative measurements of phosphorylation patterns, including neighboring CTD residues, to elucidate the CTD code.The different patterns of phosphorylation events in the heptad repeats of RNAPII CTD are critical for regulating the processivity of RNAPII, the transcription elongation rate, mRNA processing, and chromatin modification, because particular combinations of phosphorylation sites in of the CTD, so-called “CTD code”, can generate specific docking sites and determine the recruitment of specific regulatory factors at the phosphorylated CTD sites13. The overall structure of CDK12 and Cyclin K in complex is generally similar to that of other CDK complexes, with similar features including the CDK–cyclin interface, an ATP-binding pocket in the core kinase domain, and kinase activation by T-loop phosphorylation. Of interest, researchers found that human CDK12/Cyclin K expressed in a baculoviral system had only basal levels of kinase activity. However, the coexpression of yeast CAK (Cdk-activating kinase) in the expression system resulted in a significant increase in CDK12 kinase activity due to phosphorylation of Thr893 by the CAK residing in the T-loop of CDK12. Therefore, Thr893 phosphorylation in the T-loop is essential for CDK12 kinase activation. Another important structural feature is the polybasic region conserved between transcription-associated CTD kinases. The polybasic cluster (1045KKRRRQR) in CDK12 is extended from the C-terminal kinase domain and forms a helical structure in parallel orientation to the C-terminal part of the kinase domain. It was demonstrated that the truncation of this C-terminal extension, including the polybasic motif, causes a gradual loss of kinase activity, suggesting that its presence is necessary for maintaining robust catalytic activity by blocking the free exchange of ADP to ATP in the ATP-binding pocket. From these observations, potential regulatory mechanisms for CDK12 kinase activity might be based on switching of T-loop phosphorylation or protein–protein interactions in the polybasic cluster region, which may be used for a protein docking site. Therefore, further study will be important to understand how CDK12 activity is controlled from the point of view of the potential CDK12-activating kinases or the CDK12-binding proteins via the polybasic motif.The crystal structure of the CDK12/Cyclin K complex has been resolved5. However, depletion of CDK12 function in human cells affects <5% of transcription and selectively affects a uniquely defined group of genes, indicating that CDK12 must invoke a gene-targeting mechanism upon transcription activation. A parallel example was shown by the transcription activation of Nrf2 target genes in flies. Nrf2 is a master transcription factor in reactive oxygen species (ROS)-induced transcription activation. Nrf2 is rapidly induced by diverse ROS stresses to bind to the promoter of a downstream target gene to control its expression. From screening using RNAi in a fly system, Drosophila CDK12 was identified as a positive regulator of the transcription program governed by Nrf2 to promote stress resistance and overall survival under oxidative stress conditions19. Interestingly, a defined set of genes affected by CDK12 depletion differs among species. Depletion of CDK12 in flies has mostly been shown to affect redox/antioxidant genes and housekeeping genes that control metabolic activities. In humans, CDK12 is required for the transcription of DNA damage repair and DNA damage response (DDR) genes, including BRCA, ATR, FANCI, and FANCD2, which are critical for maintaining genomic stability. However, it is possible that, to some degree, CDK12 might globally regulate transcription elongation by enhancing overall Ser2 phosphorylation at the RNAPII CTD. Different ChIP-Seq experiments using CDK12-specific antibodies have consistently reported that CDK12 has genome-wide associations with promoters, protein-coding regions, and enhancers in actively transcribed genes, with extensive RNAPII signal overlap. Intensive signals of CDK12 occupancy are often found at the transcription start site (TSS), and the strongest signals of RNAPII occupancy are mostly found at promoter regions where RNAPII pauses. In this context, the polymerase-associated factor 1 (PAF1) complex, known as a general transcription elongation factor, can play an important role in the recruitment of CDK12 to genes20. In this report, PAF1 knockdown in human leukemia cells globally decreased CDK12 occupancy and reduced the levels of Ser2 phosphorylation in the CTD, outcomes consistent with the results from a yeast study21, indicating that CDK12 can play a role in general transcription elongation. In addition, a study using a CDK12-specific chemical inhibitor provided a unique perspective on these findings. Low-dose (50 nM) treatment with the CDK12 inhibitor THZ531 particularly downregulated the expression of core DDR genes, a result identical to that after genetic CDK12 depletion. However, the high-dose treatment (200 nM) of THZ531 negatively affected the expression of superenhancer-associated genes, which was not observed after the low-dose treatment22. Therefore, it is possible that two different transcription mechanisms could be regulated by CDK12; that is, CDK12 might phosphorylate unknown transcription factors that are highly specific for DDR gene expression, but the superenhancer genes might be controlled by the general mechanism of transcription elongation that is mediated according to the levels of CTD phosphorylation of multiple CTD kinases, including CDK12. In a study comparing CDK13, a homolog of CDK12, the transcription of some DDR genes depended solely on CDK12, not on CDK13, although other DDR genes showed more profound transcription defects upon the dual knockdown of CDK12 and CDK1323. The weak coincidental frequency of differentially expressed genes upon the chemical inhibition or genetic knockdown of CDK12 or CDK13 suggests that CDK12 and CDK13 have different roles in transcription elongation and target gene selection.It has been generally thought that CDK12 is globally associated with transcription elongation, similar to CDK9, based on its predominant kinase activity toward the Ser2 position of the heptad repeats in the RNAPII CTD10. Consistent with this possibility, a protein interaction network analysis of the CDK12 protein by mass spectrometry revealed that CDK12 is associated with several RNA-binding proteins in the exon junction complex (EJC), SR splicing factors (SRSFs), and the CDC5L/PRPF19 complex. Furthermore, the N-terminal RS domains in CDK12 are required for its interaction with SRSFs, allowing CDK12 to recruit RNA cleavage and polyadenylation factors at the 3′ end for the processing of the c-FOS transcript via the RS domains24. Another study showed that CDK12 and CDK13 interact with SRSFs and affect the alternative splicing of the SRSF1 gene25. Similarly, CDK13 was shown to regulate the alternative splicing of HIV by interacting with the splicing factor SRSF127. Notably, a deep bioinformatic analysis of the CDK12 transcriptome showed that CDK12 can take both gene-type and cell-type-specific control of alternative splicing, known as alternative last exon (ALE) splicing28. In breast cancer cells, ALE splicing by CDK12 regulates the expression of DDR activator ATM and DNAJB6 isoforms, which can influence tumorigenesis when CDK12 is mutated or silenced. In addition to alternative splicing, loss of CDK12 can also lead to premature termination of long transcripts with intronic polyadenylation (IPA). IPA leads to mRNA isoforms with different coding sequences or truncated transcripts that frequently result in loss of function29, as illustrated in Fig. 31, which is a detailed mechanism by which CDK12 regulates the expression of DDR and HR genes, and CDK12 mutation potentially drives tumorigenesis. To support the oncogenic role of IPA, another study demonstrated that IPA is frequently upregulated to produce truncated mRNAs and proteins encoded by tumor suppressor genes in cancer32, suggesting a novel regulatory function of CDK12 to suppress the IPA of tumor suppressor genes. In conclusion, it is obvious that CDK12 and CDK13 can regulate the gene expression of a set of genes through mRNA splicing or 3′ end processing, but the precise roles and mechanisms of CDK12 in these events remain to be elucidated; specifically, the means by which kinase activity directly controls those posttranscriptional regulators requires the identification of potential phosphorylation substrates.It is conspicuous that perturbation to CTD phosphorylation by the inhibition of either CDK9 or CDK12 has little overlapping effect on gene expression, considering that both kinases have redundant phosphorylation of the Ser2 position in CTD heptad repeats. Therefore, CDK12 might play unique roles, different from those of CDK9, in gene expression regulation. Posttranscriptional regulation serves as an additional layer of gene expression control at the RNA level after RNAPII synthesizes mRNAs. CDK12 can function posttranscriptionally, for example, in the process of RNA splicing, transcription termination, and RNA export. The initial study of CDK12 suggested that CDK12 may have potential roles in RNA splicing due to the presence of multiple RS domains that are typical of many RNA-interacting and splicing factors and its subcellular location at nuclear speckles33. In cap-dependent translation, mTOR kinase is critical for the phosphorylation of the translation repressor 4E-BP1, which is released from the mRNA–eIF4E complex, enabling translation complex assembly and further translation activation. However, the mTOR activity level is relatively low in mitosis, leading to the speculation that CDK1 can control cap-dependent translation, specifically during mitosis; thus, active CDK1 can sufficiently compensate for low mTOR activity35. Indeed, CTK1, a yeast ortholog of CDK12, plays active roles in translation regulation. CTK1 physically associates with translating ribosomes and enhances their translational accuracy