CDH5, an endothelial marker, and up-regulation of COL1A2, a mesenchymal marker, in cultured vascular endothelial cells. On the one hand, BRG1 interacted with and was recruited by Sp1 to the SNAI2 (encoding SLUG) promoter to activate SNAI2 transcription in response to Ang II stimulation. Once activated, SLUG bound to the CDH5 promoter to repress CDH5 transcription. On the other hand, BRG1 interacted with and was recruited by SRF to the COL1A2 promoter to activate COL1A2 transcription. Mechanistically, BRG1 evicted histones from the target promoters to facilitate the bindings of Sp1 and SRF. Finally, endothelial conditional BRG1 knockout mice (CKO) exhibited a reduction in cardiac fibrosis, compared to the wild type (WT) littermates, in response to chronic Ang II infusion. In conclusion, our data demonstrate that BRG1 is a key transcriptional coordinator programming Ang II-induced EndMT to contribute to cardiac fibrosis.Endothelial–mesenchymal transition (EndMT) is considered one of the processes underlying tissue fibrosis by contributing to the pool of myofibroblasts. In the present study, we investigated the epigenetic mechanism whereby angiotensin II (Ang II) regulates EndMT to promote cardiac fibrosis focusing on the role of chromatin remodeling protein BRG1. BRG1 knockdown or inhibition attenuated Ang II-induced EndMT, as evidenced by down-regulation of  Uncontrolled, excessive fibrogenesis or the failure to terminate fibrogenesis properly, however, leads to impairment of cardiac architecture, dampens heart function, and is a hallmark event in chronic heart failure2. It is generally agreed that myofibroblast cells, a unique cell type with dual abilities of contraction (hence the prefix “myo”) and laying down extracellular matrix proteins (a fibroblast-like behavior), are the primary mediator of cardiac fibrosis3. Gene signature wise, myofibroblasts are typically characterized by high levels of smooth muscle actin alpha (α-SMA), collagen type I and III, and periostin4. Because myofibroblasts are absent from the healthy myocardium, origins from which myofibroblasts arise during cardiac injury and fibrosis have been actively pursued and hotly debated with considerable amount of controversy. It can be argued that multiple lineages of cell types, including resident fibroblast cells, pericytes, myeloid cells, fibrocytes, and endothelial cells, contribute to the pool of mature myofibroblasts during cardiac fibrosis5. For instance, Zeisberg et al. exploiting a Tie2-Cre driven lineage tracing system, demonstrated that a large fraction of α-SMA-positive myofibroblasts detected in the scarring region of the murine infarct following acute myocardial ischemia (AMI) might originate from endothelial cells, likely through a process known as endothelial–mesenchymal transition (EndMT)6. Similarly, Wang et al. have reported that EndMT contributes to activation of myofibroblasts and cardiac fibrosis in mice exposed to chronic angiotensin II (Ang II) infusion, which is mediated by transglutaminase 27. It has also been suggested that the sequence-specific transcription factor Ets-1 might be responsible, at least in part, for Ang II-induced EndMT and cardiac fibrosis in mice8. In cultured endothelial cells, treatment with Ang II stimulates the down-regulation of endothelial marker genes  and up-regulation of mesenchymal marker genes  although the epigenetic mechanism is not completely understood10. EndMT and the related process epithelial–mesenchymal transition (EMT) are programmed by a host of transcription factors, among which the E-box-binding family of proteins including SNAIL, SLUG, and ZEB have been well studied11.Cardiac fibrosis, like any other organ-specific fibrogenic response, can be considered a host defense mechanism that safeguards the physiological integrity of the heart to prevent myocardial rupture and circulatory failure in the event of cardiac injury12. Recent investigations have revealed key roles for BRG1 in the regulation of cardiovascular diseases. Hang et al. have reported that postnatal deletion of BRG1 in the myocardium attenuates the development of pathological cardiac hypertrophy in response to pressure overload in mice by skewing the expression of myosin heavy chain isoforms13. We have recently found that endothelial-specific BRG1 deficiency attenuates atherosclerosis14, abdominal aortic aneurysm15, and cardiac ischemia-reperfusion injury17 in mice. Here we report that BRG1 mediates Ang II-induced EndMT in cultured cells by directly activating COL1A2 transcription and indirectly repressing CDH5 transcription. More importantly, endothelial conditional knockout of BRG1 in mice attenuates EndMT and cardiac fibrosis in mice subjected to chronic Ang II infusion.In mammalian cells, gene transcription is profoundly influenced by the epigenetic machinery, which includes histone/DNA modifying enzymes, non-coding regulatory RNAs, and chromatin remodeling proteins. Brahma-related gene 1 (BRG1) is the catalytic core of the mammalian SWI/SNF chromatin remodeling complex. BRG1 regulates gene transcription by utilizing its ATPase activity to mobilize nucleosomes and alter chromatin structure. Germline deletion of BRG1 results in developmental arrest in mice suggesting a role for BRG1 in embryogenesis18. Primary murine cardiac microvascular endothelial cells were isolated as previously described19. Angiotensin II was purchased from Sigma. SNAI2/SLUG promoter-luciferase constructs20, COL1A2 promoter-luciferase constructs21, BRG1 expression constructs22, SLUG expression constructs23, Sp1 expression constructs24, and SRF expression constructs25 have been previously described. PFI-3 was purchased from Selleck. Transient transfections were performed with Lipofectamine 2000. Luciferase activities were assayed 24–48 h after transfection using a luciferase reporter assay system (Promega) as previously described26.Immortalized human endothelial cells  and HEK293 cells were maintained in DMEM supplemented with 10% fetal bovine serum . Human primary microvascular endothelial cells (HMVEC) were purchased from Lonza and maintained in EGM-2 media with supplements supplied by the vendor; three different batches of primary cells were used in this study as previously describedSmarca4-flox mice27 and the Cdh5-Cre mice28 were crossed to make the endothelial conditional BRG1 knockout mice (CKO). The mice were housed with a 12:12 h light–dark cycle at constant room temperature, fed standard rodent diet, and allowed at least one week of acclimation before the start of the experiments. Male, 8-week-old mice were induced to develop cardiac fibrosis by Angiotensin II (1 μg/kg/min) infusion for 4 consecutive weeks using subcutaneously implanted minipumps (Alzet 2004). One day prior to the sacrifice, the mice were anesthetized using isoflurane and heart functions were evaluated by echocardiography . The mice were sacrificed.All animal experiments were reviewed and approved by the Ethics Committee on Humane Treatment of Laboratory Animals of Nanjing Medical University and were performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. The 30. Nuclear proteins were extracted using the NE-PER Kit (Pierce) following manufacturer’s recommendation. Prior to immunoprecipitation, the lysates were treated with DNase I  at 37 °C for 30 min. Specific antibodies or pre-immune IgGs (PII) were added to and incubated with cell lysates overnight before being absorbed by Protein A/G-plus Agarose beads (Santa Cruz). Precipitated immune complex was released by boiling with 1X SDS electrophoresis sample buffer. Alternatively, FLAG-conjugated beads  were added to and incubated with lysates overnight. Precipitated immune complex was eluted with 3X FLAG peptide (Sigma). Western blot analyses were performed with anti-BRG1 , anti-collagen type I , anti-α-SMA , anti-SLUG , anti-VE-Cadherin , anti-β-actin , anti-MYC , anti-FLAG , anti-GFP , anti-Sp1 , and anti-SRF  antibodies. For densitometrical quantification, densities of target proteins were normalized to those of β-actin. Data are expressed as relative protein levels compared to the control group which is arbitrarily set as 1.Whole cell lysates were obtained by re-suspending cell pellets in RIPA buffer  with freshly added protease inhibitor (Roche) as previously described32. Real-time PCR reactions were performed on an ABI Prism 7500 system with the following primers: human CDH5, 5′-TCACCTTCTGCGAGGATATGG-3′ and 5′-GAGTTGAGCACCGACACATC-3′; human PECAM1, 5′-CTGCTGACCCTTCTGCTCTGTTC-3′ and 5′-GGCAGGCTCTTCATGTCAACACT-3′; human VWF, 5′-TTGACGGGGAGGTGAATGTG-3′ and 5′-ATGTCTGCTTCAGGACCACG-3′; human COL1A1, 5′-TCTGCGACAACGGCAAGGTG-3′ and 5′-GACGCCGGTGGTTTCTTGGT-3′; human COL1A2, 5′-GTGGCAGTGATGGAAGTGTG-3′ and 5′-AGGACCAGCGTTACCAACAG-3′; human VIM, 5′-GAGAACTTTGCCGTTGAAGC-3′ and 5′-TCCAGCAGCTTCCTGTAGGT-3′; human BRG1, 5′-TCATGTTGGCGAGCTATTTCC-3′ and 5′-GGTTCCGAAGTCTCAACGATG-3′; human SNAI2, 5′-CACCATGCCGCGCTCCTTCCTGGTC-3′ and 5′-TCAGTGTACACAGCAGCCAGA-3′; human SNAIL, 5′-GAAAGGCCTTCAACTGCAAA-3′ and 5′-TGACATCTGAGTGGGTCTGG-3′; human ZEB1, 5′-GCACCTGAAGAGGACCAGAG-3′ and 5′-TGCATCTGGTGTTCCATTTT-3′; mouse Cdh5, 5′-TCAACGCATCTGTGCCAGAGAT-3′ and 5′-CACGATTTGGTACAAGACAGTG-3′; mouse Pecam1, 5′-GACTCACGCTGGTGCTCTATGC-3′ and 5′-TCAGTTGCTGCCCATTCTCA-3′; mouse Col1a2, 5′-GCCACCATTGATAGTCTCTCC-3′ and 5′-CACCCCAGCGAAGAACTCATA-3′; mouse Vim, 5′-CGGAAAGTGGAATCCTTGCA-3′ and 5′-CACATCGATCTGGACATGCTGT-3′; mouse Snai2, 5′-CGAACTGGACACACACACAG-3′ and 5′-AAAGGAGAGTGGAGTGGAGC-3′. Ct values of target genes were normalized to the Ct values of housekeekping control gene  using the ΔΔCt method and expressed as relative mRNA expression levels compared to the control group which is arbitrarily set as 1.RNA was extracted with the RNeasy RNA isolation kit (Qiagen). Reverse transcriptase reactions were performed using a SuperScript First-strand Synthesis System (Invitrogen) as previously described44. In brief, chromatin in control and treated cells were cross-linked with 1% formaldehyde. Cells were incubated in lysis buffer  supplemented with protease inhibitor tablet and PMSF. DNA was fragmented into ~200 bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-BRG1 , anti-Sp1 , anti-SRF , , anti-SLUG , anti-ZEB1 , anti-SNAIL , anti-acetyl H3 , anti-acetyl H4 , anti-histone H3 , or pre-immune IgG. For re-ChIP, immune complexes were eluted with the elution buffer , diluted with the re-ChIP buffer , and subject to immunoprecipitation with a second antibody of interest.ChIP assays were performed essentially as described beforeHistologic analyses were performed essentially as described before. Briefly, paraffin-embedded sections were stained with picrosirius red (Sigma-Aldrich) according to standard procedures. Pictures were taken using an Olympus IX-70 microscope . Quantifications were performed with Image J by two independent assessors. For each animal, at least three slides with ~5 fields for each slide were included for quantification.p < 0.05 were considered statistically significant.One-way ANOVA with post-hoc Scheff´e analyses were performed by SPSS software . Unless otherwise specified, values of CDH5, PECAM1, and VWF) and simultaneous up-regulation of mesenchymal marker genes  in immortalized vascular endothelial cells (EAhy926) as well as primary human microvascular endothelial cells (HMVECs) indicative of an EnMT-like process. BRG1 depletion, however, partially reversed the Ang II-induced EndMT by blocking CDH5 down-regulation and COL1A2 up-regulation. Next, the endothelial cells were treated with Ang II in the presence or absence of a