Precise regulation of gene transcription is of great importance to development and diseases. Promoter-proximal transcriptional pause is a key and general mechanism to precisely control transcription in metazoans. Subsequent to transcription initiation and synthesis of a short RNA, RNA polymerase II (Pol II) usually pauses at the promoter-proximal regions, standing by for further signals to be released into the productive elongation stage. Fine regulation of Pol II pausing and release is achieved by the concerted action of many negative and positive elongation factors, including the super elongation complex (SEC). Recent studies suggested that phase-separated assemblies of transcription regulatory complexes could provide a general biophysical basis for the dynamic regulation of transcription in response to various cellular needs, though direct evidence at endogenous level in living cells is still largely lacking. Here, we summarize and discuss latest advances in understanding how phase separation contributes to RNA polymerase II-mediated transcription, with a focus on transcriptional pause and release.RNA Pol II-mediated transcription begins with the assembly of the pre-initiation complex (PIC), which contains polymerase, general transcription factors (TFs), and a large number of coactivators. The final essential step in the assembly of PIC is the recruitment of transcription factor IIH (TFIIH), which not only initiates transcription by opening the double-stranded DNA but also phosphorylates Ser5 and Ser7 residues of Pol II C-terminal domain (CTD) through its catalytic subunit cyclin-dependent kinase 7 (CDK7) . Ser5-phDrosophila melanogaster. A large amount of Pol II was detected between nucleotides −12 and +65 of the HSP70 gene under the nonheat shock condition  and 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF). NELF comprises four subunits: NELF-A, NELF-B, NELF-C/D, and NELF-E, while DSIF is composed of SPT4 and SPT5. Similar to Pol II CTD, the C-terminal region of SPT5 has a repeat region that can be phosphorylated by both TFIIH and positive transcription elongation factor b (P-TEFb). The release of paused Pol II into productive elongation is triggered by P-TEFb-containing complexes . By inteIn vitro transcriptional elongation assays showed that the DSIF and NELF complexes can arrest Pol II around the promoter. In the presence of DSIF, NELF can effectively inhibit transcription in vitro . These phase-separated biocondensates allow biomolecules to rapidly move inside and exchange with outside factors, enabling effective bioreactions . Transcrcis-regulatory elements that activate transcription under physiologically relevant conditions.Weak and multivalent interactions among proteins bearing low-complexity domains (LCDs) or intrinsically disordered regions (IDRs) are believed to be one of the main driving forces for phase separation . Many euPhase separation is also involved in the release of paused Pol II into productive elongation stage through dynamic exchange of related factors at the promoter-proximal regions. The histidine-rich domain of CCNT1, a subunit of P-TEFb, forms phase-separated condensates to enrich RNA Pol II, thereby leading to Pol II CTD hyperphosphorylation and enhancing transcriptional elongation activity . In addiPol II CTD is intrinsically disordered and lowly complex. A recent study has shown that the switch from hypophosphorylated to hyperphosphorylated state of Pol II CTD drives the transition of Pol II from transcription initiation to RNA splicing condensates . RNA splMisregulation in promoter-proximal Pol II pause and release can lead to developmental abnormalities and human diseases, including cancers. A recent study has suggested that unblending of transcriptional condensates via the expansion of amino acid repeats in TFs may cause the disease synpolydactyly . Fusion Transcriptional regulation is a highly complex process. It is challenging to fully understand the regulatory processes of transcriptional pause, release, and productive elongation. The phase separation model helps in understanding how different factors, including Pol II, TFs, coactivators, chromatin regulators, and RNA-binding proteins, are recruited to maintain the homeostasis of gene expression, and how cells rapidly change their gene expression programs in response to signaling. However, we have to be aware that direct measurements are still lacking regarding the capabilities and properties of phase-separated condensates under endogenous conditions and also the causal relationship between LLPS and transcriptional regulation . Deciphe[We are grateful to the members in Lin & Luo Lab for critical discussion. This work was financially supported by grants from the National Key R&D Program of China  and the National Natural Science Foundation of China ]