through phosphorylation of the ribosomal protein Rps2, and the loss of CTK1 function causes a decrease in translation efficiency and growth defects36. A follow-up study in yeast extensively demonstrated that depletion of CTK1 impaired the formation of the 80S initiation complex, resulting in attenuated cap-dependent translation initiation; however, the translation of internal ribosome entry site (IRES)-containing mRNAs was not affected37. We recently reported that, in mammalian cells, human CDK12 can directly phosphorylate 4E-BP1 and control translation of a set of mitotic genes, as shown by a genome-wide analysis11. In contrast to yeast CTK1, which stimulates mRNA translation globally, our next-generation sequencing (NGS)-based ribosome-profiling analysis (Ribo-Seq) was used in our study, and it revealed that CDK12 is necessary for protein synthesis of a group of 200 selective genes. Remarkably, the majority of CDK12 target genes essentially functioned in mitotic progression. From the mechanistic view of translation control, the phosphorylation status of 4E-BP1 was dramatically altered by the depletion of CDK12 in the cells, a finding corroborated by the ability of the affinity-purified CDK12 to phosphorylate 4E-BP1 in the reconstituted in vitro kinase assay. Moreover, this kinase activity was profoundly enhanced by phosphorylation priming by the mTOR kinase complex, strongly suggesting that a certain set of mTOR-dependent genes requires CDK12 activity for efficient translation, as modeled in Fig. 10, suggesting multiple phosphorylation events affected the protein during mitosis. Other evidence for mitotic-specific phosphorylation was shown by anti-CDK12 immunoprecipitates that were detected by the MPM-2 monoclonal antibody, a phospho-epitope-specific antibody that recognizes a number of phosphorylated proteins in mitosis10. Therefore, it is possible that the activity of CDK12 might be regulated by cell cycle-specific phosphorylation patterns, although it is not clear whether critical phosphorylation sites, such as those in the T-loop of CDK12 are regulated similarly throughout the cell cycle. Therefore, it is necessary to investigate whether the kinase activity of CDK12 or cyclin K can fluctuate through the cell cycle, and the search for potential upstream kinases for CDK12 phosphorylation at the onset of mitosis is required. Another question regarding the role of CDK12 in translation control is its predominant nuclear localization, where translation events do not usually occur. How does CDK12 cooperate with mTOR to phosphorylate 4E-BP1 and regulate translation, which is assumed to occur mainly in the cytosol? Several subunits of the mTOR complex are partly localized to the nucleus. For example, 4E-BP1 and eIF4E can move in and out of the nucleus, controlling the nuclear export of specific mRNAs39. In this respect, CDK12 may phosphorylate 4E-BP1 in the nucleus and even function cotranscriptionally to remodel the cap complex of nascent target mRNAs and facilitate transcription by phosphorylation of the RNAPII CTD. In summary, little is known about the involvement of CDKs in translation. However, studies on certain CDKs, including human CDK12 and yeast CTK1, showed that these kinases play critical roles in translation control through the phosphorylation of translation machinery components. Therefore, further investigation into CDKs is needed to understand how they affect gene expression throughout translation.Translational control is another layer of gene expression regulation that controls protein synthesis from a given mRNA species. In general, translation is controlled through two different ways: global control, whereby the translation of most mRNAs in the cell is affected, and mRNA-specific control, in which the translation of a specific mRNA or defined group of mRNAs is regulated without affecting the overall protein synthesis or translation efficiency of the cells. It is also thought that global control mainly occurs through the regulation of general translation factors by modifications, such as phosphorylation, whereas mRNA-specific control is mostly mediated by regulatory protein complexes that bind to specific mRNAs; these complexes can recognize particular elements that are usually encrypted in the 5′ and/or 3′ untranslated regions (UTRs) of the target mRNAs, which form specific secondary structures of these mRNAs. It is well known that several kinases play important roles in both global and mRNA-specific translational control, although the roles of CDKs in translational regulation have not been well defined. An early report showed that mitotic CDK1 can phosphorylate 4E-BP1 in a cell cycle-dependent mannerBRCA mutations, discovered by the Cancer Genome Atlas (TCGA) project. The analysis identified CDK12 as one of the recurrently mutated genes in 3% of HGSOC patients40. These CDK12 mutations were sporadic, but most of these mutations were either nonsense, indel, or missense mutations in the protein kinase domain, suggesting potential loss of CDK12 function in this type of cancer. We now understand that CDK12 is a universal regulator of DDR and HR gene expression, including BRCA1. Since BRCA1 defects are closely associated with susceptibility to breast cancer, ovarian cancer, and prostate cancer, CDK12 mutation has been similarly related to the susceptibility of these cancers, with a defect in the double-strand break repair mechanism mediated by DDR and HR defined as “BRCAness”42. In addition, it is well known that BRCA1-defective cancers are intrinsically sensitive to poly (ADP-ribose) polymerase (PARP) inhibition43. The advent of PARP inhibitor monotherapy established a novel concept44, termed synthetic lethality, whereby a specific defect in the cell creates another specific susceptibility to induce cell death. Therefore, the synthetic lethal relationship based on PARP1 inhibition could be applied to CDK12 mutants, similar to its application to BRCA1 mutants45, and CDK12 inhibition might provide a rationale for the use of combination therapy with a PARP inhibitor in CDK12 mutation-related cancers. Further study of CDK12 mutants in HGSOC demonstrated that most CDK12 mutations abolish the CDK12/Cyclin K interaction, resulting in the inactivation of CDK12 kinase, defects in the expression of HR genes, impairment of DNA double-strand break repair via HR, and reinforced sensitivity to the PARP inhibitor46. To support this finding, CDK12 was identified in a synthetic lethality screening, in which CDK12 depletion conferred sensitivity to PARP inhibition as a phenocopy of BRCA deficiency45. Interestingly, the majority of CDK12 mutations were shown to be mutually exclusive in BRCA1 and BRCA2 mutants, strongly indicating that CDK12 may act as an upstream effector in the same pathway in which BRCA1 and BRCA2 function as tumor suppressors45. In support of this hypothesis, CDK12 mutants drive defects in the expression of key DDR genes, including BRCA1, in ovarian cancer46. Therefore, we now consider CDK12 an upstream factor in the BRCA and HR gene response pathway, and CDK12 mutations, as well as BRCA mutations, can be therapeutically used to predict the sensitivity to PARP inhibitors in cancer treatment.The role of CDK12 in human cancer was first indicated in high-grade serous ovarian cancer (HGSOC), a disease characterized by a relatively high frequency of 47. In addition to this observation, the CDK12 gene is frequently amplified in HER2-positive tumors48. The HER2 amplicon usually contains the CDK12 gene because CDK12 is a neighboring gene to HER2 in the human genome. In this circumstance, CDK12 is overexpressed by copy number amplification, and its activity is accordingly upregulated. It is intriguing that tumors with BRCA mutations are more likely to be HER2-negative49, implying that high activity of CDK12 in the HER2 amplicon in HER2-positive breast cancers maintains BRCA function and genome stability during tumor development. If high CDK12 activity has these functions, then how can HER2-positive breast cancer cells develop tumors without disrupting genome stability? It is possible that high CDK12 activity can upregulate DDR genes to promote rapid DNA replication50. In another possibility, CDK12 might serve as a key factor for the expression of important oncogenes. For example, MYC is a strong oncogene that is highly deregulated in most human cancers. It was demonstrated that chemical inhibition of CDK12 reduced tumor growth with the concurrent abrogation of MYC expression in patient-derived xenograft experiments52. In support of these observations, CDK12 is necessary for the 3′ end processing of MYC transcription7 and ribosome protein synthesis that induce MYC-driven anchorage-independent growth11. In addition, CDK12 was found to have a synthetic lethal relationship with MYC53 and EWS/FLI rearrangements in Ewing sarcoma54, indicating that CDK12 is an essential component in oncogene-driven tumorigenesis. Thus, it is possible that CDK12 can cooperate with oncogenic transcription factors, such as MYC and EWS/FLI, to drive oncogenic transcription programs by modulating RNAPII activity as a transcriptional cofactor55. In this scenario, CDK12 might be a promising therapeutic target for the cancers described -positive breast cancers, there is a significant correlation between CDK12 overexpression and tumor aggressiveness56. Although CDK inhibitors have adverse cross-reactivity due to the close similarity of the core kinase domains among CDKs, two CDK12-specific inhibitors were recently identified as having highly potent inhibition activity against a CDK12-mediated gene expression program and were shown to be potential anticancer agents. The first CDK12-specific inhibitor, THZ531, was derived from a covalent CDK7 inhibitor, THZ122. This small molecule is a potent inhibitor of the kinase activity of both CDK12 (IC50 of 158 nM) and CDK13 (IC50 of 69 nM) and can also covalently target Cyc1039, which is located outside of the CDK12 kinase domain and is highly conserved between yeast and the human CDK12 family. A