small-molecule BRG1 inhibitor (PFI-3). PFI-3 treatment antagonized Ang II induced down-regulation of CDH5 expression and up-regulation of COL1A2 expression in a dose-dependent manner . As shown in Fig. ner Fig. . These dCDH5 promoter  normalized CDH5 expression in endothelial cells  for the sequence-specific transcription factor SRF between −500 and −100 of the COL1A2 promoter. We therefore investigated a potential interplay between SRF and BRG1 in mediating Ang II-induced COL1A2 transcription in endothelial cells. Co-immunoprecipitation assay confirmed that BRG1 and SRF interacted with each other in HEK293 cells , which functions as a repressor of endothelial gene (CDH5) transcription. On the other hand, BRG1 interacts with SRF to activate the transcription of collagen type I (COL1A2), a mesenchymal marker. More important, endothelial-specific deletion of BRG1 significantly ameliorated Ang II-induced EndMT and cardiac fibrosis in mice. It should be noted that there are a few caveats with regard to the proposed model. First, it remains unknown why BRG1 selectively activates the transcription of SLUG (SNAI2), but not SNAIL or ZEB1, to promote the loss of endothelial markers because SNAIL48 and ZEB149 have been shown to mediate EndMT in different settings. This preference could be attributed to a combination of specific cell types, stimuli, and sequence-specific transcription factors that recruit BRG1. For instance, ZEB1, but not SNAIL or SLUG, is activated by Wnt signaling to promote EMT in the kidneys50. SNAIL, but not ZEB1 or SLUG, is found to be specifically up-regulated in poorly differentiated and mesenchymal-like breast cancer cells51 and colon cancer cells52. The redundancies among different EndMT/EMT factors are an intriguing issue53 and our data suggest that BRG1 might be a contributing factor but this hypothesis deserves further attention. Second, BRG1 may contribute to Ang II-induced EndMT via alternative mechanisms. Accumulation of reactive oxygen species (ROS) is considered a key driving force of EndMT54. It has been found that BRG1 modulates intracellular ROS levels by transcriptionally activating the genes involved in ROS synthesis27. In fact, we have previously shown that activation of NADPH oxidase 4 (NOX4) gene transcription in endothelial cells by BRG1 may be responsible for TGF-β-induced EndMT and liver fibrosis40. The latter observation is not at variance but rather consistent with the present data because Ang II stimulation can trigger ROS accumulation and simultaneous up-regulation of collagen type I expression56. Third, BRG1-mediated EndMT may not fully account for Ang II-induced cardiac fibrosis in vivo. Chronic inflammation is one of the major culprits for cardiac fibrosis58. We have recently shown that BRG1 deficiency in endothelial cells attenuates inflammation in the vessel wall59 and in the kidneys60, which can be partly explained by the fact that BRG1 activates the transcription of adhesion molecules  and chemokines  to promote macrophage trafficking. Alternatively, BRG1 may regulate the expression of endothelial-derived humoral factors to influence cardiac fibrosis. For instance, BRG1 has been shown to activate the transcription of endothelin (ET-1), a pro-fibrogenic factor61, and repress the transcription of eNOS (NOS3), an anti-fibrogenic factor62, in endothelial cells. Therefore, attenuation of cardiac fibrosis in the CKO mice may be construed as a consequence of skewed balance between endothelial-derived pro-fibrogenic and anti-fibrogenic factors. It should be pointed out that BRG1 may be able to directly repress CDH5 transcription to promote EMT/EndMT-like processes. Sánchez-Tilló E et al. have reported that BRG1 can form a complex with ZEB1 to directly bind to the CDH5 promoter to repress its transcription and promote β-catenin nuclear trans-location in several different cancer cells63. The discrepancies in our observations and those by Sánchez-Tilló E et al. likely reflect the complex nature regarding BRG1-mediated transcription regulation in different cells and different circumstances. A ChIP-seq analysis with anti-BRG1 antibodies aiming to examine the dynamic association of BRG1 with target promoters during EndMT will likely provide additional mechanistic insight on the precise role BRG1 plays in regulating this process. Finally, Ang II not only promotes cardiac remodeling and fibrosis but vascular remodeling and hypertension. Ang II-induced endothelial dysfunction including altered endothelial gene expression is noted in model animals and in humans. Therefore, it would be of great interest to determine whether BRG1 deficiency normalizes blood pressure in the model as described in Fig. Endothelial plasticity is essential to embryonic development. Aberrant and inadvertent activation of EndMT, however, is associated with the pathogenesis of a myriad of human diseases including cardiac fibrosisein Fig. . BRG1 onSNAI2 and COL1A2 by modulating histone status. Eviction of histones from the chromatin, as observed here, during nucleosome mobilization is one of the better understood functionalities of BRG1. For instance, regulation of eNOS transcription in endothelial cells exposed to hypoxia involves BRG1-mediated histone eviction from the eNOS promoter64. We have previously shown that eviction of histones from the CRP promoter by BRG1 underlies its trans-activation by free fatty acids in hepatocytes43. Of note, recent studies have portrayed a scenario wherein BRG1 relies on its interactions with histone-modifying enzymes to regulate the transcription of target genes. The observation that the status of histone H3/H4 acetylation is associated with SNAI2/COL1A2 transcription compels a natural question as to whether specific histone acetyltransferases may participate in EndMT and cardiac fibrosis through BRG1. A series of independent studies have shown that E1A-associated protein 300 (p300), a well-documented binding partner for BRG1, regulates EndMT by potentiating the TGF-β-signaling pathway65. Although not investigated in the present study, other histone-modifying enzymes that play a role in EndMT, including histone deacetylase 3 (HDAC3)67, histone H3K4 methyltransferase WDR568, histone H3K27 methyltransferase EZH269, and histone demethylase JMJD2B40, can all interact with BRG1 in various settings. A comprehensive mapping of BRG1-dependent histone modifications on a genomewide scale will likely provide novel insights on the epigenetic regulation of EndMT.We show here that BRG1 contributes to Ang II-induced trans-activation of SNAI2 promoter and COL1A2 promoter, respectively, raising the possibility that targeting Sp1 and/or SRF in endothelial cells may phenocopy the BRG1 CKO mice in the model of Ang II-induced cardiac fibrosis. Both Sp1 and SRF have been demonstrated to contribute to myofibroblast maturation in the context of cardiac fibrosis although cell-specific roles remain undetermined71. We have previously shown that BRG1 mediates EndMT induced by TGF-β, ROS, and hypoxia72. In light of our new findings as summarized here, targeting the endothelial BRG1–Sp1–SRF cluster may be considered as a reasonable approach in the intervention of cardiac fibrosis.BRG1 relies on sequence-specific transcription factors to participate in locus-specific transcriptional regulation and, by extension, pathogenesis of human diseases. Our data indicate that BRG1 is recruited by Sp1 and SRF to the Supplementary Figure legendsnSupplementary Figure S1Supplementary Figure S2


SMO, PTCH1, GLI1, GLI2, and GLI3, were determined in 33 cancer cell lines and normal prostate cells and tissues. We performed a computational analysis of the upstream region of the SMO gene to identify the regulatory elements. (3) Results: Three potential CpG islands and several putative SMO promoter elements were identified. Luciferase reporter assays mapped key SMO promoter elements, and functional binding sites for SP1, AP1, CREB, and AP-2α transcription factors in the core SMO promoter region were confirmed. A hypermethylated SMO promoter was identified in several cancer cell lines suggesting an important role for epigenetic silencing of SMO expression in certain cancer cells. (4) Discussion: These results have important implications for our understanding of regulatory mechanisms controlling HH pathway activity and the molecular basis of SMO gene function. Moreover, this study may prove valuable for future research aimed at producing therapeutic downregulation of SMO expression in cancer cells.(1) Background: The hedgehog (HH) signaling pathway is a key regulator of embryonic patterning, tissue regeneration, stem cell renewal, and cancer growth. The smoothened (SMO) protein regulates the HH signaling pathway and has demonstrated oncogenic activity. (2) Methods: To clarify the role of the HH signaling pathway in tumorigenesis, the expression profile of key HH signaling molecules, including  The hedgehog (HH) pathway is one of the key signaling pathways regulating embryonic patterning, tissue regeneration, stem cell renewal, and cancer growth ,2,3,4. CThe switch between active and inactive states of the HH pathway involves rapid translocation of SMO. The SMO protein is the key positive regulator of the HH pathway, and GLI family proteins play a critical role in the regulation of HH signaling pathway activity. Despite a strong link between SMO expression, HH pathway activity, and cancer development, the basis for SMO gene regulation has not been well characterized. Therefore, an investigation of the mechanisms controlling the expression of SMO and additional HH pathway genes may provide valuable insight into HH signaling alterations associated with cancer development. SMO also is the major target for pharmaceutical agents that modulate HH pathway activity ,18,19, sDNA methylation of HH pathway genes is a potential regulatory mechanism in the progression of cancers. Several epigenetic factors that act on the HH signaling pathways have been associated with cancer initiation and progression ,24. It wIn the current study, we have developed a qRT-PCR method to accurately determine the expression levels of SMO, PTCH1, GLI1, GLI2, and GLI3 in a panel of cancer cell lines. Different SMO expression patterns in the cancer cell lines led us to characterize SMO gene regulatory elements. The SMO 5′-flanking region and Exon 1 were analyzed in silico, revealing that the region surrounding the SMO transcriptional start site (TSS) has an extremely high GC content (70%+) that prevents its PCR amplification by traditional methods. We used a touchdown PCR method to amplify SMO promoter fragments and determined their promoter activity using a dual luciferase assay. EMSA analysis identified binding sites for the transcription factors, SP1, AP1, CREB, and AP-2α, which likely play an important role in SMO transcriptional activity in cancer cells. To gain insight into the epigenetic regulation of SMO, bisulfite sequencing PCR (BSP) and methylation-specific PCR (MSP) were carried out to determine the methylation status of the potential SMO promoter region. The relationship between the methylation status and SMO mRNA expression was analyzed.To gain a greater understanding of the transcriptional regulation of the HH pathway components, a careful analysis of mRNA levels for key HH signaling genes was conducted. Quantification of mRNA expression levels using a real-time PCR method is increasingly used to determine the activity of HH signaling genes. However, most studies use a relative RT-PCR method, which is less precise and does not provide meaningful comparisons of gene expression between different cell lines. Therefore, accurate quantitation of HH pathway mRNA expression is necessary. In this study, we developed and validated a standard curve based on a Taqman qRT-PCR method to measure key HH signaling genes, including SMO, PTCH1, GLI1, GLI2, and GLI3.