Reversible lysine methylation is essential for regulating histones and emerges to critically regulate non-histone proteins as well. Here we show that the master transcription factor OCT4 in pluripotent stem cells (PSCs) was methylated at multiple lysine residues. LSD1 that is highly expressed in PSCs can directly interact with and demethylate OCT4 at lysine 222 (K222) in the flexible linker region. Reduced LSD1 activity led to the methylation of OCT4-K222 that diminished the differentiation potential of PSCs while facilitating proteasome-independent degradation of OCT4 proteins. Furthermore, site-specifically replacing K222 with phenylalanine to mimic the constitutively methylated lysine promoted the ‘locked-in’ mode engagement of the OCT4 PORE-homodimers that tightly bind to and block the transcription of multiple PORE-motif-containing target genes regulating cell fate determination and cell junction organization, and thereby reducing the pluripotency of PSCs. Thus, LSD1-mediated demethylation of OCT4 plays a crucial role in restricting the ‘locked-in’ mode binding of OCT4 PORE-homodimers to the PORE-motif-containing genes and thereby maintaining their transcription to safeguard the pluripotency of PSCs. Both ESCs derived from the inner cell mass of the mammalian preimplantation blastocyst3 and their malignant counterpart ECCs derived from teratocarcinomas4 are cultured in the Petri dish indefinitely as a unique tool for modeling cell fate determination. In contrast, iPSCs are acquired by enforced expression of certain pluripotency transcription factors, such as OCT4, SOX2, KLF4, and c-MYC (OSKM), in differentiated somatic cells6. The core stemness circuitry comprising OCT4, SOX2 and NANOG is known to be essential for maintaining the pluripotency and self-renewal PSCs via forming a positive feedback transcriptional regulatory circuit to suppress differentiation8, and to coordinate with other transcription factors, epigenetic regulators and signaling pathways in cell fate determination7. However, little is known about the precise mechanisms underlying the interplay between pluripotency transcription factors and epigenetic regulators in establishing and maintaining the pluripotency of PSCs.Cell fate determination during embryogenesis depends on the properties of self-renewal and pluripotency possessed by pluripotent stem cells (PSCs) including embryonic stem cells (ESCs), embryonal carcinoma cells (ECCs) and induced pluripotent stem cells (iPSCs) that have the potential to differentiate into three embryonic germ layers  and are valuable for basic research, regenerative and translational medicinePOU5F1 gene), a member of the class 5 POU (Pit-Oct-Unc) family of transcription factors, is one of the most important pluripotency transcription factors and pioneer transcription factors participating in ESC maintenance, zygotic gene activation and cellular reprogramming10. OCT4 proteins specifically bind to the canonical octamer motif (consensus sequence ATGC(A/T)AAT) of target gene enhancer or promoter regions by the cooperation of the POUS  and POUH AAT) domains connected with a flexible linker region10. This flexible linker of 17 amino acids (N213-A229) allows human OCT4 proteins to form either monomers recognizing the MONO motif (ATGC(A/T)AAT), or OCT4/SOX2 heterodimers binding to the SORE (sox oct recognition element) motif (CATTGTAATGCAAAA), or homodimers in PORE  motif (ATTTGAAATGCAAAT)- or MORE  motif (ATGCATATGCAT)-binding configuration, depending on the positioning of POUS and POUH domains relative to each other9, and therefore plays a crucial role in regulating the target gene recognition and transcriptional activity of OCT412. An early study utilizing PORE- and MORE-motif containing luciferase reporters indicated the potential regulation of murine Oct4 homodimer configurations by PKA-mediated phosphorylation of S229 site at the POUH domain13, but validation of the conclusion with sufficient number of target genes in the context of PSCs was lacking. A