crystal structure study virtually demonstrated that the short region neighboring Cyc1039 forms an ATP cleft outside the canonical kinase domain of CDK12. This unique feature enables THZ531 to induce highly selective inhibition of CDK12/CDK13, compared to CDK7 or CDK9. Inhibition of CDK12 by THZ531 can induce anti-proliferation of cancer cells by inducing apoptosis (IC50 of 50 nM in Jurkat cells), presumably due to transcriptional stress, similar to its predecessor, THZ1. The other CDK12 inhibitor, SR-4835, was derived from structure-guided optimization of small molecules with high affinity for CDK12 and CDK13. In contrast to THZ531, the inhibitory mode of SR-4835 is ATP-competition via hydrogen bonding with the hinge region of the kinase core domain, which makes SR-4835 highly selective for CDK12 and CDK13, among kinases23. The pharmacological inhibition of CDK12 by both inhibitors suppresses the expression of DDR genes, leads to a global decrease in the Ser2 phosphorylation of the CTD and triggers apoptosis. Intriguingly, the SR-4835 study showed that the treatment induced less toxicity in primary cells than it did in tumor cells. The cause of this tumor-specific cytotoxicity is unclear. It is possible that CDK12 might govern some cancer-specific transcription programs. In this circumstance, cancers might depend on CDK12 activity for cell proliferation, and its inhibition may result in cancer-specific cell death.CDKs are known to play significant roles in cell cycle progression and gene expression regulation, and they are highly active in human cancers. The development of CDK inhibitors is of great interest for clinical purposes, mainly for cancer treatments. Therefore, the identification of CDK inhibitors has been passionately pursued to treat cancers by preventing cancer cell proliferation. For example, the FDA approved the CDK4/6 inhibitor palbociclib for the treatment of hormone receptor-positive, HER2-negative metastatic breast cancer57. In turn, BRCA-deficient cells would utilize another, error-prone DNA repair pathway mediated by PARP. Therefore, synthetic lethality is achieved when cells are no longer able to repair DNA damage because of the dual inhibition of BRCA and PARP, induced either genetically or pharmacologically. However, this synthetic lethality has limitations in cases involving BRCA wild-type cancers, the genetic reversion of a BRCA mutant that mediates BRCAness in cancer to a form that restores HR activity and resistance to PARP inhibitors58. CDK12 inhibition may overcome this limitation by eliminating restored HR and forcing the cancer cells to acquire the BRCAness phenotype59. This possibility makes targeting CDK12 a compelling strategy to overcome the drawbacks of PARP inhibitors when clinically applicable.In addition to the DDR genes involved in HR, PARP has an important function in DNA damage repair by maintaining genomic stability and cancer cell survival. As we discussed previously, PARP inhibitors have attracted intense interest because they can induce synthetic lethality in cancer cells harboring BRCA mutations, which is defined as “BRCAness”; defects in HR repair are mostly induced by BRCA1 or BRCA2 lossCDK12, a RNAPII CTD kinase, is involved in multiple biological processes, not only in transcription elongation but also splicing, translation, and DDR gene regulation. Additionally, its function is often lost in advanced recurrent cancers, but its inhibition shows promising therapeutic potential in the treatment of BRCA mutant cancers or triple negative breast cancers. Although we have recently discovered CDK12 functions, its mechanisms in diverse functions have been elucidated. Since CDK12 is a protein kinase, the identification of its substrates, including non-CTD substrates, should be addressed in the search for novel functions. For example, CDK12 might target unknown transcriptional factors for phosphorylation to prevent internal polyadenylation and maintain the fidelity of DDR gene transcription. Together with identification of potential substrates, investigations into CDK12-associated factors that regulate CDK12 activity will be instrumental, as they were, for example, for endogenous inhibitors in the similar cases of HEXIM in P-TEFb. CDK12 is a uniquely long protein with several structural features; therefore, it possible that the long arm region of CDK12 can interact with critical regulatory factors involved in gene transcription or translation. The in vivo study of CDK12 is not well elucidated due to lethality by the CDK12 knockout. Therefore, conditional modulation of CDK12 loss or gain of function in a mouse model may lead to insights into the mechanism of CDK12 and its tumor suppressor or oncogenic roles in tumorigenesis in subsequent studies; these studies will broaden the understanding of the functions and molecular mechanisms of CDK12.Since CDK12 mutations and alterations in the genome were found in various tumors, therapeutic approaches using CDK12 inhibition should be developed for cancer treatment. Recent CDK12-specific inhibitors are very useful tools to study the therapeutic application of CDK12, especially in combination therapy with PARP inhibitors to enhance drug sensitivity and promote chemotherapy. In conclusion, CDK12 inhibitors can effectively sensitize cancer to PARP inhibition, overcoming primary and acquired resistance, and clinical trials (NCT01434316) and the development of more powerful CDK12-specific inhibitors are expected to be promising.


Epigenetic modifications regulate normal physiological, as well as pathological processes in various organs, including the uterus and placenta. Both organs undergo dramatic and rapid restructuring that depends upon precise orchestration of events. Epigenetic changes that alter transcription and translation of gene-sets regulate such responses. Histone modifications alter the chromatin structure, thereby affecting transcription factor access to gene promoter regions. Binding of histones to DNA is regulated by addition or removal of subunit methyl and other groups, which can inhibit or stimulate transcription. Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of polycomb repressive complex 2 (PRC2) that catalyzes tri-methylation of histone H3 at Lys 27 (H3K27me3) and subsequently suppresses transcription of genes bound by such histones. Uterine EZH2 expression exerts a critical role in development and function of this organ with deletion of this gene resulting in uterine hyperplasia and expression of cancer-associated transcripts. Elucidating the roles of EZH2 in uterus and placenta is essential as EZH2 dysregulation is associated with several uterine and placental pathologies. Herein, we discuss EZH2 functions in uterus and placenta, emphasizing its physiological and pathological importance. It is becoming increasingly apparent that DNA sequences alone do not dictate gene activity or disease risk. Instead, these processes are regulated by interaction of genes with various factors in the intracellular and extracellular environments. Environmental factors may cause phenotypic effects by inducing mutations in DNA, but commonly they influence gene activity through epigenetic changes. Epigenetics is a term coined by the British developmental biologist Conrad Waddington in 1942, which defines a layer of information that exists beyond that encoded and inherited in the DNA sequence itself [3) to a cytosine nucleotide preceding a guanine nucleotide (CpG). Areas of the genome that contain high numbers of these sites are known as CpG islands. These CpG islands frequently occur in or near promoter regions of genes [Various epigenetic modifications have been described, including DNA methylation  and chroof genes . The ncRNA are functional RNAs that are not translated into a protein; some of these have regulatory effects at the transcriptional or post-transcriptional levels. They are classified by size, and there are three major short non-coding RNAs ; microRNA (miRNA), and piwi-interacting RNA (piRNA)), as well as long non-coding RNA (lncRNAs). The miRNAs and siRNAs interact as a regulatory network, often binding to mRNA to promote its degradation, thus inhibiting further translation . siRNAs Within chromosomes, DNA is encased by members of a protein family called histones to form nucleosomes. Chromatin functionality is modified by assembly and disassembly of these nucleosomes, and histones are key in this . HistoneMethylation commonly occurs on histones H3 and H4 on specific lysine (K) and arginine (A) residues. Lysine (K) can be mono, di and tri-methylated, which dictates the biological outcome . In contEnzymes that catalyze these fundamental epigenetic alterations are identified as “writers” and “erasers” . WritersPolycomb repressive complex 1 and 2 (PRC1 and PRC2) function as epigenetic repressors ; PRC2 isDysregulation of H3K27 methylation is associated with multiple pathologies, including tumorigenesis and metastasis . NeoplasIn this review, we will discuss potential roles of EZH2 in regulating normal physiological responses in the uterus and the placenta, which forms an intimate association with the uterus during pregnancy. To help understand EZH2’s role in the uterus and other organs, transgenic mouse models have been created in the past few years, and the resulting phenotypic changes in these mice will be described. Lastly, we will consider the role of EZH2 in uterine diseases.Three primary mechanisms of EZH2 action have been characterized. In the canonical pathway, the action of the PRC2 complex requires EZH2, which results in trimethylation of H3K27, i.e., H3K27me3 formation. The closely related PRC1 family ubiquitinates histones and consists of heterogeneous variants, which contain CBX (chromobox-containing protein) and signal through the canonical pathway or lack CBX proteins and signal through non-canonical pathways. PRC1 plays important roles during embryology but also disease formation. There is also recent evidence that PRC1 can function as a transcriptional activator . For furEzh1 is believed to have