(−1/slope)] −1 [2R is greater than 0.99.The results produced a broad linear dynamic range of detection of at least six logs and a small quantitative variation produced by triplicate analysis. The slope of the curve was used to determine the reaction efficiency. Efficiency = [10 ope)] −1 . The effThe expression levels of HH signaling components were determined in 33 cancer cell lines using the quantitative Taqman RT-PCR method. Expression levels of the HH pathway genes, the HH signaling receptors PTCH and SMO, and the target transcription factors GLI2 and GLI3, varied significantly among the cancer cell lines . The SMOp = 0.040), and between PTCH1 and GLI3 . No correlation was found between the expression levels of SMO/PTCH or SMO/GLI1  were all hypermethylated in the amplification region . Five additional CNS were identified, but all had a lower p value that was greater than 0.005. A 924 bp region consisting of the full length 5′-UTR and exon 1 regions, along with 293 bp of upstream and 20 bp of intron 1 sequence, is shown in SMO genes, and 95% between human and rhesus.The global genomic sequence comparison showed significant highly conserved regions among the three genes immediately upstream of the transcriptional start site  returned 336 potential TFBS, distributed over the entire sequence. Furthermore, we performed an analysis with the PromoterInspector program and found a 1028 bp potential promoter region within this sequence, located from position −508 to +520 bp. By using the ModelInspector program, 25 models were identified, including SMAD-MIT, SMAD-AP1, YY1-SMAD, ETF-AP1, SP1-ETS. SP1F-NF1, IKRS-AP2, EGR-SP1, SP1-KLFS, GATA-SP1, CAAT-CAAT, NFKB-SP1, and SP1-CAAT.SMO gene TSS using the MethPrimer program , to produce five constructs , and the luciferase activities of these constructs were measured.The potential promoter region upstream of the nstructs , based onstructs . PlasmidThe highest promoter activity was observed in MCF7 cells, and moderate activity was found in PC3 and BT549 cells. In MCF7 cells, increased promoter activity was detected upon removal of 459 bp of 5′ sequence up to position −500 bp (relative to the transcription initiation site), indicating the presence of negative regulatory element(s) in the region from −959 bp to −500 bp in MCF7 cells. When truncated to −470 bp, the promoter activity returned to the full-length promoter activity, and deletion of additional sequence to either −400 or −293 further reduced promoter activity, suggesting the presence of positive regulatory element (s) in the region −500 bp to −293 bp. In PC3 and BT549 cells, the 5′ truncations had little effect and maximal activity was observed with the PGL3-SMO-400/+25 construct. The PGL3-SMO-500/+25 construct exhibited the highest promoter activity in MCF7 cells, therefore, this reporter vector was used for subsequent 3′ deletion analysis B.SMO promoter.To further identify the 3′ boundary of the core promoter, three plasmids were generated sharing the same 5′ boundary at position −500, and variable 3′ ends from +50 to −15. In contrast to the results from the 5′ deletion analysis, luciferase activity with the 3′ deletions showed similar effects in all three cell lines. The promoter activities were comparable between the PGL3-SMO-500/+25 and PGL3-SMO-500/+15 in the three cell lines, while the addition of 25 bp of 3′ sequence to +50 resulted in a decreased activity B, indicaSMO promoter, and both negative and positive regulatory regions can affect the promoter activity of the SMO gene depending on cell context.Taken together, these results demonstrated that the region between −500 and +15 bp is important for the transcriptional activity of the SMO promoter sequences in nuclear extracts of PC3, BT549 and MCF7 cells.  family of transcription factors, leading to hyperproliferation of epithelial cells .SMO promoter is the abundance of CpG dinucleotides and multiple SP1 binding sites (7 × SP1 sites) close to the TSS. SP1 has been widely described as a general transcription factor involved in the transcription of gene promoters that lack a TATA box. CpG-rich promoters bound by DNA sequence-specific transcription factors including SP1 have the highest expression level, and deletion of SP1 binding sites results in significantly decreased promoter activity [CpG islands represent a common epigenetic element that regulates transcription at many promoters through methylation-induced silencing. In this study, the most striking feature revealed by in silico analysis of the activity ,34.SMO promoter region, and the strongest promoter activity was identified in the 5′-UTR region around −500/+25 that contains a high GC content, is CpG rich and lacks a canonical TATA box. Evaluation of the predicted SP1 consensus sites revealed that the SMO-472P (−472 to −444 bp) and SMO-422P (−422 to −398 bp) regions are functional in binding to the SP1 transcriptional factor as shown in EMSA/supershift experiments. In addition, inducible TF factors binding to AP1 and AP2 sites were also identified in the promoter region and may play a role in modulating SMO expression.We have isolated and cloned DNA fragments containing the predicted SMO transcription was characterized in 33 cancer cell lines. We determined the mRNA expression of the major HH pathway genes, SMO, PTCH1, GLI1, GLI2, and GLI3 and found the highest level of mRNA expression was observed in the SMO gene, but their corresponding proteins were very rare or produced weak signals in normal breast epithelium [SMO, PTCH1 and GLI1 in the NCI60 cell line panel  showed a significant positive correlation  between SMO and GLI2, supporting the conclusion that SMO gene regulation is important in HH pathway regulation (The epigenetic regulation of ithelium . We havene panel . SMO exp tissues . Expressgulation .SMO expression in seven cell lines derived from breast (5 of 9), stomach (1 of 1), and colon (1 of 1) cancer tissues was correlated with a high level of gene methylation. The level of SMO mRNA was negatively correlated with the methylation status of the SMO promoter , whereas eight prostate cancer cell lines had no detectable methylation in the SMO gene. Whether the degree of SMO methylation correlates with the tissue specificity remains to be explored. By using a ChIP-sequencing approach, specific histone mark Histone 3 Lysine 4 Acetylation (H3K4Ac) peaks have been confirmed in the proximal promoter of the SMO and GLI1 genes, demonstrating the expression of these genes was regulated by the removal of H3K4Ac mediated by Histone Deacetylase 3 (HDAC3) [SMO promoter region and their biological impact using ChIP-seq or site-directed mutagenesis in future studies.DNA methylation is a major epigenetic regulatory mechanism of gene expression and is involved in the progression of cancer . The abspromoter . In thispression . Moreove (HDAC3) . It willSMO and GLI2 in our study. This is consistent with previous reports that identified overexpression of SMO and GLI2 in progesterone receptor (PR) negative breast cancers and gastric cancers [PTCH1 is an established tumor suppressor gene and developmental regulator. Although the role of GLI3 as a negative regulator of HH signaling is well established in the context of normal development, its role in cancer has largely been ignored [GLI3 expression is positively correlated with PTCH1 levels. This result is indirectly supported by the finding of increased expression of PTCH and GLI3 in cancerous tissues and correlated with the increased proliferating index of Ki-67 in breast cancer. Moreover, PTCH1 haploinsufficiency is associated with distinct autosomal dominant syndromes [The full mechanistic details of HH signal transduction are still under investigation. Abnormal HH activation has been implicated in tumorigenesis in a wide variety of tumors, and SMO and GLI play a critical role in this pathway. GLI2 is suggested to function primarily as a transcriptional activator, and GLI3 as a repressor . A signi cancers ,40. Over cancers ,41. PTCH ignored ,43,44. Iyndromes . Previouyndromes ,47. ThesSMO gene were performed with the ECR Browser, and the publicly available web-based tool mVista [http://www.cbrc.jb/research/db/TFSEARCH.html).Identification and sequence analysis of evolutionarily conserved regions (ECRs) of the l mVista  using thAll cell lines were purchased from American Type Culture Collection  and grown according to the ATCC instructions. Total RNA from human prostate tissues was obtained from Clontech . 5-Azacytidine  was freshly prepared in PBS before use. A vehicle control consisting of culture medium alone was included in the analysis. MCF7 and PC3 cells were pre-cultured for 24 h, then treated with 1 µM 5-Aza for 72 h. Cells were collected by centrifugation, then genomic DNA and RNA were extracted and analyzed.SMO, PTCH1, PTCH2, GLI1, GLI2, and GLI3 from full length cDNA . The copy number of plasmid cDNA was calculated by optical density according to the exact molar mass derived from the sequences. Serial dilutions were made to obtain 10e1 to 10e7 copies. The slope and intercept were calculated for each run using a linear regression analysis of the log copy number versus threshold cycle (Ct) value for both target genes and 18S rRNA standard curves [Total cellular RNA was isolated and purified by RNeasy columns  according to the manufacturer’s instructions with on-column and in-solution DNaseI digestion. RNA quality and quantity were determined using Agilent RNA 6000 Nano Chip . cDNA synthesis was carried out using Random Hexamer primer, Taqman Reverse Transcription Reagents kit . Taqman real time RT-PCR primers and probes for target genes were designed by using the Primer Express software. SMO Fwd: 5′-GAGACTCGGACTCCCAG-3′; Rev: 5′-GTATACGGCACACAGCAG-3′ and probe: 5′(FAM)-TCGGGCCTCCGGAAT-(MGB)3′. PTCH1 Fwd: 5′-GCATAGGAGTGGAGTTCA-3′; 5′-CCCTGCGGTTCTTGTC-3′ and probe 5′(FAM)-TTGGCCTTTCT-(MGB)3′. GLI1 Fwd: 5′-GTCTCAAACTGCCCAGC-3′; Rev: 5′-CGTTCAAGAGAGACTGGG-3′ and Probe: 5′(FAM)-TCCCACACCGGTACCA-(MGB)3′. PTCH2, (Assay ID = Hs01085642_ml), GLI2 (Assay ID = Hs00257977_ml) and GLI3 (Assay ID = Hs00609233_ml). Taqman real time RT-PCR was used to determine the expression profile, with a 18S rRNA plasmid as the standard reference gene using primers Fwd: 5′-CCGAAGCGTTTACTTTGAAAAAA-3′; Rev: 5′-TTCCATTATTCCTAGCTGCGGTAT-3′ and probe 5′(VIC)-AGTGTTCAAAGCAGGCC-(MGB)3′. The PCR reactions were performed in 20 µL final volume containing 5 ng of cDNA, 1 × Master Mix , 900 nM of each primer and 200 nM of each probe, respectively. The thermal cycling conditions are 40 cycles of PCR amplification  . All assays were performed in triplicate, and each plate contained the same standard and positive quality control sample. For each unknown sample, the copy number of each gene is calculated using linear regression analysis from their respective standard curves. The relative mRNA expression level of target genes was normalized by the following formula: (copy number of target gene)/(copy number of 18S rRNA) × 10e6. The standard curves were generated using a dilution series of plasmids containing d curves .http://www.urogene.org/methprimer/). The primer sequences used for methylation analysis are summarized in ® PCR SuperMix . Thermal cycling conditions were 95 °C for 2 