functional study showed that the short α-helix in the linker segment of murine Oct4 could modulate its reprogramming potential by serving as a protein–protein interaction site12. Furthermore, the intramolecular interaction between the murine Oct4 linker residues and the POUH RK residues constrained the nonspecific binding of the POUH domain to random DNA sequences to ensure the specific recognition and binding of the OCT4-binding motifs, and it was speculated that post-translational modifications (PTMs) of critical residues in the OCT4 linker may impact on the mode of OCT4-DNA interactions14. In the present study, by systematically deciphering and comparing multiple PTM profiles of OCT4 protein in PSCs and other cellular contexts, we identified OCT4-K222 as a conserved crucial residue in the linker region that can be regulated by reversible methylation/demethylation modification. The demethylation of OCT4-K222 by LSD1 played an essential role in safeguarding the pluripotency of PSCs by restricting the ‘locked-in’ mode binding of OCT4 homodimers and allowing for the transcription of a group of PORE-motif-containing genes (PORE genes).Octamer-binding transcription factor 4   and somatic cancer cells (U87), with the untreated recombinant OCT4 proteins as control. Consistent with the above results, OCT4-K222 was demethylated in the cellular contexts of both ESCs and ECCs, while OCT4-K222 in U87 cells was mono-methylated and untreated recombinant OCT4 was di-methylated Fig. . Given Obona fide demethylase for OCT4-K222. As expected, when the U87 cell lysate was omitted, recombinant LSD1 proteins were unable to demethylate the di-methylated OCT4-K222  to block synthesis of new proteins. CHX treatment revealed that OCT4 proteins in control cells infected with scramble shRNA had a half-life of approximately 4 h which was increased to 6 h by LSD1 shRNA  can form an intramolecular salt bridge with negatively-charged D166 (numbered D29 in the structure) that may help to stabilize the PORE-mode OCT4 homodimers  with DNA probes containing various OCT4-binding motifs. Among all the tested OCT4 variants, the K222D, when present as monomers, had the lowest binding affinity to NANOG- and OCT4-SORE motifs . These OCT4-binding motif-luciferase reporter constructs were co-transfected with OCT4 variant genes into HeLa cells, and the mRNA levels of the transcribed luciferase as well as the OCT4 variants were determined by qRT-PCR. In the first set of experiment where different OCT4-binding motif-luciferase reporter constructs were co-transfected with the WT OCT4 construct only, the level of luciferase transcription driven by the 3MORE was the highest, followed by that of 3PORE, 3MONO, and 3SORE  that only differs the PORE motif (ATTTGAAATGCAAAT) by one single nucleotide  and H1 cells  subjected to similar treatments. The expression of SOX2, NANOG, GATA4, GATA6, NKX3.1 and HES4 was reduced by OCT4-K222F and, to a lesser extent, by OCT4-K222A in H9 cells Fig. D,E, cons22. It is widely used for inducing the differentiation of cultured PSCs into embryoid bodies and neural ectodermal cells. To track transcriptional changes of the PORE genes during RA-induced differentiation of ECCs, NCCIT cells were treated with RA for 0, 3, and 9 days, respectively. Most PORE genes showed time-dependent suppression in transcription , consistent with the assumption that transcription of the PORE genes is positively associated with the self-renewal and pluripotency of PSCs.Retinoic acid (RA) is a vitamin A metabolite that is essential for early embryonic development and promotes stem cell neural lineage specification at relatively high concentrationsWe further investigated the effects of two LSD1 inhibitors, 2PCPA and OG-L002 which presumably increase the OCT4-K222 methylation, on RA-induced differentiation of NCCIT cells. NCCIT cells were pre-treated with LSD1 inhibitors and RA for 1 h followed by adding in CHX and a further incubation for 12 h . Cells treated with the two LSD1 inhibitors showed similar patterns of OCT4 protein degradation and H3K9 methylation . CHX treatment per se appeared to elevate the expression of mesendoderm marker genes . Interestingly, the overall transcription patterns of the lineage-specific markers in NCCIT cells treated with the more potent LSD1 inhibitor OG-L002 were similar to those in H9 cells transfected with the OCT4-K222F mutant, supporting the assumption that