arisen by gene duplication from Ezh2. Ezh1, in some cases, may compensate for the loss of Ezh2, and can also potentially have a role in cancer. However, our focus is on EZH2, as there is strong evidence to suggest that it can be affected by xenoestrogens and dysregulation of EZH2 leads to a variety of cancers, including uterine cancer, as detailed below [The core components of PRC2 are EZH2, embryonic ectoderm development (EED), suppressor of zeste 12 (SUZ12), and retinoblastoma (Rb)-associated protein 46/48 (RbAp46/48) . EZH2 ised below . Wassef ed below  provide The SUZ12 protein aids nucleosome recognition  and RbApPhosphorylation of EZH2 on serine 21 by protein kinase B (AKT) inhibits PRC2-mediated H3K27 enzymatic activity. However, this phosphorylation is seemingly necessary for PRC2-independent functions of EZH2 . ThreoniLastly, EZH2 can alter gene expression in a PRC2-independent manner, which entails the methylation of non-histone targets or interaction with other transcription factors to activate downstream genes. For example, EZH2 functions as a co-activator of estrogen receptor 1  and promotes the transcription of its target genes. Thus, EZH2 inhibits gene expression by altering chromatin structure, but it can also act as a coactivator and promote transcription. Whether EZH2 acts to repress or stimulate transcription largely relates to its association with other proteins. EZH2 regulation itself occurs through post-translational modifications, including the phosphorylation of various amino acids within the protein.Ezh2 induces early embryonic lethality. The null mouse embryos implant in the uterus but fail to correctly develop between gestation day (GD) 6.5 and 8.5. Normal embryonic germ layers fail to differentiate, and abnormal development of mesoderm cells in extraembryonic cells are observed [The global deletion of observed . DetermiEzh2 gene [Pgr) promoter [Ezh2 gene were generated as described in Ezh2cKO and WT female mice receiving vehicle (V) or 17β–estradiol (E2) [Krt5), keratin 15 (Krt15), the maternally-imprinted gene- H19, leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5), and cell division cycle associated 5 (Cdca5). Fang et al. [Ezh2cKO uteri relative to WT controls [Ezh2cKO mice, and may be involved in the uterine epithelial proliferation seen even following ovariectomy in these animals. To determine the role of EZH2 in the uterus, we have generated a conditional knockout mouse lacking EZH2 in progesterone receptor (PGR)-expressing cells in the uterus and other organs, such as the ovary, oviduct, pituitary gland, and mammary gland. . To prodzh2 gene , were obpromoter  provide iol (E2) . Conditig et al.  also shocontrols . Based oEzh2cKO uteri supports a fundamental role for this protein in restraining uterine epithelial proliferation, as these mice had uterine luminal and glandular epithelial hyperplasia [Ezh2cKO model by Fang et al. [Disrupted EZH2 expression has been implicated in several uterine diseases, including endometriosis, uterine fibroids, endometrial cancer, and other proliferative disorders ,43,44,45erplasia . Similarg et al. , who addThe EZH2 mutations found in endometrial and other cancers are predominantly gain of function mutations ,48,49. AARID1A. The reduction in this tumor suppressor gene presumably leads to unrestrained expression of cancer-promoting genes [These studies indicate that EZH2 dysregulation increases risk for cancer or other proliferative diseases, depending upon which histone protein marks and corresponding genes are affected by this epigenetic modifier. For instance, if EZH2-induced methylation of H3K27 normally represses a transcription factor or proto-oncogene, EZH2 loss may result in expression of that gene and downstream genes. For proto-oncogenes, loss of EZH2 may lead to unrestrained transcription of these genes, with resulting neoplastic transformation. However, specific ones related to endometrial cancer remain to be identified. Conversely, for tumor suppressor genes, EZH2 overexpression could suppress these genes and increase susceptibility to tumorigenic changes. For example, EZH2 overexpression in endometrial cancer causes a downregulation of the tumor suppressor gene ng genes . Thus, bAR) [FOXO1) [ESR1, CDH1, and PGR, using human endometriotic tissues.While we have not examined these mice at later ages to determine if their uterine pathologies become more severe, it is clear that women with endometrial hyperplasia are at risk for endometrial cancer ,46. TranAR) , E-cadhe [FOXO1) . Colón-C [FOXO1)  identifiThe placenta is a transient organ that is essential during pregnancy, in that it allows for communication between mother and fetus and exchange of nutrients, gases, and waste. Defects in placental development lead to a number of disorders, such as preeclampsia, recurrent miscarriage, stillbirth, and intrauterine growth restriction, that impact the mother, embryonic or fetal growth, and/or later offspring health . The plaN-acetylglucosamine transferase (Ogt), an X-linked gene that has low expression in placentas of male fetuses under maternal stress conditions [Ezh2 deletion, combined with prenatal stressors , would result in similarly impaired neurodevelopment in females. To generate placenta-specific Ezh2 conditional knockout mice, a previously developed transgenic mouse model was used [Ezh2 conditional knockout in the uterus. this model, a regulatory sequence that is responsible for placenta-specific gene expression of aromatase P450 (CYP19), already defined previously in the human, was used to drive Cre expression [Ezh2 expression was ablated from the trophoblast lineages of the placenta by GD 11.5, the age by which CYP19-Cre is expressed in all trophoblast cells. While the trophoblast-specific Ezh2cKO did not impact bodyweight or cause neuroendocrine dysregulation in females, the combination of trophoblast-specific Ezh2 knockout and prenatal stress led to later pathological changes [Ezh2 deletion made females more vulnerable to prenatal stress. A conditional knockout mouse model has been created to help understand the role of EZH2 in the mouse placenta. Nugent et al.  found thnditions , contribnditions . The invwas used  that is pression . Using t changes . SpecifiCYP19-Cre transgene is expressed in all cells derived from trophoblast stem cells by GD 11.5. Therefore, it is unclear what the impact would be if Ezh2 expression were reduced in trophectoderm cells of the blastocyst that give rise to the chorionic portion of the placenta. As the placenta is composed of multiple trophoblast types with diverse functions, the impact of deleting Ezh2 in specific trophoblast cell populations, such as invasive trophoblast cells that are important for anchoring the placenta to the uterine wall, spongiotrophoblast and trophoblast giant cells that are important for steroid and peptide hormone production, and glycogen trophoblasts that serve as an energy reserve for the placenta [Ezh2 deletion affected the histology of this organ and trophoblast differentiation. Placenta-specific Ezh2cKO mice may have defects in placenta structure, weight, or nutrient transport functions that could impact females in ways not assessed in this study. Similarly, different types of maternal stressors could lead to different phenotypes observed in the placenta and resulting offspring. As noted above, the placenta , would aRND3 [HSD11B2 [ANGPTL4 [Excess glucocorticoids in pregnancy is another stressor that can lead to neuroendocrine dysregulation. In the placenta, 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) inactivates glucocorticoids, contributing to establishment of the glucocorticoid barrier, thus regulating fetal glucocorticoid exposure ,72,73. TRND3 , and HSD[HSD11B2 . The int[ANGPTL4  was repoLv et al.  found thEzh2cKO mice provide an experimental model to elucidate the complex roles of EZH2 in uterus and placenta. Although select genes affected by EZH2 have been identified in these organs, the global gene targets remain uncertain. Therefore, these transgenic mouse models, coupled with advanced ‘omics technology, such as spatial transcriptomics and chromatin immunoprecipitation sequencing, will further our understanding of how loss of Ezh2 in the uterus and placenta affects transcriptomic profiles in specific cell-types. Such knowledge will have important clinical ramifications in understanding uterine and placental regulation by EZH2 in health and disease.EZH2 is a critical epigenetic modifier acting predominately through the PRC2 complex to affect the H3K27me3 histone protein methylation mark. Uterine EZH2 plays a critical role in regulating epithelial proliferation; both under and overexpression of this protein may result in hyper-proliferative conditions. Similarly, EZH2 dysregulation is linked to placenta-associated pregnancy disorders. The