min, followed by 35 cycles of 95 °C for 15 s, 56 °C for 30 s, 72 °C for 10 s, and a final extension at 72 °C for 5 min. For sequence analysis, the PCR products were subcloned into a pCR2.1 vector using a TOPO TA Cloning kit  according to the manufacturer’s instructions. At least 12 clones were sequenced in an ABI 3730 DNA Sequencer  for each cell line tested. Methylation analysis was performed using BiQ Analyzer software [DNA was extracted using the QIAamp DNA Mini Kit . Bisulfite modification of 1µg of genomic DNA was performed with the EpiTect Bisulfite Kit  as described by the manufacturer. Primers for BSP and identification of predicted CpG islands in the SMO promoter region were carried out with the assistance of Methyl Primer Express Software v1.0 (Applied Biosystems) and MethPrimer . Water blanks and PCR mixtures without template were also used as experimental controls in each assay. The primers were listed in SMO gene was examined using actin as the internal control for DNA quantification. The beta actin gene contains no CpG dinucleotides and is not affected by DNA methylation status or bisulfite treatment. The primers for quantitative MSP are the same as the normal MSP shown in The bisulfite–converted genomic DNA was amplified using fluorescence-based real-time MSP using FastStart SYBR Green Master Kit (Roche). Methylation of the ® PCR SuperMix Kit . A modified touchdown PCR was performed with the following cycling conditions: The templates were denatured at 94 °C for 3 min, and then 20 cycles composed of 20 s at 95 °C, 30 s annealing with a stepwise reduction of annealing temperature from 68 °C to 58 °C decreasing by 0.5 °C every cycle, and an elongation step of 4 min at 72 °C. Twenty additional cycles were then performed at 94 °C 20 s, 58 °C for 40 s, and 72 °C for 40 s. The standard PCR program was 35 cycles at 94 °C 20 s, 58 °C for 40 s, and 72 °C for 40 s. All PCR products were analyzed by electrophoresis on a 1.0% agarose gel stained with ethidium bromide.PCR was carried out in a volume of 50 µL containing 100 ng of genomic DNA, 20 pmol of each primer and 3% Dimethyl Sulfoxide  using PlatinumSMO promoter constructs, including five deletions from the 5′ side and three deletions on the 3′ side, were created by PCR using the primers shown in A series of truncated 5 cells per well in a 24-well plate the day before transfection and incubated overnight at 37 °C in 5% CO2. For each well, 5 µL of HilyMax transfection reagent  was diluted in 30 µL of growth medium without serum and incubated at room temperature for 5 min. The DNA mixture containing 2 µg of the specific reporter construct plus 20 ng of Renilla luciferase pRL-SV40 control DNA was then added to each well, and incubated at room temperature for 20 min. Luciferase activity was assayed at 48 h using the Dual-Luciferase Reporter Assay System  according to the manufacturer’s instructions. Measurement of the firefly luciferase activity of the SMO promoter constructs was normalized relative to the activity of the Renilla luciferase produced by the pRLSV40 control vector and each construct was tested in triplicate in at least three independent experiments.Two breast cancer cell lines, MCF7 and BT549, and prostate cancer cell line PC3, were used for the analysis of promoter constructs. The cells were plated at 1 × 10Nuclear extracts were prepared from PC3, BT549 and MCF7 cells using the CellLytic NuCLEAR extraction kit . Six double-stranded DNA oligonucleotide probes corresponding to −500 to −357 bp of SMO promoter were synthesized (SMO, PTCH1, GLI1, GLI2, and GLI3 were assessed by Pearson’s correlation coefficient using GraphPad Prism 7 software. All p values reported were two-tailed, with significance defined as p < 0.05.The correlation of mRNA expression levels of SMO gene. The SMO promoter has been characterized and its major regulatory elements, including multiple CpG islands and SP1 binding sites, were identified. A correlation between SMO/GLI2 and PTCH1/GLI3 expression was observed. Moreover, SMO expression is correlated with the degree of CpG island methylation. Our results reveal a central role for epigenetic regulation of SMO gene transcription that may be exploited for the development of new therapeutic strategies to treat hedgehog-driven tumors.In conclusion, our data provide strong experimental and computational evidence for genetic and epigenetic regulatory mechanisms of the


Long non-coding RNAs (lncRNAs) have important regulatory functions across eukarya. It is now clear that many of these functions are related to gene expression regulation through their capacity to recruit epigenetic modifiers and establish chromatin interactions. Several lncRNAs have been recently shown to participate in modulating chromatin within the spatial organization of the genome in the three-dimensional space of the nucleus. The identification of lncRNA candidates is challenging, as it is their functional characterization. Conservation signatures of lncRNAs are different from those of protein-coding genes, making identifying lncRNAs under selection a difficult task, and the homology between lncRNAs may not be readily apparent. Here, we review the evidence for these higher-order genome organization functions of lncRNAs in animals and the evolutionary signatures they display. The three-dimensional (3D) organization of DNA in the cell nucleus has become a significant subject of study, particularly its influence on gene regulation. Recent advances in chromatin conformation capture (3C) techniques, computational, and modeling approaches have made its study feasible on a genome-wide scale, giving insight into the structure and the dynamics of chromatin folding in space and time. Nuclear 3D organization has multiple levels and varies between cell types and biological conditions. For instance, chromosomes are subdivided into topologically associating domains (TADs) within which chromatin loops bring together regulatory elements and target loci separated in the linear genome . These cIn addition to DNA and histones, RNA is a major component of the cell nucleus . High-thThere has been a long debate on whether most lncRNAs are functional or not . This diThe conservation signals in lncRNAs can differ from those typically found in protein-coding genes . For insin vivo across the genome  is highly conserved from human to zebrafish , encode transcription factors crucial for patterning along the anterior-posterior axis. Numerous ncRNAs are transcribed from the human HOX loci, and their expression relates to differential histone marks and transcriptional accessibility  lncRNA is transcribed from the boundary between domains with differential chromatin marks at the HOXC locus but acts in trans repressing transcription of coding and non-coding genes on the HOXD locus  located downstream of HOTAIR promotes transcription of the lncRNA. This loop is disrupted by the recruitment of hepatocyte nuclear factor 4-α (HNF4α), a master regulator of epithelial differentiation, to the HDE  present contradictory results. On the one hand, the deletion of the HoxC cluster, including mHotair, did not affect HoxD silencing in vivo , transcribed from the 5' end of the HOXA locus in mammals and conserved in avians  complex, driving H3K4me3 (HOTTIP and its association with CCCTC-binding factor (CTCF), which delineates active and inactive TADs within the HOXA cluster, also influence the expression of HoxA genes  is a lncRNA involved in pluripotency, hematopoiesis, and adipogenesis (Firre accumulates across a ~5 Mb domain around its transcription site on the X chromosome (Firre transcription (Firre with Heterogeneous Nuclear Ribonucleoprotein U (HNRNPU), through a 156-bp repeating RNA domain  are conserved to different extents across Firre orthologs in mammals. Firre is also required for the super-loop formation of the inactive X chromosome (Xi), H3K27me3 deposition, and the localization of the Xi to the perinuclear region , to direct the active priming of the promoters of immune genes, necessary for a rapid and robust pro-inflammatory response as part of trained immunity , engages in chromosomal contacts with CXCL chemokine genes belonging to the same TAD, but UMLILO does not have enhancer-RNA-like characteristics. In contrast to other IPLs, UMLILO is not conserved in mice and only partially conserved in pigs, suggesting that IPLs are not essential across species, but have a complementary role in ensuring robust gene expression. UMLILO has short conserved sequence motifs and interacts with WDR5 through its conserved exon 3, directing WDR5/MLL1 to chemokine gene promoters, mediating H3K4me3. Transcription of chemokines in UMLILO knockdown cells was restored by insertion of another WDR5-binding lncRNA, HOTTIP, under the control of the UMLILO promoter  locus. ESR1 is strongly upregulated in cancerous cells undergoing estrogen deprivation. A cluster of ncRNAs, ESR1 locus enhancing and activating non-coding RNAs (Eleanors), are transcribed from introns in a large chromatin cluster within a TAD that contains the ESR1 locus , induced repression of the rest of the Eleanors and the genes within the TAD, including ESR1  despite almost no sequence conservation outside an eight nucleotide-long conserved patch of microhomology (Examples of this conundrum are dosage compensation lncRNAs in nogaster . Detailenogaster . Importaensation . Furtherhomology .antisense to Igf2r RNA non-coding (Airn), required for paternal-specific silencing of imprinted genes in the insulin-like growth factor 2 (Igf2r) cluster , and this association is necessary for the insulator activity of CTCF in vivo (The non-coding isoform of the steroid receptor RNA activator (SRA), ervation . ncSRA ftructure . ncSRA a in vivo . The fun in vivo .X-inactive specific transcript (Xist), essential to the establishment of X chromosome inactivation, interacts with proteins such as the polycomb repressive complex 2  proteins. Domains outside these interaction zones are not conserved and lack structure (Dosage compensation lncRNAs (see next section) show patches of structural conservation of biological importance. The Repeat A (RepA) region of  2 PRC2; , ATRX ch 2 PRC2; , and SHA 2 PRC2; . RepA wafunction . lncRNAstructure .HOTAIR has also been shown to have a complex secondary structure, with some evidence of conservation in mammals acquired from computational methods  and is expressed exclusively from the inactivated X  found in marsupials, chicken, and fish suggests that Xist emerged through pseudogenization of Lnx3, possibly by the insertion of tandem repeats from transposable elements , which is transcribed from and coats the paternal chromosome (Xist and Rsx adds to the notion of dosage systems rapidly evolving from ancient silencing mechanisms common to all eukaryotes through the use of lncRNAs (Xist by non-coding elements located at its own and the neighboring TAD and the impact of this 3D conformation on the regulatory landscape adds another layer of complexity to the mechanisms for dosage compensation (Interestingly, in marsupials, X-chromosome inactivation is imprinted, tissue-specific, and somewhat incomplete compared to eutherians, and thought to be achieved by female-specific expression of the lncRNA romosome . The ind lncRNAs . The disensation .roX1 and roX2 lncRNAs mediate the upregulation of genes on the single male X chromosome to equalize expression of the two X chromosomes in females. roX1 and roX2 associate to the MSL proteins, forming the MSL complex that localizes to numerous specific sites along the male X (via spatial proximity