OCT4-K222 methylation is likely a common target for these two experimental settings.23, O-GlcNAcylation24, sumoylation25, ubiquitination27, and acetylation28. Although methylation of non-histone proteins at lysine residues emerges to influence their stability and functionality, regulate their interaction with DNA and partner proteins, and prevent other PTMs at the same lysine residues29, systematic identification and in-depth functional characterization of methylated residues in OCT4 proteins is still lacking. In this study, we identified multiple methylated lysine and arginine residues in OCT4 proteins in the cellular contexts of PSCs and somatic cancer cells, and demonstrated that LSD1 can maintain the un-methylated status of the crucial OCT4-K222 residue to prevent the ‘locked-in’ mode engagement of the OCT4 PORE-homodimers and thereby allowing for the transcription of the PORE genes.Accumulating evidence indicates that the protein stability, transcriptional activity, subcellular localization, protein–protein interaction of OCT4 are comprehensively and delicately regulated by a variety of PTMs including phosphorylation30. It represses a variety of genes via demethylation of H3K4me1/me230. In human ESCs, high levels of LSD1 are required to suppress H3K4 methylation at the regulatory regions of differentiation genes and thereby maintaining their undifferentiated state, and downregulation of LSD1 during differentiation can favor the resolution of the bivalent domains towards H3K4 methylation and gene activation17. Interestingly, when forming a complex with androgen receptor, LSD1 changes its substrates to H3K9me2 and can regulate gene activation through demethylation of H3K9me1/me231.LSD1 was the first identified histone lysine-specific demethylase that demethylates H3K4 or H3K9 in a reaction utilizing flavin adenosine dinucleotide (FAD) as a cofactor32, such as p5333, DNMT134, STAT335, E2F136, and HIF1α37. Given the finding that there is a significant overlap of genome-wide distribution of OCT4 binding sites with the LSD1 binding sites17, and OCT4 is associated with different subunits of the CoREST and NuRD complexes40, OCT4 may recruit LSD1 to target genes via direct or indirect interactions. In this study, we demonstrated direct interaction between LSD1 and OCT4 in PSCs, and the methylated OCT4-K222 site can be demethylated by LSD1 both in vitro and in vivo. Recently, it was shown that murine Oct4 can interact with Lsd1 to inhibit its catalytic activity in F9 ECCs, leading to retention of H3K4me1 and a “primed” chromatin state at pluripotency gene enhancers41. It remains to be seen if such inhibitory mechanism is also present in ESCs, and if this may represent a feedback regulation between OCT4 and LSD1.Besides histone family proteins, LSD1 is able to demethylate lysine residues at a growing number of non-histone substratesS domain with POUH domain that functions as a protein–protein interaction interface and plays a critical role during reprogramming by recruiting epigenetic modifiers to OCT4 target genes42. Secondly, it is highly conserved across many species despite its neighboring residues are not conserved. Thirdly, it is the only positively charged residue in the whole linker region. Based on the solved and modeled crystal structures12, the positively charged K222 (numbered as K85 in the cited reference) may be important for forming the intramolecular salt-bridge with the negatively-charged D166 (D29 in the cited reference) to facilitate forming the OCT4 PORE homodimers. Fourthly, although in this study methylation appears to be the only PTM for K222, the information in the PhosphoSitePlus database provided by Cell Signaling Technology, Inc. indicated that murine Oct4-K215 (corresponding to human OCT4-K222) can be ubiquitinated. Therefore, different PTMs may crosstalk at the OCT4-K222 site. For instance, methylation of K222 would be expected to block its ubiquitination, which may well explain our observation that K222 methylation induced by LSD1 ablation promotes the proteasome-independent degradation of OCT4, supporting the notion that methylation increases the stability of proteins by competing with ubiquitination43. As methylation of a lysine residue does not modify the side chain's positive charge43, the above-mentioned intramolecular salt-bridge between K222 and D166 is unlikely to be disrupted by K222 methylation. However, since the phenylalanine