Background: Dysregulation of Pit-Oct-Unc family transcription factors has been implicated in esophageal squamous cell carcinoma (ESCC). In this study, we evaluated the expression and promoter methylation status of Octamer (OCT) transcription factor genes in human ESCC clinical specimens to investigate the mechanism underlying this observation along with the clinical significance.Methods: Total DNA or RNA was extracted from ESCC tissue specimens and the mRNA level of genes encoding the transcription factors OCT1, OCT2, OCT3/OCT4, OCT5, OCT7, OCT9, and OCT11 were evaluated by quantitative PCR. The DNA methylation status of gene promoters was assessed by bisulfite pyrosequencing and next-generation sequencing. The relationship between the expression of these transcription factors and ESCC proliferation was investigated in vitro and in vivo with the colony formation assay and a mouse xenograft tumor model, respectively. We also examined the correlation between OCT gene expression and promoter methylation and clinicopathologic characteristics of ESCC.Results:OCT1 was upregulated whereas OCT4, OCT6, and OCT11 were downregulated in ESCC compared to non-tumor tissue. OCT2, OCT7, and OCT9 were undetected in all samples. OCT1, OCT6, and OCT11 levels were negatively correlated with the methylation of their respective promoters, but there was no relationship between OCT4 expression and promoter methylation status.Conclusion: Changes in promoter methylation rate underlie the observed alterations in OCT1, OCT6, and OCT11 expression in ESCC, whereas another mechanism is likely responsible for the dysregulation of OCT4. Pit-Oct-Unc (POU) family transcription factors mediate the transcription of downstream genes that are necessary for the maintenance of pluripotency in embryonic stem cells but also promote the proliferation or stemness of human cancers by binding to the octamer sequence motif (AGTCAAAT consensus sequence) of gene promoters. Octamer (OCT) transcription factors belonging to the POU family have been shown to play a regulatory role in cancer cells Methylation of gene promoters is an epigenetic modification that regulates gene transcription A series of 150 surgically resected fresh ESCC and their corresponding adjacent normal tissue samples were collected at the First Affiliated Hospital of Zhengzhou University  and snap-frozen in liquid nitrogen from 2012 to 2016 . No patiin vitro experiments and in DMSO along with polyethylene glycol 400 or Tween 80 for use in mice. The agents were prepared according to a published protocol We used three ESCC cell lines  that have also been described in our previous work Genomic DNA was extracted from ESCC or non-tumor tissue using the QIAamp DNA Mini Kit  according to the manufacturer's protocol. DNA concentration and quality were determined using a spectrophotometer . The samples were subjected to bisulfite treatment with the ZYMO EZ DNA Methylation-Gold Kit  according to the manufacturer's protocol. The promoter region of each target gene - i.e., the sequence 2000 bp upstream of the transcription start site—was obtained by searching the NCBI database. Methyl Primer Express v1.0 software (Thermo Fisher Scientific) was used to identify and predict CpG sites in the promoter sequence and primers were designed for PCR amplification of this region. The amplified product, which was about 218 bp in size, was purified using magnetic beads for sequencing library construction , and a barcode was added for second-generation sequencing on the Iontorrent PGM platform (Life Technologies). If the sequencing result of the OCTs' promoter region after BSP treatment is C, it indicates that the CpG site was methylated; if the sequence of the OCTs promoter region sequencing after BSP treatment was T, it indicated that the methylation of the CpG site was not occurred. The methylation rate of OCT gene promoter sequences was calculated as the number of methylated CpG sites divided by the total number of CpG sites. The forward and reverse primers used for BSP and NGS experiments were as follows: Oct1/POU2F1, 5'-ATTGAGGGYGTTGTTTTAGTT-3' and 5'-CCTCAAAAAAACTCCACC-3'; Oct4/POU5F1, 5'-GTGGTTAGGTATTTTGGGAGGT-3' and 5'-CAAACTAAACTCR AACTCCC-3'; Oct6/POU3F1, 5'-TYGAGATTTTTTTTTTTTGGAATT-3' and 5'-AACRA TTCTACAATCCTACRC-3'; and Oct11/POU2F3, 5'-TTGTAATTTTAGGGAAG TTTAATTGA-3' and 5'-CTCAAATTCTCTTATCCCTAATTAAA-3' .OCT1, OCT2, OCT3/OCT4, OCT5, OCT7, OCT9, and OCT11. The forward and reverse primers used for qPCR were as follows: Oct1/POU2F1, 5'-GAAACGCACCAGCATAGAGACC-3' and 5'-GGCGGTTACAG AACCAAACACG-3'; Oct2/SLC22A2, 5'-GAGATAGTCTGCCTGGTCAATGC-3' and 5'-GTAGACCAGGAA TGGCGTGATG-3'; Oct4/POU5F1, 5'-CCTGAAGCAGAAGA GGATCACC-3' and 5'-AAAGCGGCAGATGGTCGTTTGG-3'; Oct6/POU3F1, 5'-GTGTTC TCGCAGA CCACCATCT-3' and 5'-CGCGA TCTTGTCCAGGTTGGTG-3'; Oct7/POU3F2, 5'-GTGTTCTCGCAGACCACCATCT-3' and 5'-GCTGCGATCTTGTCTATGCTCG-3'; Oct9/POU3F4, 5'-GTGTTCTCGCAGACCACCATCT-3' and 5'-GCGATCTTGTCAAT GCTGGTCG-3'; and Oct11/POU2F3, 5'-GCTGGAGAAGTTTGCCAAGACC-3' and 5'-GTGAGATGGTGGTCTGGCTGAA-3'. The β-Actin gene (5' CACCATTGG CAATGAGCGGTTC-3' and 5'- AGGTCTTTGCGGATGTCCACGT-3') was used as an internal control for calculating the relative expression level of target genes.RNA was extracted from paired ESCC and non-tumor clinical specimens using the PARIS Kit (Thermo Fisher Scientific) and reverse transcribed using Multiscribe reverse transcriptase (Thermo Fisher Scientific) according to the manufacturer's protocol. qPCR was performed as previously described 3 cells/well); the colony formation assay was performed as previously described 546) was measured and used to calculate colony formation rate according to the following formula: [(OD546 of the control group) - (OD546 of the treatment group)]/(OD546 of the control group)] × 100%. The results were used to determine half-maximal inhibitory concentration (IC50) values ESCC cells were cultured in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum. For colony formation experiments, the cells were treated with various concentrations of antitumor agent  for 48 hOCT1 sequence or a siRNA targeting OCT1. OCT1 protein level was detected by western blotting , with GAPDH serving as a loading control.Total protein was extracted from ESCC cells according to a published protocol in vivo, we established a subcutaneous xenograft tumor model in nude mice. Cultured ESCC cells were subcutaneously injected into the mice All experiments with mice were approved by the Institutional Animal Care and Use Committee of Zhengzhou University  and were performed in accordance with the 1986 UK Animals (Scientific Procedures) Act and associated guidelines. To examine the growth of ESCC cells P < 0.05 was considered significant. IC50 values of the tested agents against ESCC cells were calculated using Origin v6.0 software .Statistical analyses were performed with SPSS v9.0 software . Differences between groups were evaluated for statistical significance by two-way analysis of variance with Bonferroni correction. OCT2, OCT7, and OCT9 were not expressed in either type of sample .The expression of OCT transcription factors in ESCC and paired non-tumor tissue was examined by qPCR. e Figure . MeanwhiOCT1, OCT4, OCT6, and OCT11 expression was dysregulated in ESCC specimens, we examined the methylation status of their promoters  compared to non-tumor tissue  .Given that OCT1, OCT6, and OCT11 were negatively correlated with transcript levels in both types of sample  of OCT transcription factors and methylation of their gene promoters in ESCC and non-tumor tissues. We found that the methylation rates of e Figure . HoweverOCT1 was the most highly expressed OCT transcription factor in ESCC specimens and our previous finding that higher levels of OCT4 were significantly associated with higher tumor grade in ESCC OCT1 and OCT4 expression and clinicopathologic characteristics of the tumors specimens. We found that OCT1 levels were elevated for higher (more aggressive) T stages of ESCC  compared to lower T stages   Figure A. MoreovC Figure B and pooC Figure B, a trenC Figure C, D.OCT4, there was no relationship between mRNA level and T stage of ESCC . In contrast, Het-1A, non-tumor human esophageal epithelial cells, and two other patient-derived ESCC cell lines (Nos. 4 and 5) expressed a low level of endogenous OCT1. We used KYSE70, KYSE140, and KYSE450 cells and patient-derived ESCC cell line Nos. 1-3 for OCT1 knockdown and patient-derived ESCC cell line Nos. 4 and 5 for OCT1 overexpression experiments (We used ESCC cell lines to examine the function of OCT1 in ESCC. eriments .OCT1 expression and examined cell growth in vitro using ESCC cell lines and patient-derived cells expressing high endogenous levels of OCT1 (OCT1 in Het-1A cells and patient-derived cells (No. 4 and 5) with low endogenous OCT1 expression  in ESCC and the mechanism of OCT4's expression in ESCC needed for the further research. OCT1 may be a useful therapeutic target for ESCC treatment. However, functional redundancy among OCT proteins could contribute to the failure of single-target inhibitors; it is therefore worthwhile to determine the expression levels of all OCT family members.ESCC is one of the most aggressive neoplasms and has poor clinical outcome OCT1 overexpression increased proliferation in Het-1A cells whereas OCT1 knockdown in ESCC cell lines or patient-derived ESCC cells with high endogenous OCT1 expression had the opposite effect. Thus, modulating the expression of OCT1 either directly or indirectly by targeting its epigenetic modifiers is a potential strategy for overcoming the drug resistance of ESCC cells.There have been few studies to date investigating the role of OCT1 in ESCC OCT1 expression or promoter methylation was related to both T stage and histological grade. OCT transcription factors are key regulators of the proliferation, metastasis, and drug resistance of human cancer cells and are therefore promising targets of antitumor drugs.We previously demonstrated that OCT4 was expressed in ESCC clinical specimens and that OCT4 level was unrelated to T stage but was positively associated with histological grade of ESCC OCT gene promoters directly by BSP and NGS. In contrast, traditional methylation-specific PCR can only detect the presence or absence of methyl marks at CpG sites in single clones OCT4 promoter was lower in ESCC compared to non-tumor tissue but that this was unrelated to transcript expression. Additional studies are needed to clarify the clinical significance of this observation.Methylation of gene promoters is a regulatory mechanism that is typically associated with transcriptional repression in vitro for a long time Moreover, the previous studies mainly used existing current cell lines, but recent studies have shown that tumor cells are affected by tumor microenvironment and other factors in tumor tissues, which are different from current cell lines cultured OCT1, OCT6, and OCT11 in ESCC tissue. These results indicate that targeting these proteins or the factors regulating their methylation/demethylation - especially that of OCT1 - may be an effective strategy for the treatment of ESCC.Changes in promoter methylation status are responsible for the altered expression of Supplementary figures and tables.Click here for additional data file.