from high-affinity sites – enriched at TAD boundaries – to other regions (Xist, whose activity is limited to the chromosome from which it is expressed (roX transgenes target the X chromosome in trans and rescue roX1 and roX2 mutant males (lncRNAs are also the effectors of dosage compensation in drosophilids, but they differ in both origin and mechanism to those in mammals. Here, the e male X , mediati regions . Contrarxpressed , roX trant males .Xist in mammals, Rsx in marsupials, and roX1 and roX2 in flies suggests that lncRNAs may be one of the fastest mechanisms to evolve novel epigenetic controls. As these lncRNAs participate in dosage compensation but have emerged independently in several lineages, they are extraordinarily difficult to identify as functionally convergent. Additional examples of functionally equivalent lncRNAs with no evolutionary relationship may likely have gone undetected.The independent origin of via modeling or direct RNA-seq evidence, followed by comparison across taxa (Distinctly, lncRNAs have emerged as an additional layer of complexity involved in shaping the three-dimensional organization of the genome by interacting and modifying the structure of chromatin. Several lncRNAs affect chromatin conformation and display a combination of conservation signals that may be difficult to identify solely by looking at traditional genomic conservation metrics (summarized in oss taxa . In the oss taxa . One of oss taxa , 2020.in silico studies with experimental approaches in the context of spatiotemporal gene expression programs is crucial to further assess the impact of these ncRNAs on modulating genome architecture, including their specific contribution to the complexity and evolution of animal gene regulation.In the context of studying novel lncRNAs, its unique conservation signatures, albeit more difficult to detect, are excellent ways to identify potentially functional lncRNA candidates and give a first insight on their possible mechanisms of action. They can also help guide the search for homologous mechanisms in other species. Complementing All authors participated in writing and reviewing the manuscript and approved the final version for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Gene expression is controlled in a dynamic and regulated manner to allow for the consistent and steady expression of some proteins as well as the rapidly changing production of other proteins. Transcription initiation has been a major focus of study because it is highly regulated. However, termination of transcription also plays an important role in controlling gene expression. Transcription termination on protein-coding genes is intimately linked with 3′ end cleavage and polyadenylation of transcripts, and it generally results in the production of a mature mRNA that is exported from the nucleus. Termination on many non-coding genes can also result in the production of a mature transcript. Termination is dynamically regulated—premature termination and transcription readthrough occur in response to a number of cellular signals, and these can have varied consequences on gene expression. Here, we review eukaryotic transcription termination by RNA polymerase II (RNAPII), focusing on protein-coding genes. In eukaryotes, RNA polymerase II (RNAPII) is responsible for the transcription of protein-coding genes as well as many non-coding genes. It is highly regulated to allow the constitutive production of “housekeeping” genes as well as the dynamic transcription of regulatory genes in response to signals. Transcriptional control is essential in almost every cellular process, including during development, growth, and stress.1 During transcription initiation, the preinitiation complex (PIC) assembles on promoter sequences and the promoter DNA strands are separated, allowing RNAPII to access the DNA template strand and begin RNA synthesis. RNAPII is then able to escape the promoter region, leaving many initiation factors behind.4 The transition from initiation to elongation is highly regulated: in metazoans, RNAPII pauses close to the promoter and can either be released to continue transcribing or can be removed from the gene by promoter-proximal premature termination, attenuating gene expression.5 After RNAPII has transcribed the remainder of the transcript, including a polyadenylation signal (PAS) sequence (most commonly AAUAAA), termination takes place.6Transcription can be divided into three phases: initiation, elongation, and termination .1 During8 This pre-mRNA cleavage defines the end of the 3′ untranslated region (UTR) and is coupled to transcription termination. If cleavage occurs too early, it results in truncated gene products. On the other hand, if it does not occur in a timely manner, it results in transcriptional readthrough, causing interference with initiation on downstream genes or the production of extended 3′ UTRs that may contain elements that alter transcript stability. Thus, both premRNA cleavage and transcription termination must be tightly controlled.At the 3′ ends of genes, pre-mRNAs are cleaved and polyadenylated to generate a mature transcript.9 including termination mediated by the Integrator complex (covered in an accompanying review),5 the ZC3H4-WDR82 restrictor complex,12 and microprocessor.13In this review, we focus on transcription termination at the 3′ ends of protein-coding genes. We provide details on the prevailing models of transcription termination, its coupling to 3′ end processing, how it is regulated, and cases of deregulation in disease. Alternative mechanisms also facilitate termination on non-coding genes,14 To enable the coupling of mRNA processing and transcription, the capping, splicing, and 3′ end processing machineries physically interact with RNAPII.15 Cryoelectron microscopy (cryo-EM) and biochemical studies have shown that all three of these RNA processing machineries bind next to the RNA exit tunnel of RNAPII.18 In this position, they can monitor the nascent transcript and proceed with processing as soon as the relevant sequences have been transcribed.19 If binding of these complexes to RNAPII is mutually exclusive, it could promote ordered and coordinated pre-mRNA processing.Pre-mRNAs undergo 5′ capping, splicing, and the addition of a polyadenylate (poly(A)) tail at their 3′ end before they are exported from the nucleus as mature mRNAs. These processing events generally occur co-transcriptionally: as RNA emerges from RNAPII, it is recognized by processing factors.23 Before transcription begins, the CTD is hypophosphorylated. Soon after initiation, Ser5 and Ser7 are phosphorylated by Kin28/CDK7 within TFIIH  of Rpb1, the largest subunit of RNAPII. The CTD is an unstructured polypeptide composed of 26- or 52-heptad repeats in yeast and human, respectively, with a consensus sequence of Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Differential post-translational modifications of the CTD, including phosphorylation, proline isomerization, and glycosylation, provide binding sites for transcription and pre-mRNA processing regulators throughout the transcription cycle.in TFIIH . Ser5 ph26 At this time, Ser5 is dephosphorylated by the Ssu72/SSU72 phosphatase.28 During transcription elongation, Bur1/CDK9 and Ctk1/CDK12 phosphorylate the CTD of RNAPII at Ser2 to recruit transcription elongation and splicing factors. CDK9 also phosphorylates Spt5 and the XRN2 torpedo exonuclease to modulate elongation and termination, respectively.29 At the 3′ end of genes, phospho-Ser2 recruits termination factors and the 3′ end processing machinery.30At the transition from transcription initiation to elongation, the Spt5/SPT5 subunit of the DSIF elongation factor is phosphorylated by Bur1/CDK9 of the P-TEFb complex, facilitating release of RNAPII from its promoter proximal pause site.32 In yeast, Tyr1 phosphorylation is proposed to prevent premature recruitment of termination factors during elongation.33 When RNAPII transcribes the PAS near the end of the 3′ UTR, Tyr1 phosphorylation is removed by the Glc7 phosphatase along with its regulatory subunit, Ref2, to allow phospho-Ser2-mediated recruitment of 3′ end processing and termination factors.37 In mammals, Tyr1 phosphorylation is associated with promoter antisense transcription38 and Tyr1 itself has been implicated in transcription termination.39 Still, the identity of the Tyr1 kinase remains unknown and Tyr1 phosphorylation is difficult to detect.Although Ser2 and Ser5 phosphorylation are most prevalent, Tyr1, Thr4, and Ser7 can also be phosphorylated.40 and histone pre-mRNA processing41 in metazoans, and small nucleolar RNA (snoRNA) termination in yeast.42 Ser7 phosphorylation, on the other hand, functions to recruit the Integrator complex to small nuclear RNA (snRNA) genes in metazoans,44 plays a role in RNAi-mediated heterochromatin formation in fission yeast,45 and primes the CTD as a substrate for other CTD kinases and phosphatases.46Thr4 phosphorylation is involved in transcription termination47 alongside genetic perturbations will be required.In summary, phosphorylation of the CTD and transcription factors is thought to strongly influence transcription and pre-mRNA processing. Still, the exact role of each modification is difficult to discern due to the essentiality and pleiotropy of the factors involved. In addition to the post-translational modifications described above, it is likely that there are many additional substrates of the kinases and phosphatases that play a major role in gene expression. To fully understand the function, prevalence, and importance of specific phosphorylations in termination, sophisticated mass spectrometry experiments8 CPF/CPSF recognizes the PAS sequence in RNA and also contains the endonuclease activity.48 RNA recognition is thought to activate the endonuclease  to cleave the nascent transcript 10–30 nucleotides downstream of the PAS, most often after a CA dinucleotide  in yeast, or cleavage and polyadenylation specificity factor (CPSF) in humans.cleotide . The pol53 In humans, RNAPII can continue hundreds or thousands of nucleotides beyond the PAS before termination takes place. However, PAS recognition, pre-mRNA cleavage, and transcription termination are intimately coupled in all species examined.In yeast, transcription termination occurs soon after the PAS has been transcribed, thereby preventing transcriptional interference on downstream genes, which are generally in close proximity.37 Because CPF phosphatase activity contributes to transcription termination, CPF couples RNA recognition, 3′ end processing, and transcription termination.In addition to an endonuclease and poly(A) polymerase, the multi-subunit CPF complex in yeast also contains two protein phosphatases: Ssu72 and Glc7. Ssu72 dephosphorylates Ser5 and Ser7 of the RNAPII CTD during transcription elongation, while Glc7 dephosphorylates Tyr1 at the 3′ end of genes— possibly in response to the recognition of the PAS sequence in RNA.54 Indeed, the role of phosphatases in 3′ end processing and transcription termination is likely to be conserved throughout eukaryotes (see below).The human orthologs of Glc7 and Ssu72  are not constitutive subunits of the CPSF complex. However, PP1 and its regulatory subunit PNUTS, as well as SSU72, associate with a post-cleavage CPSF complex purified from human cell extracts.58 This dependence on the PAS led to the development of two  models for transcription termination: the allosteric (or anti-terminator) model and the torpedo model, which are described in detail below59 .64 The