Drosophila, it has been proposed that transcription is fundamental for TAD organization while the participation of genetic sequences bound by architectural proteins (APs) remains controversial. Here, we investigate the contribution of domain boundaries to TAD organization and the regulation of gene expression at the Notch gene locus in Drosophila. We find that deletion of domain boundaries results in TAD fusion and long-range topological defects that are accompanied by loss of APs and RNA Pol II chromatin binding as well as defects in transcription. Together, our results provide compelling evidence of the contribution of discrete genetic sequences bound by APs and RNA Pol II in the partition of the genome into TADs and in the regulation of gene expression in Drosophila.Chromosomes are organized into high-frequency chromatin interaction domains called topologically associating domains (TADs), which are separated from each other by domain boundaries. The molecular mechanisms responsible for TAD formation are not yet fully understood. In  Drosophila, transcription is thought to be required for TAD formation, while the role of architectural proteins remains controversial. Here, the authors find that deletion of domain boundaries at the fly Notch locus results in TAD fusion and long-range topological defects, loss of architectural protein and RNA Pol II chromatin binding, and transcription defects.In  At the sub-megabase scale, chromosomes are organized into high-frequency chromatin interaction domains termed topologically associating domains (TADs) separated from each other by domain boundaries6. The presence of TADs has been described in different species suggesting that they represent a common feature of genome organization7. From a transcriptional perspective, TADs can facilitate the establishment of specific regulatory landscapes by bringing into proximity enhancers and promoters, while at the same time, precluding unspecific regulatory communication with sequences outside the domains9. Disruption of TAD organization by genetic manipulation of domain boundaries can result in ectopic regulatory communication of sequences leading to gene misexpression and significant consequences in cell physiology and organismal development13.In eukaryotes, the genome is non-randomly folded in the nuclear space14. Manipulation of the CTCF binding motif at domain boundaries or the acute degradation of CTCF or subunits of the Cohesin complex leads to loss of TADs and alterations in gene expression18. In Drosophila, TAD boundaries are detected at chromatin accessible regions enriched for active histone marks and occupied by multiple APs including CP190, BEAF-32, M1BP, Su(Hw), and CTCF among others21. Analysis of high-resolution Hi–C datasets suggests that in the fly, genome domains emerge as a consequence of the transcriptional activity of sequences within the domain while APs modulate interactions between domains22. Genome-wide inhibition of transcription in embryos and cell lines results in a significant decrease in TAD boundary insulation; however, TADs do not disappear24. On the other hand, depletion of BEAF-32, an AP enriched at TAD boundaries, results in minor changes in genome organization19. Then, whether TADs in Drosophila are the result of self-aggregation of chromatin regions with similar transcriptional states or emerge as a consequence of the activity of discrete genetic elements bound by APs acting as chromatin insulators remains controversial.TAD boundaries in mammals are occupied by CTCF and the Cohesin complexNotch gene locus in Drosophila provided one of the first evidence linking chromatin insulation with domain formation and gene expression26. Deletion of a ~900-bp region upstream of the Notch promoter in the mutant allele fa(swb) results in loss of the interband 3C6-7 and fusion of the band containing Notch with the upstream band harboring the kirre locus26. This cytological effect leads to a reduction in Notch expression and an eye phenotype in adult mutant flies26. In Drosophila, the bands observed in polytene chromosomes correspond to TADs, and inter-band regions have a close correspondence with TAD boundaries28, which suggest that the Notch locus is a TAD itself and the 5′ intergenic region is a domain boundary.The Notch locus in Drosophila. We show that Notch is organized into two TADs with boundaries overlapping the 5′ intergenic region and an intronic enhancer. We generated CRISPR-Cas9 mediated deletions of the Notch domain boundaries and evaluated their topological effect by in nucleus Hi–C. Consistent with cytological data, removal of the entire 5′ intergenic region results in the fusion of the first domain of Notch with the upstream TAD. Furthermore, we uncovered that portions of the 5′ intergenic region, with binding sites for specific APs, act as discrete chromatin insulators between the upstream gene kirre and Notch, with removal of all regions necessary for TAD fusion. In all cases, topological disorganization of the first domain of Notch is accompanied by loss of APs and RNA Pol II occupancy at the disrupted boundary, reduction in transcription of the exons that reside within the affected domain and changes in the transcription levels of genes located in adjacent TADs. Removal of the intragenic enhancer resulted in the fusion of the two Notch domains and decreased transcription along the locus. Moreover, in wild-type cells, this genomic element is also the anchor of a megabase-sized domain, which is lost upon enhancer deletion, and results in changes in transcription of the genes located inside the domain. Together, our data provide compelling evidence on the contribution of domain boundaries to TAD formation and the control of gene expression in Drosophila.Here, we study the contribution of domain boundaries to TAD organization and the regulation of gene expression at the Notch, we performed in nucleus Hi–C using a 4-cutter restriction enzyme (MboI) in the embryonic cell line S2R+ in triplicate (see Methods)29 reaching a minimum of 89% valid pairs per replicate  comprises from the start of the gene to exon six while Domain 2  contains the remaining 4 exons  while the dnc locus is contained within a ~150-kb TAD (termed dnc domain). kirre domain-2 is adjacent to the 5′ end of Notch while the dnc domain is next to the 3′ end of the locus . The second boundary (termed B2) is located at intron five and the start of exon six and demarcates the transition between D1 and D2 domains of Notch  bound by more than 8 APs and RNA Pol II    matches the sequence between exon 6 to the end of the Notch gene  Fig. . The B2 31) Fig. . The enhNotch domain boundaries by qChIP. Consistent with ChIP-seq data, CTCF binding is only detected at the B1 boundary while the H3K27ac histone mark is ~10 times more enriched at the enhancer and exon 6 of the B2 boundary than at the B1 boundary , and the enrichment of histone marks H3K4me3, H3K27ac, and H3K27me3 at ary Fig. . In contary Fig. .Drosophila, TADs first emerge around the onset of Zygotic Genome Activation (ZGA)32. To get insight into the dynamics of domain formation at the Notch locus during early development, we analyzed public in nucleus Hi–C data24. A domain boundary at the 5′ intergenic region of Notch (B1) is detected at nuclear cycle 13 (nc13) before ZGA while a second domain boundary (B2) between intron five and exon six is detected at nc14 concomitant with ZGA. Both boundaries partition the Notch locus into two domains 35, and Zelda36. We found that the establishment of boundaries at Notch strongly correlates with the progressive gain of chromatin accessibility and the binding of Zelda up to nc14  and the male CNS-L3 cell line BG3 (ref. 42) and compared Notch 3D organization with the one observed in the male embryonic cell line S2R+. We observe two domains spanning the Notch locus in the male derived cell lines (S2R+ and BG3) in contrast with a single domain organization of Notch in the female derived cell line Kc167  and the male cell lines (S2R+ and BG3), with H4K16ac being highly enriched at the Notch region encompassing the D2 domain detected in male cells (exon 6–exon 9) while the histone post-translational modification H3K27me3 is enriched at the same region in female cells and overall depleted in male cells . Also, the correlation between the different mutants and the wild-type samples is high (0.89–93) which suggest only local topological effects upon