PP2A phosphatase also antagonizes elongation by dephosphorylating RNAPII and SPT5. However, PP2A is recruited by Integrator near gene promoters and it acts on distinct residues from PP1-PNUTS.65Efficient transcription elongation requires phosphorylation of both the Spt5/SPT5 elongation factor and Ser2 of the RNAPII CTD.67 Tyr1 phosphorylation in yeast is thought to prevent premature recruitment of termination factors, thereby acting as an “anti-terminator.”33 Selective dephosphorylation of Tyr1 by Glc7/PP1 therefore allows phospho-Ser2 to recruit termination factors, including Pcf11 and Rtt103.70 In mammals, phospho-Thr4 is also enriched on RNAPII located beyond the PAS, and this phosphorylation is dependent on CPSF73.71 This suggests that Thr4 is modified after recruitment of the 3′ end processing machinery and, potentially, as a result of pre-mRNA 3′ cleavage. Phosphorylation patterns of the CTD therefore likely couple pre-mRNA cleavage to transcription termination via the recruitment of termination factors.A decrease in the efficiency of RNAPII elongation is paralleled by other changes that promote termination. For example, the transcription of a functional PAS and high levels of phospho-Ser2 at the 3′ ends of genes promote recruitment of the 3′ end processing machinery and termination factors.16 Thus, the oligomerization state of RNAPII could also contribute to the slowing of transcription in the allosteric model of termination.At transcription termination, phosphatase activity reverts RNAPII complexes to a hypophosphorylated state, perhaps resembling those at the beginning of transcription in having little elongation competence. It was recently reported that the dephosphorylation of yeast RNAPII results in the formation of an RNAPII dimer that likely cannot bind transcription elongation factors.73 Pcf11, an accessory factor of the 3′ end processing machinery, has been reported to dissociate elongation complexes from DNA in vitro.74 Like some other 3′ processing factors, Pcf11 binds both the RNAPII CTD via phospho-Ser2 and RNA. Interestingly, Pcf11 is of low abundance compared with other 3′ end processing factors and may act more selectively than core CPFs. In agreement with this, Pcf11 depletion more strongly affects the transcription of closely spaced genes, where it presumably acts to prevent transcriptional interference.40 Notably, Pcf11 can only terminate stalled elongation complexes, which is compatible with the slowing of RNAPII beyond the PAS, as described above.Many pro- and anti-termination factors indirectly influence transcription by creating the conditions for efficient termination—specifically, by modulating RNAPII elongation capacity. For example, the Paf1 complex modulates the elongation capacity of RNAPII to influence elongation, release from promoter-proximal pause, and termination.76 Sequence elements may also contribute to RNAPII termination in some cases, as U-tracts and other motifs are enriched at the ends of terminated nascent RNA.79 Such sequences may cause termination on their own but could also improve the process, for example, by facilitating RNAPII stalling.Stalling or slowing of transcription is a conserved feature of termination. For example, in prokaryotes, factor-independent termination relies on primary and secondary structures within the RNA to arrest RNA polymerase, and eukaryotic RNAPIII can terminate after the transcription of four or more U’s.80 This may involve the trigger loop of RNAPII that orients incoming nucleotides. However, it remains unclear whether this is a direct effect on RNAPII conformation and, if so, how this would be mediated. In summary, in the allosteric model, multiple factors, including phosphorylation states and sequence context, are likely to slow RNAPII elongation and promote termination.Finally, the analysis of semi-purified transcription complexes led to a proposal that transcription of the PAS results in conformational changes to RNAPII itself.59 The major nuclear 5′-3′ exonuclease is XRN2, and its inactivation or depletion causes termination defects at almost all protein-coding genes.83 The torpedo exonuclease in budding yeast is Rat1, and it functions in complex with the Rai1 and Rtt103 proteins.34Terminating without prior 3′ end processing might be dangerous and could release immature (non-processed) pre-mRNA from RNAPII that may ultimately be dysfunctional. Coupling PAS cleavage to termination ensures completion of RNA maturation prior to RNAPII release. This is the basis of the torpedo model for termination, whereby PAS cleavage exposes the RNAPII-associated RNA to a 5′-3′ exonuclease that chases down the polymerase to terminate it.84 Moreover, placing XRN2-resistant structures downstream of the PAS impairs termination, suggesting that RNA directly connects XRN2 activity and RNA polymerase.63 As discussed above, transcription of the PAS slows RNAPII—this likely facilitates termination by XRN2. Indeed, there is a correlation between polymerase speed and the site of termination. RNAPII mutants that are slowed tend to terminate at upstream sites, whereas faster RNAPII variants terminate further downstream.82In the torpedo model, Rat1/XRN2 co-transcriptionally degrades the 3′ cleavage product continuously until it reaches RNAPII. In agreement with this, XRN2 initiates degradation at the cleavage site and XRN2 depletion stabilizes RNA downstream of the PAS.82 Moreover, antisense oligonucleotides (ASOs) can direct RNaseH1-dependent RNA cleavage and subsequent XRN2-dependent transcription termination.86 This is not dependent on a PAS, again suggesting that pre-mRNA cleavage, but not the 3′ end processing machinery itself, is required for XRN2-dependent termination. It is possible that ASOs direct RNA cleavage more rapidly than a PAS and negate the need to slow RNAPII. Alternatively, RNA cleavage itself could trigger RNAPII slowing. Importantly, not every co-transcriptionally formed 5′ phosphate promotes XRN2-dependent termination: XRN2 elimination does not affect termination at snRNA and histone genes, although 3′ end processing at both transcript classes involves an endonuclease81 (see below). Interestingly, inactive XRN2 impairs the degradation of histone 3′ end cleavage products and mildly affects subsequent termination, perhaps by loading onto the 5′ end of the cleaved RNA and blocking access to a redundant exonuclease.87 Finally, XRN2 is implicated in the promoter-proximal termination of RNAPII and, although the nature of its entry site is not defined, possibilities include the 5′ end of incompletely capped RNA or the new end generated by Integrator cleavage.87XRN2-dependent termination can also occur in a PAS-independent manner, for example, in response to RNaseP activity at NEAT1 and MALAT1 non-coding genes.82 it seems likely that their activity forms a key part of this process. Analogous mechanisms are present in bacteria, some of which utilize the exonuclease activity of RNaseJ1 for termination or the helicase activity of Rho.89 Structures are available for the latter, which, together with biochemical studies,90 suggest Rho activity pulls on RNA or causes forward translocation of the polymerase. Even so, RNA degradation may not be sufficient for termination because Rat1 activity cannot dislodge E. coli RNA polymerase from DNA in vitro and nuclear Xrn1 cannot promote termination in yeast cells lacking Rat1.92 This suggests that Rat1 (and XRN2) may form specific contacts that are important for terminating eukaryotic RNAPII. Additional factors discussed above may also improve the process. Interestingly, structures of Rho-dependent termination demonstrate that NusG bridges Rho with RNA polymerase.90 NusG is the homolog of Spt5, which plays a key role in RNAPII elongation competence, as outlined above.A major question is how XRN2 causes RNAPII to release the template. The torpedo model originally implied that exonuclease activity would forcefully dislodge RNAPII, but it remains unclear whether this is the case or how it may occur. Because inactive mutants of Rat1/XRN2 cannot support efficient termination,The allosteric and torpedo models are compatible with each other. Transcription of the PAS allows the 3′ end processing machinery to assemble on RNA. This would activate the 3′ endonuclease and protein phosphatase activities, resulting in cleavage of the pre-mRNA and changes in the phosphorylation state of the CTD . This wo96 As such, interfering with the function of these factors causes premature mRNA 3′ end processing and transcription termination in human cells.97 This is best-characterized in the case of U1 snRNA, which masks intronic PAS sites in a process called telescripting.95 Telescripting is independent of the role of U1 in splicing and is enabled by the fact that U1 snRNA levels exceed those of other spliceosomal snRNAs, presumably allowing it to occupy additional sites within introns and suppress nearby PASs. Consistently, core components of U1 snRNP  crosslink together with cleavage factors at intronic PAS sequences that are activated when U1 snRNA telescripting is inhibited.98During transcription elongation, premature transcription termination and the use of intragenic PAS sequences is inhibited. Specifically, splicing factors, U1 snRNA, SCAF4, and SCAF8, as well as the kinase CDK12, inhibit the action of the 3′ end processing machinery at intronic PAS sites.99 This arrangement underpins the elongation to termination transition by allowing recognition and processing of the correct PAS. Intriguingly, both the 3′ end processing machinery and U1 snRNP interact directly with RNAPII near the RNA exit channel.18 A physical interplay between these complexes at this site could influence telescripting.A critical issue is how telescripting is alleviated to allow correct PAS usage at the ends of genes. In internal exons, there is normally another 5′ splice site (that can recruit U1 snRNP) downstream of a given 3′ splice site, which may promote telescripting in the subsequent intron. However, at the end of the gene, the terminal 3′ splice site is instead followed by a PAS, which may favor 3′ end processing over further telescripting.100 As mentioned above, XRN2 does not play a major role in termination at histone genes in humans. Thus, allosteric termination may be more prominent on histone genes, although it is worth noting that CPSF73 can act as an exonuclease that may substitute for XRN2.101 While CPSF binds upstream of the PAS cleavage site at protein-coding genes, U7 snRNA tethers the histone cleavage complex downstream of the cleavage site, which could better-position CPSF73 to initiate degradation of the downstream RNAPII-associated product and potentially serve as an alternative torpedo exonuclease.In addition to the poly(A)-dependent mechanism, transcription can terminate via a variety of alternative pathways. Human histone mRNAs are processed by a machinery that contains U7 snRNA and also shares some subunits with CPSF, including the CPSF73 endonuclease.102 Despite this, termination at snRNA genes is insensitive to XRN2 and so could employ an allosteric mechanism or an alternative exonuclease. INTS11 is highly similar to CPSF73, although it is unknown whether it has exonuclease activity. Integrator also mediates premature transcription termination at protein-coding genes that controls their ultimate output.105 Transcription termination by the Integrator complex is covered in detail in a parallel review by Adelman and colleagues.5Primary snRNA transcripts are also processed endonucleolytically via the Integrator complex, within which INTS11 provides cleavage activity.106 However, in budding yeast, termination and 3′ end processing of short non-coding RNAs, including snRNAs and snoRNAs, occurs via a PAS-independent mechanism involving the Nrd1-Nab3-Sen1 (NNS) complex.108 Recruitment of Nrd1-Nab3 to elongating RNAPII is mediated through the recognition of phospho-Ser5 on the CTD of RNAPII.110 Sequence elements on nascent pre-snoRNAs also contribute to Nrd1-Nab3 recruitment.111 Sen1, a 5′-3′ ATP-dependent superfamily 1 helicase, is recruited through multiple contacts with RNAPII and the Nrd1-Nab3 heterodimer.113 Sen1 likely promotes termination of short non-coding RNA genes similar to Rho and XRN2, whereby the activity of a processive enzyme tracks down RNAPII to disengage it from chromatin.114 Sen1-mediated termination is sensitive to RNAPII elongation rates, in agreement with a requirement for slowed transcription for efficient termination.115In fission yeast, termination of snoRNA genes is dependent on the CPF endonuclease, the torpedo exonuclease, and, in some circumstances, the exosome, which is proposed to act as a “reverse torpedo” in a 3′-5′ direction.116 The APT complex contains both of the CPF phosphatases, and these are required for transcription termination on non-coding RNAs, likely by dephosphorylating Sen1 and the RNAPII CTD.118 Similarly, phosphatases within the Integrator complex play an important role in termination.119 Other components of the cleavage and polyadenylation machinery also play a role in poly(A)-independent termination. For example, Pcf11 contributes to termination on snoRNAs, possibly in concert with Rtt103, which binds phospho-Ser2 and phospho-Thr4.122 Thus, it appears that although cleavage and polyadenylation activities are only required on a subset of RNAPII-transcribed RNAs, the phosphatase activities as well as the accessory factors (CF IA) are more generally required for the termination of RNAPII transcripts.Interestingly, in budding yeast, six subunits of CPF (the phosphatase module) associate with the Syc1 protein to form the associated with Pta1 complex (APT), which contributes to transcription of non-coding RNAs.Alteration of transcription termination in response to cellular cues likely regulates gene expression . Mountin123 DoGs are continuous with their associated upstream mRNA transcripts and do not represent de novo transcription initiation events. They are depleted of strong PAS sequences, are retained in the nucleus, and are not translated.124 Thus, DoGs represent bona fide readthrough transcripts. DoGs do not appear to be unstable and instead show half-lives similar to the median mRNA half-life (1 h). They have been detected in both poly(A)− and poly(A)+ mRNA fractions of cells undergoing osmotic shock. The production of DoGs is linked to reduced Integrator function.125During heat, osmotic, or oxidative stress, transcription termination is impaired and gives rise to “downstream of gene” transcripts (DoGs).126 This has been interpreted as DoGs being under modes of stress-specific regulation, and potentially playing a role in recovery from stress conditions. Moreover, because some DoGs remain tethered to chromatin, one hypothesis is that DoGs function globally in maintaining nuclear integrity during stress.123 Alternatively, transcription readthrough itself may regulate downstream genes by transcription interference.127 Interestingly, DoGs tend to be more prevalent if there is a downstream gene in close proximity (irrespective of orientation).126 Systematic studies that rescue the termination defects that give rise to DoGs will be critical in assessing their function and understanding how they are generated.It has been proposed that DoGs carry out important physiological functions during cellular stress responses. When cells are removed from stress conditions, DoGs show stress-specific recovery rates as they return to basal pre-stress levels.128 This is largely mediated by the viral RNA-binding protein ICP27, which interacts with CPSF and disrupts the assembly of a functional complex.129 Surprisingly, ICP27 can also bind GC-rich sequences upstream of the PAS, which paradoxically results in the recruitment of CPSF and premature transcription termination.131 The molecular details of the dual function of ICP27 remain unclear. Further structural and biochemical studies using the recently reported reconstituted CPSF complex51 could help explain how ICP27 can function to both aid and antagonize CPSF function and transcription termination.During herpes simplex virus 1 (HSV-1) infection, transcription termination is impaired.132 The termination defect is mediated, at least in part, by the non-structural viral protein, NS1A, which directly interacts with the CPSF30 subunit of CPSF.134 Exactly how NS1A inhibits CPSF activity is not understood. It is clear, however, that NS1A interferes with the binding of CPSF to its RNA substrate.134 Biochemical and structural studies show that two copies of NS1A interact with two copies of the RNA-binding zinc fingers of CPSF30. This interaction may disrupt CPSF30 assembly into CPSF or it may induce the dimerization of CPSF to favor an inactive form of the complex. In addition, the influenza A viral polymerase stabilizes the NS1A-CPSF30 interaction135 and interacts with the Ser5 phosphorylated form of RNAPII near the 5′ end of genes, where it can sequester CPSF away from the site of 3′ end processing.137During influenza A infection, host gene expression is disrupted by defects in transcription termination and the depletion of transcribing RNAPII near the 5′ ends of genes.132 Thus, cellular stress may generally lead to transcription termination defects that primarily affect transcripts lacking strong PAS sites.124 Further studies are required to understand whether these defects in transcription termination are specifically induced or whether they are the general consequence of cellular stress.Divergent influenza strains with orthologs of NS1A that are not predicted to interact with CPSF30 also impair transcription termination, leading to transcription readthrough reminiscent of DoG production in oxidative stress.138 In clear cell renal cell carcinoma, readthrough correlates with mutations in the SETD2 gene, which methylates histone H3 lysine 36 (H3K36me3), suggesting that there is a link between transcription termination and chromatin marks of active transcription.138 Readthrough transcription can lead to novel chimeric fusion transcripts or circular isoforms,139 which arise from trans-splicing and back-splicing events between adjacent co-directional genes.140 It is likely that chimeric fusion transcripts are a general feature of transcription termination defects,141 and may even produce functionally novel proteins.Transcription termination defects have also been observed in a number of cancers, and this can correlate with poor prognosis.142 START transcripts appear to repress the expression of adjacent convergent genes and promote the senescent gene expression program and permanent cell proliferation arrest. START RNAs are stable transcripts and arise from increased readthrough transcription due to a defect in RNAPII slow down within the termination window. Although it remains unclear why elongating RNAPII fails to slow down, the H2A histone variant H2A.Z appears to play a role in repressing START RNAs.In cells undergoing oncogene-induced senescence, termination defects between a small subset of convergent genes gives rise to senescence-triggered antisense readthrough (START) RNAs.143 Many of the prematurely terminated genes contain multiple introns and strong intronic PAS sequences, consistent with impaired telescripting.96 Surprisingly, heat shock also increases the transcription rate of elongating RNAPII.143 A faster moving RNAPII could miss the termination window near the 3′ ends of genes, giving way to runaway transcription elongation complexes.94 This would be reminiscent of the way weak splice sites can be ignored when elongation is fast.144Telescripting may also be regulated by external inputs. For example, heat shock causes premature transcription termination as well as termination defects at the 3′ ends of genes.145 would reduce U1 snRNA transcript levels, impair telescripting, and increase intronic cleavage and polyadenylation, followed by premature transcription termination at protein-coding genes. During HSV-1 infection, on the other hand, ICP27-mediated recruitment of CPSF to PAS sites near GC-rich regions would cause premature termination independently of telescripting.131 A stress-induced shift in U1-snRNP abundance could also tip a delicate balance between telescripting and transcription termination.143 Finally, the heat-responsive master regulator heat-shock factor 1 (HSF1), directly interacts with the 3′ end processing machinery in a heat-shock-dependent manner,146 and may recruit CPSF to heat-responsive genes, which are typically shorter, have fewer introns, and are not subject to telescripting.96A tantalizing idea that emerges from these studies is that cellular stressors, disease states, and viral infections may cause early recognition of the PAS and premature transcription termination through independent mechanisms. For example, cap snatching of the 5′ end of the U1 snRNA148 Studies of the kinase and phosphatase components of the transcription cycle have given critical insight into the importance of RNAPII elongation control. It seems certain that the substrate repertoire of these is much broader than currently appreciated and identification of new targets will further improve our understanding of termination. Similarly, SPT5 is central to RNAPII elongation, but we do not understand how its phosphorylation influences critical protein-protein interactions. Lastly, is XRN2 the only “torpedo” or does CPSF73 (and even INTS11) also have torpedo exonuclease activity in certain contexts?Termination has been the least-studied phase of the transcription cycle, perhaps because it happens after RNAPII is perceived to have already done its important jobs. As this review highlights, we are now beginning to understand its mechanisms, as well as appreciate that termination plays important roles in gene expression beyond evicting RNAPII from chromatin. Still, there are many interesting questions remaining. Although recent data unify the allosteric and torpedo models, we still do not know how RNAPII is dislodged from its template. Cryo-EM offers the most exciting avenue to pursue this, having already illuminated critical RNA processing mechanisms in detail, including prokaryotic transcription termination.119 That this process is sensitive to external inputs such as heat shock suggests an underappreciated regulatory capacity that will be interesting to elucidate in the future. CPSF is already known to be targeted by influenza and HSV, as well as being targeted in cancer, but there may be more ways of conditionally influencing transcription termination. It is likely that altered 3′ end processing and transcription termination are as highly regulated and as prevalent in disease as changes in splicing. Understanding how transcription terminates has led to a transformation in our understanding of the transcription cycle in general. Far from being at the end, we may be just beginning to understand its many roles in gene regulation.Once RNAPII escapes the promoter, complete elongation across a gene was once thought inevitable. However, premature termination is commonplace and, on many genes, it may seal the fate of the majority of elongating RNAPIIs.