deletion of small DNA fragments (<800 bp), as expected  removes M1BP DNA-binding site but not CTCF DNA-binding site as well as the ChIP-seq peak summits of CTCF, Su(Hw), Pita and BEAF-32  upstream of the PPR of Notch removes the CTCF DNA-binding site but not M1BP as well as the ChIP-seq peak summits of CTCF, Ibf1 and Ibf2   Fig. . TherefoNotch (5pN-Δ343) resulted in a dramatic loss of intra-domain interactions at the D1 domain  accompanied by a significant increase in interactions with the kirre domain-2  and therefore in TAD fusion  and a decrease in the intra-domain interactions for the D2 domain   Fig. . A similAPs Fig. . Therefo5pN-Δ102) leads to a marked decrease in the binding of RNA Pol II and CTCF, despite not removing the binding site for this protein  shows an even more pronounced decrease in RNA Pol II occupancy and loss of CTCF binding, consistent with the removal of its binding site  using nuclear extracts of S2R+ cells. Mutation of the CTCF binding site results in a sharp decrease in shift and therefore in binding of nuclear proteins to that sequence, suggesting that the CTCF binding motif is necessary for the recruitment of regulatory proteins including RNA Pol II  or show an increase (mutant 5pN-Δ183)  Fig. . In partn 6 Fig. . TherefoNotch. Therefore we evaluated the transcriptional level of kirre-full, kirre-med, and rst in all the B1 boundary mutants  are differentially expressed between wild-type and the enhancer mutant   Fig. . Interes05) Fig. , suggestNotch is a genetic element with two topological activities: it actively insulates the D1 and D2 domains of Notch while at the same time is responsible for the formation of the Nenh-mega-domain. Remarkably, loss of the Nenh-mega-domain impairs gene expression of genes located inside the domain, strongly suggesting that genome topology can impact gene expression.Taken together our data suggests that the intronic enhancer of Notch gene locus in Drosophila. We provide evidence that discrete genetic sequences occupied by APs and RNA Pol II are potent chromatin insulators that actively partition the genome into Topological Domains. Furthermore, partial disruption or complete removal of the domain boundaries alter genome topology, transcription, and the recruitment of APs and RNA Pol II. These findings have implications in our understanding of the mechanisms that promote genome organization and the control of gene expression as discussed below.Here we analyzed the topological and transcriptional consequences of TAD boundary disruption at the 2. Our collection of CRISPR-Cas9 mediated deletions of domain boundaries at the Notch locus provide evidence on the existence of autonomous genetic elements bound by APs and Pol II that act as chromatin insulators essential for TAD formation.Whether domain boundaries are autonomous discrete genetic elements mediating the formation of TADs is a subject of intense debatekirre and Notch is a modular chromatin insulator constituting a domain boundary. We uncovered that non-overlapping portions of the intergenic region, with binding sites for specific APs and RNA Pol II, act as discrete modules that restrain interactions of the kirre and the Notch genes, with the removal of all modules necessary for TAD fusion  where deletion of a ~0.9-kb region spanning the 5′ region of Notch results in loss of an interband and fusion of the 3C7 band containing Notch with the upstream band26. Also, reporter assays in transgenic flies and cytological evidence support an autonomous role for the 5′ intergenic region of Notch as a chromatin insulator as the ectopic insertion of this sequence is sufficient and necessary to split a band into two, forming an interband in polytene chromosomes46. In the case of the intragenic enhancer boundary, deletion of a ~2-kb region results in a dramatic increase of ectopic interactions between Notch domains and loss of a ~1-Mb domain downstream of Notch , and therefore, before transcription at the locus  and with the binding of proteins like RNA Pol II (nuclear cycle 12), the general transcription factor TBP, and the pioneering factor Zelda  resulted in a major decrease in transcription within the D1 domain (>80%) but just in discrete topological changes mainly detected as a reduction in intra-domain interactions for the D1 domain  have a stronger effect in RNA Pol II recruitment than deletion of the Promoter Proximal Region (5pN-Δ102) which contains a binding site for M1BP ) were cultured at 25 °C in Schneider’s Drosophila Medium (Thermofisher) supplemented with 10% FBS (Biowest) and penicillin/streptomycin (Invitrogen).53. The following antibodies were obtained from commercial sources: anti-H3K4me3 (Abcam #8580), anti-H3K27ac (Abcam #4729), anti-H3K27me3 (Abcam #6002), anti-RNA Pol II pSer2 (Abcam #5095), anti-RNA Pol II pSer5 (Abcam #5408), anti-IgG mouse (Millipore #12-371), and anti-IgG Rabbit (Millipore #12-370).The following antibodies were used for ChIP: dCTCF polyclonal antibodies were generated by New England Peptide by immunizing rabbits with a peptide corresponding to the first 20 aminoacids of dCTCFDesign and cloning of sgRNAs into pAc-sgRNA-Cas9. For design and evaluation of guide sequences for the CRISPR-Cas9 system we used the CRISPROR software (http://crispor.tefor.net/)54 and the Drosophila melanogaster dm6 version of the genome. The genomic regions used for guide design were chrX:3,134,000-3,134,869 and chrX:3,160,210-3,163,651 which correspond to the 5′ region of Notch and the intronic enhancer, respectively. Selected guide sequences had among the lowest off-target scores. Sequences for all guides used in this study are provided in Supplementary Table Notch do not target the sequence containing the major TSS sites annotated for Notch and the guides for the intronic enhancer are located at least 100 bp away from the nearest exon. Oligonucleotides for guides were cloned into pAc-sgRNA-Cas9 (Addgene #49330) as described55. Integrity of cloned guides was evaluated by Sanger Sequencing. Plasmids were expanded and purified using a Qiagen MiniPrep kit prior to transfection.Transfections. CRISPR-Cas9 mediated deletions were generated by transfecting two plasmids each with a specific guide targeting each side of the region of interest  was used for transfection using manufacturer guidelines (https://worldwide.promega.com/techserv/tools/FugeneHdTool/). A 4:2 FugeneHD:Plasmid ratio was used for all transfections. Transfected cells were placed at 25 °C and 72 h after transfection cells were re-suspended in fresh medium supplemented with 5 μg/mL of Puromycin (Sigma) and transferred into a well of a 12-well plate. After 3 days (6 days post-transfection), cells were re-suspended in fresh medium supplemented with 5 μg/mL of Puromycin (Sigma) and transferred into a well of a 6-well plate. After 3 days (9 days post-transfection) an aliquot of cells was used for DNA extraction by phenol-chloroform and PCR genotypification using specific primers spanning the desired deletions  and 100 μl of medium with cells were added per well of 96-well plate. Cells were incubated at 25 °C for at least 3 weeks. Single clones were expanded and an aliquot of cells was used for DNA extraction by phenol-chloroform and PCR genotypification using specific primers spanning the desired deletions  and 5′ overhangs were filled with Biotin-14-dATP (Invitrogen). Hi–C libraries were sequenced on a HiSeq 2500 platform 50 pb paired-end.Generation of mutant genomes in silico. Individual in silico mutant genomes were built for all CRISPR mutants to allow for precise mapping of reads without gaps. The sequence of breakpoints generated at deletion sites for each CRISPR mutant (as determined by Sanger Sequencing) were used to replace the wild-type sequence for the resulting mutant sequence in the chromosome X using R. A specific index for each mutant genome was build using Bowtie2 (ref. 57). Also, mutant genomes were used to generate mutant-specific MboI digestion files for subsequent analysis using HiC-Pro digest_genome.py.Data processing. Mapping, filtering, correction and generation of Hi–C matrices were done using HiC-Pro58. Briefly, read pairs were mapped independently to the wild-type or mutant genomes generated in silico using Bowtie2 with HiC-Pro default parameters. After filtering, valid-pairs were used to generate raw and ICE normalized matrices at 1 kb, 5 kb, 20 kb, and 100 kb bin resolution. To correct for differences in sequencing