Total chromatin isolated from cells can be directly analyzed using MS or further fractionated into transcriptionally active and inactive chromatin prior to MS-based analysis. Newly formed chromatin that is assembled during DNA replication can also be specifically isolated and analyzed. Furthermore, capturing specific chromatin domains facilitates the identification of previously unknown transcription factors interacting with these domains. Finally, in recent years, advances have been made toward identifying proteins that interact with a single genomic locus of interest. In this review, we highlight the power of chromatin proteomics approaches and how these provide complementary alternatives compared with conventional affinity purification methods. Furthermore, we discuss the biochemical challenges that should be addressed to consolidate and expand the role of chromatin proteomics as a key technology in the context of gene expression regulation and epigenetics research in health and disease.Regulation of gene expression is essential for the functioning of all eukaryotic organisms. Understanding gene expression regulation requires determining which proteins interact with regulatory elements in chromatin. MS-based analysis of chromatin has emerged as a powerful tool to identify proteins associated with gene regulation, as it allows studying protein function and protein complex formation in their   •An overview of proteomics methods to study chromatin and gene regulation.•Strength and limitations of the different approaches are highlighted.•An outlook on the outstanding challenges for chromatin proteomics.•Future directions for chromatin proteomics are discussed. MS-based analysis of chromatin has emerged as a powerful tool to identify proteins associated with gene regulation. Total chromatin isolated from cells can be directly analyzed using MS, further fractionated into transcriptionally active and inactive chromatin, enriched for specific compartment or regions, and potentially used for single-locus isolation. This review highlights recent advances and discusses current challenges that should be addressed to further advance the field of chromatin proteomics. Chromatin, which is present in every eukaryotic cell, is a complex assembly of DNA and proteins, which plays a central role in the regulation of gene expression. Gene expression regulation involves recruitment of proteins  to regulatory regions in the genome, such as promoters and enhancers. Consequently, these TFs recruit a multitude of factors including chromatin modifying and chromatin remodeling complexes that, depending on their function, mediate either transcriptional activation or silencing of a gene. To comprehend how gene expression is regulated, chromatin research aims to identify all TF-binding sites, how TFs and regulatory protein complexes are recruited to those sites, and how this affects the local chromatin environment. To determine genome-wide binding for chromatin-associated proteins of interest, CHromatin Immuno Precipitation followed by sequencing and cleavage under targets and release using nuclease are currently the most widely used methods , 2. ThesGenome-wide profiling technologies are crucial for our understanding of gene regulation. However, these techniques do not provide an unbiased and comprehensive view of chromatin since they are limited to studying one or a few proteins at a time and are thus limited with respect to their ability to identify novel chromatin regulatory proteins. In addition, genomics techniques (1) are slightly biased toward active chromatin regions ; (2) comversus diseased cells. Purifying chromatin followed by MS-based profiling allows identification of the chromatin-bound proteome in a cell-type or disease-specific manner. The advantage of this approach compared with MS-based analysis of whole cell proteomes, or even nuclear proteomes, is that it allows measuring chromatin-associated factors that are typically low abundant and difficult to identify without extensive sample fractionation prior to LC–MS (A), which proved highly informative in defining novel TFs associated with overexpression of the oncogene c-Myc . As such, ChEP has been successfully used to study the chromatin of a multitude of cell types of different organisms . The use of nuclear isolation prior to crosslinking and maintaining chromatin in soluble form together may result in lower contamination from cytoplasmic proteins crosslinked to chromatin. Thus, while technically more challenging than ChEP because of the requirement for ultracentrifugation, DEMAC has the potential to enable the characterization of the chromatin-associated proteome with low levels of contaminants. DEMAC is therefore a valuable addition to the toolbox available for studying the chromatin-associated proteome in different cellular contexts.While native chromatin purifications have been adopted to obtain new insights into various biological processes , 11, 13,Fig. 1s e.g., , 17, 18), 18B). As . An orthogonal approach is to expose native nuclei to different concentrations of salts, as TF binding is largely driven by electrostatic interactions (F). A third approach to separate euchromatin and heterochromatin, called gradient-seq, uses a crosslinking approach comparable to DEMAC and makes use of ultracentrifugation of crude chromatin fragments in a sucrose gradient. This approach separates molecules depending on their sedimentation rate, which is mainly determined by the size of the molecules . This is in contrast to a cesium chloride gradient, through which molecules diffuse based on their protein–DNA ratio. The gradient-seq method has been used to study difficult-to-sonicate heterochromatin regions, and MS-based analyses of this fraction revealed good concordance with previously described proteins associated with heterochromatic regions , which rely on incubating cells for a short period (10–20 min) with thymidine analogs that are incorporated into newly synthesized DNA strands. In case of isolation of Proteins On Nascent DNA method, the analog is 5-ethynyl-2′-deoxyuridine, which can be covalently linked to biotin–azide after crosslinking, allowing enrichment of nascent chromatin using streptavidin beads. The Nascent Chromatin Capture protocol relies on integration of biotin–2′-deoxyuridine, 5′-triphosphate, which after cell lysis and chromatin isolation can be used directly as a handle for nascent chromatin isolation using streptavidin-coated beads. These protocols have for example revealed the important role of FAM111A as a replication factor for proliferating cell nuclear antigen loading and consequently progression of the cell cycle (H). The resulting so-called DNA-mediated chromatin pull-down workflow is easy to perform with limited reagents, provided that the cell type of interest is proliferating.When chromatin is isolated from cells or tissues, the sample contains cells that are in different phases of the cell cycle and thus represents a mixture of “old” and “new” chromatin. An open question in the field remains how exactly new chromatin is formed during replication, how DNA replication is integrated with chromatin dynamics, and which chromatin structures and proteins are transmitted to newly formed chromatin. Several research groups have thus taken up the task to specifically isolate and characterize newly synthesized chromatin strands that are generated during replication. This has resulted in two analogous methods called isolation of Proteins On Nascent DNA and nascent chromatin capture , 32 Fig, H and Ill cycle . These pll cycle  Fig. 1HHH and I,Altogether, many workflows have been developed to study the chromatin proteome, and the choice of method should be tailored to the research question, the available infrastructure in the laboratory, the amount of biological material available, and whether the study focuses on cultured cells or tissue material .Table 1Oin vivo.Analysis of the chromatin proteome is highly informative and can be used to identify candidate regulatory chromatin factors for cell types of interest as well as their dynamics. How these TFs act to regulate cell type–specific gene expression can be further investigated using genomics tools such as CHromatin Immuno Precipitation followed by sequencing or cleavage under targets and release using nuclease. This can be combined with TF perturbation followed by gene expression profiling, which yields a global picture as to if, and to some extent how, this TF is relevant for a specific cell type. However, to understand in detail how a TF functions, it is important to understand the chromatin environment in which it is functional, to identify relevant PPIs, to determine how local chromatin features define PPIs, and how this is affected by a (disease-induced) mutation in the protein itself or the DNA it associates with. A wide range of methods are currently available to study protein–protein and protein–DNA binding of chromatin factors. These assays frequently use crude nuclear extracts, followed by either affinity purification (AP) of a TF of interest using antibodies or introduced protein tags such as GFP . AlternaDrosophila . These methods use analogous principles as mChIP but only require an antibody for a protein of interest and a cell type that can be obtained in sufficient quantities, making them implementable in virtually every laboratory, and as such, these assays are frequently used nowadays. While antibody-based methods may also be subject to biases caused by nonspecific binding of antibodies or blocking of the epitope recognized by the antibody, they have the advantage of being able to target post-translational modifications on histones or other proteins.These so-called ChIP–MS workflows rely on introducing tagged proteins into cells. However, this may be challenging in cell types that are hard to transfect or already fixed and could introduce artifacts because of (over)expression of the tagged protein. This required adaptations of the ChIP–MS workflow to use antibodies to a protein of interest, resulting in methods referred to as chromatin proteomics (ChroP) , quantitHowever, these ChIP-based approaches also have some limitations. At first, application of these methods does not involve a chromatin isolation step but rather uses sonicated nuclei as input for the IP. This results in a higher degree of contamination from “hitchhiker” proteins binding to the highly charged DNA backbone  and alloC). ChIP–SICAP has been successfully used to identify proteins associated with the pluripotency network in embryonic stem cells, revealing TRIM24 as a novel pluripotency-associated protein  method was developed . This apFig. 2 protein . While t protein  Table 2C. ChIP–SD). Furthermore, since bait-proximal proteins become biotinylated, denaturing conditions can be used during cell lysis and subsequent affinity enrichment. As the biotin–streptavidin interaction is extremely strong, the IP and the washes can therefore be performed in the presence of high concentrations of salt and detergents, which strongly reduce the number of contaminants. The most commonly used enzymes are an engineered soybean ascorbate peroxidase called APEX2, and a promiscuous mutant of the Escherichia coli biotin ligase BirA, referred to as BioID, which has recently been derived to an enhanced and much faster enzyme called (mini)TurboID  enabling the identification of partners that may potentially be missed by using a faster enzyme. While such assays can be used for any protein of interest, they are particularly useful to investigate the local proteome of chromatin-bound proteins  when these are in close proximity in vivo. In such cases, the biotin ligase can be “split” over the two chromatin proteins, and only when these are close together, the enzymatic activity is reconstituted, which then results in biotinylation of the proximal environment . Finally, proximity biotinylation can also be performed by horseradish peroxidase (HRP), which is an enzyme that can be coupled to antibodies. By targeting an antibody–HRP conjugate to proteins inside cells, a proximal proteome for any protein of interest can be obtained, provided a specific antibody is available . This approach has already been shown to be applicable to chromatin factors . Of theproteins , 59, 60.ironment , 62, 63.ironment    , 77, thu9 cells) , 79, butFinally, a much-discussed challenge in the chromatin field is single-locus proteomics. The biology of a single locus in the genome such as a promoter or an enhancer can only be completely understood if the proteome of that locus is known. While approaches are available that target repetitive elements such as telomeres in a reproducible manner , 77, andRecent developments in the chromatin proteomics field have allowed analysis of the entire chromatin proteome or subfractions thereof. These approaches, also when integrated with genomics approaches, will form a strong basis to discover novel TFs associated with cellular homeostasis, development, and disease.versus the chromatin fraction is thus an important goal for the future.Epigenetic proteins and complexes are frequently studied by determining their composition and architecture in crude cell extracts or in solution. However, the behavior of proteins is often different as soon as they are taken out of their native contexts, underlined by the existence of distinct versions of protein complexes on and off chromatin . In the It is further likely that the 3D organization of protein complexes changes when they become associated with a chromatin template. It would therefore be highly informative to determine the topology and the 3D structure of proteins and protein complexes bound to chromatin. One approach would be to combine crosslinking–MS (XL–MS) with any of the chromatin-domain enrichment procedures described previously. While potentially biochemically challenging, XL–MS has readily been shown to be applicable to intact nuclei , providiAnother important aspect of chromatin biology that is becoming increasingly apparent is phase separation. The process of phase separation yields liquid condensates that can confine protein and nucleic acids in separate membraneless compartments. Recent advances have demonstrated that reconstituted chromatin fibers can phase separate and that this can be modulated by histone modifications . In addiWhile this review mainly focuses on TFs, the epigenome is highly complex. Many different RNA molecules are associated with the chromatin, and the DNA, as well as RNA and proteins associated with it, can be chemically modified. In terms of chromatin extraction, informative assays will be to use the techniques described in this review to assess whether proteins and RNA possess specific modifications when bound to chromatin (compartments). This information will be required to advance our understanding of chromatin regulation at the local scale, and how this is for example dynamic in cell fate transitions or perturbations.In summary, because of improvements in hardware, software, and methodology, we envision that chromatin proteomics will take up a place in routine epigenetic research. Future improvements aimed at downscaling will allow obtaining global and local chromatin proteomes, even when small amounts of input material are available. Finally, efforts aimed at increasing throughput will allow screens to investigate how drugs or compounds modulate the  chromatin proteome.The authors declare no competing interests.