depth matrices were normalized by the smallest number of valid-read pairs .Compartment analysis. Compartments were identified using HOMER62runHiCpca.pl with parameters –res 10000 –window 50000. Peaks of enriched for the histone mark H3K4me3 in S2R+ cells (GSM2259985) were identified using MACS2 (ref. 63) and used to assign sign to the PCA1 results. PCA1 score bedgraph files were displayed using IGV60Differential analysis of Hi–C datasets. HiCcompare64 was used to identify differences in chromatin interactions with default parameters. Pairwise comparisons between wild-type and CRISPR mutants was performed using raw contact matrices at 5 and 20 kb resolution as input. Differential contacts in regions of interest were plotted using the Washington Epigenome Browser.Hi–C data processing of public datasets. Publicly available Hi–C datasets from Drosophila embryos were obtained from the European Nucleotide Archive (ENA) database under the following accession numbers: embryos nuclear cycle 12 (ERR1533189-199), embryos nuclear cycle 13 (ERR1533200-209), embryos nuclear cycle 14 (ERR1533226-236), embryos 3–4 h (ERR1533170-181), and embryos nuclear cycle 14 triptolide treatment (ERR1912894-899). Publicly available Hi–C datasets from Drosophila cell lines were obtained from the Gene Expression Omnibus (GEO) database under the following accession numbers: Kc167 cells  BG3 cells (GSM3475690 and GSM3475691). Datasets were processed as described using Bowtie 2 and HiC-Pro. Heatmaps from contact matrices were generated from HiCPlotter.65 and DESeq2 (ref. 66). Briefly, we estimated transcript-level abundance for each dataset using Salmon with a specific index for the Drosophila melanogaster transcriptome (dm6) and options –validateMappings and --gcBias. Quantification data from Salmon was then imported into R and we created an input table with gene level quantification data as input for DESeq2. We tested for differential expression (DE) between wild-type and the CRISPR enhancer mutant using DESeq2. We called DE at the gene level with a fold-change of at least 0.5 and a corrected p-value of <0.05. Abundance levels for genes located at the Nenh-mega-domain were Z-score transformed and used to create a heatmap with pheatmap using R. To create signal tracks from RNA-seq data, sequencing reads were mapped against the Drosophila melanogaster genome (dm6) using Bowtie2 with default parameters. SAM files were converted into BAM files using samtools view. BAM files were sorted (samtools sort) and indexed (samtools index). deepTools67 v3.3.0 was used to calculate Pearson Correlation and PCA for all datasets. BAM files from replicates were merged due to high correlation (Pearson Correlation > 0.9) and BAM files from merged experiments were used to create signal tracks with bamCoverage –normalizeUsing BPM. Signal tracks for wild-type and the CRISPR enhancer mutant data were visualized using IGV.Total RNA was extracted from wild-type and enhancer mutants in duplicate using TRIzol reagent (Thermofisher) following manufacturer’s instructions. Total RNA libraries for all samples were generated and sequenced by Novogene. Two independent libraries per condition were sequenced in a HiSeq 4000 platform paired-end 150 bp. RNA-seq data was analyzed using SalmonRP49 was used as a control. Data was analyzed by the ΔΔCt method68. Statistically significant differences in gene expression between the wild-type and the CRISPR mutants were computed using a T-test  and the Graphpad Prisma Software 7.0.Total RNA was extracted from wild-type and CRISPR mutants at least in duplicate using Trizol and following manufacturer’s instructions. RNA was used directly for qRT-PCR using the KAPA SYBR FAST One Step kit (KAPA Byosystems) in at least two technical replicates per sample using a StepOne Real-Time PCR System. A list of all primer sets used in this study are provided in Supplementary Table 69. For each IP, the following amount of antibody was used: 5 μg/IP for anti-CTCF, anti-IgG rabbit and anti-H3K27me3; 2 μg/IP for anti-H3K4me3, anti-H3K27ac, anti-RNA Pol II pSer2, pSer5 and anti-IgG mouse. Purified DNA was used for region-specific quantification by qPCR using SYBR Green in duplicate per ChIP. Mean values for all the regions analyzed in different conditions were expressed as fold-enrichment compared over IgG. Statistically significant differences between wild-type samples and CRISPR mutants were computed using a T-test  and the Graphpad Prisma Software 7.0. ChIP primers are listed in Supplementary Table Chromatin immunoprecipitation (ChIP) using wild-type cells and all CRISPR mutants was performed in duplicate as previously described70. In particular, nuclear protein was extracted using ~40 × 106Drosophila S2R+ cells. For super-shift assays 2 μg of dCTCF or IgG rabbit antibody were used. Oligo-sequences used for EMSA assays are listed in Supplementary Table Electrophoretic Mobilitiy Shift Assay (EMSA) was performed as previously described with minor modificationsDrosophila embryos were obtained from the Gene Expression Omnibus (GEO) database. Raw data for S2/S2R+ cells was obtained under the following accession numbers: CP190 (GSM1015404), SuHw (GSM1015406), Mod (GSM1015408), CTCF (GSM1015410), Ibf1 (GSM1133264), Ibf2 (GSM1133265), BEAF32 (GSM1278639), Pita (GSM1313420), ZIPIC (GSM1313421), RNA Pol II (GSM2259975), H3K4me1 (GSM2259983), H3K4me3 (GSM2259985), H3K27ac (GSM2259987), MSL2 (GSM2469507), H4K16ac (GSM2469508), M1BP (GSM2706055), GAF (GSM2860390), Input (GSM1015412). Raw data for Kc167 cells was obtained under the following accession numbers: CTCF (GSM762842), BEAF32 (GSM762845), Ibf1 (GSM2133766), Ibf2 (GSM2133767), Pita (GSM2133768), ZIPIC (GSM2133769), Su(Hw) (GSM762839), GAF (GSM2133762), CP190 (GSM762836), RNA Pol II (GSM1536014), and H3k27ac (GSM890121). ATAC-seq data from Drosophila cell lines was obtained from GEO under the following accession numbers: Kc167 (GSM3381113), S2 (GSM3381126).Publicly available ChIP-seq data for S2/S2R+ cells and Drosophila embryos was obtained under the following accession numbers: Zelda nc8 (GSM763060), Zelda nc13 (GSM763061), Zelda nc14 (GSM763062), TBP MBT , TBP post-MBT , TBP pre-MBT (GSM1022912), RNA Pol II-pSer5-nc12 (GSM1536376), RNA Pol II-pSer5-nc13 (GSM1536379), RNA Pol II-pSer5-nc14early (GSM1536382), RNA Pol II-pSer5-nc14mid (GSM1536384), RNA Pol II-pSer5-nc14late (GSM1536386), and RNA Pol II-pSer5-nc13ZeldaKD (GSM1536390). Raw data from Drosophila embryos was obtained from ENA under the following accession numbers: RNA Pol II-pan nc14 (ERR1912880 and ERR1912881), RNA Pol II-pSer5 nc14 (ERR1912882 and ERR1912883), RNA Pol II-pan nc14-triptolide (ERR1912890 and ERR1912891), and RNA Pol II-pSer5 nc14-triptolide (ERR1912892 and ERR1912893). ATAC-seq data from Drosophila embryos was obtained from GEO under the following accession numbers: ATAC-nc11-3 h(GSM2219678), ATAC-nc11-6 h(GSM2219681), ATAC-nc11-9 h(GSM2219684), ATAC-nc12-3 h(GSM2219687), ATAC- nc12-6 h(GSM2219690), ATAC- nc12-9 h(GSM2219693), ATAC- nc12-12 h(GSM2219696), ATAC- nc13-3 h(GSM2219700), ATAC- nc13-6 h(GSM2219703), ATAC- nc13-9 h(GSM2219706), ATAC- nc13-12 h(GSM2219709), ATAC- nc13-15 h(GSM2219712), and ATAC- nc13-18 h(GSM2219715).Raw data for Drosophilamelanogaster genome (dm6) using Bowtie2 with default parameters for single and paired reads. Mapped reads were filtered by map quality (-q 30) using samtools (sammtools view). Bam files were sorted (samtools sort) and indexed (samtools index). Duplicates were removed with Pickard. Bam files were imported to deeTools v3.3.1 to create signal tracks with bamCoverage –normalizeUsing CPM. Signal tracks for all data were visualized using IGV. Peak calling for Architectural Proteins in S2/S2R+ cells was performed using MACS2 (ref. 63) callpeak function with option –call-summits and default parameters.Reads were mapped against the http://www.modencode.org/ under the following accession numbers: H4K16ac-Kc167 (318), H4K16ac-S2 (319), H4K16ac-BG3 (316); H3K27ac -S2 (296), H3K27ac -BG3 (295); H3K27me3 -Kc167 (5136), H3K27me3-S2 (298), and H3K27me3-BG3 (297). Wig files were visualized using IGV.ChIP-chip signal files (.wig) were retrieved from 71 and the JASPAR72 Insect Database using as a threshold p-value < 0.0001.Identification of binding sites for insulator proteins was done using FIMOFurther information on research design is available in the Peer Review FileSupplementary InformationReporting Summary