to mimic methylated lysine does not carry any charge, K222F mutant is expected to lose the salt-bridge interaction with D166. Nevertheless, our biochemical data indicated that K222F stabilized the OCT4 PORE-homodimers and strengthened their ‘locked-in’ mode engagement onto the PORE genes and thereby continuously suppressed their transcription. Further structural-based studies are warranted to better understand the detailed molecular mechanisms for such a ‘locked-in’ mode engagement. Paradoxically, K222A mutant that has reduced protein stability and diminished binding with the PORE genes also suppressed their transcription to some extent. These seemingly contradicting results may be reconciled by hypothesizing that a dynamic ‘on-and-off’ mode binding of the OCT4 PORE-homodimers to the PORE genes is required for their normal transcription.Among multiple methyl-lysine sites identified in OCT4, K222 is of particular interest for the following reasons: firstly, it is situated at the highly flexible linker region (N213-A229) connecting the POU11, the canonical PORE motif sequence (ATTTGAAATGCAAAT) has been analyzed mainly for its interaction with the OCT4 homodimers structurally and biochemically45. Surprisingly, few studies have been reported to systematically investigate the physiological functions of the PORE genes and their regulation by OCT4 in PSCs. We show here that the PORE genes are mainly involved in cell fate determination, cytoskeleton/cell junction organization, and a variety of related signaling pathways that are crucial for PSC self-renewal. Importantly, we reveal that the ‘locked-in’ mode engagement of the OCT4 PORE-homodimers inhibits rather than activates the transcription of most PORE genes in PSCs, and LSD1 can prevent such engagement by maintaining the un-methylated status of the crucial OCT4-K222 residue . Taken together, we provide evidence in this study that LSD1-mediated demethylation of OCT4-K222 plays a crucial role in safeguarding the pluripotency of PSCs by restricting the ‘locked-in’ mode engagement of the OCT4 PORE-homodimers to maintain the transcription of the PORE genes. These results reveal a novel interplay between an epigenetic regulator and a pluripotency factor, and exemplify how a site-specific PTM of OCT4 critically regulates the pluripotency of PSCs.Since its first characterization over two decades ago47. All cells were maintained in a 37 °C incubator with 5% CO2, and free of mycoplasma contamination based on routine tests every 3 months. H1 and H9 cells were cultured in mTeSR1 medium from STEMCELL . 293 T, HeLa and U87 cells were transfected with GenEscort II Transfection Reagent reagents  as described from WISGEN. H1 and H9 cells were transfected with Lipofectamine 3000 Transfection Reagent  as described in the instructions provide by Thermo-Fisher. In some experiments, cells were treated with 20 μg/ml CHX, 5 μM MG-132, 200 μM Chloroquine, 10 μM Retinoic acid (RA), 100 μM Tranylcypromine (2-PCPA) HCl and 50 μM OG-L002 (DMSO as the vehicle) either individually or in combination.The sources and culture conditions for 293 T, HeLa, HepG2, U87, HT29, LO2, HUVEC, MCF7, H1, H9 and NCCIT cells were described in detail previouslyThe anti-Oct-3/4 (C-10) mouse antibody , anti-Oct-3/4 (H-134) rabbit antibody , anti-LSD1 (B-9) antibody  and anti- Histone H3 (C-16) antibody  were purchased from Santa Cruz Biotechnology. Anti-DYKDDDDK-tag mouse antibody  and anti-DYKDDDDK-tag rabbit antibody , anti-His antibody , anti-GAPDH (HRP) antibody , goat anti-mouse IgG antibody (H&L) [HRP]  and goat anti-rabbit IgG antibody (H&L) [HRP]  were from GenScript. Anti-Myc-tag mouse antibody , anti-Myc-tag rabbit antibody  and di-methyl-histone H3 (Lys9) antibody  were from Beyotime. Anti-pan-methylated lysine(di-/mono-methyl) antibody  was from Abcam. Anti-mouse IgG DyLight 488  and anti-mouse IgG DyLight 594  were from Jackson ImmunoResearch. Cycloheximide  and MG-132  were purchased from Calbiochem. Anti-FLAG M2 Magnetic Beads  was from Sigma-Aldrich. Pierce protein A/G agarose  was from Thermo-Fisher. Ni–NTA agarose resin was from QIAGEN . Chloroquine  was from Sangon Biotech. Retinoic acid (RA)  was from Sigma-Aldrich. Tranylcypromine (2-PCPA) HCl  and OG-L002  were from Selleck.15, which was applied to infect and transfect the cells for 48–96 h as described in detail previously15. The OCT4 variants  were generated from OCT4 WT (His-OCT4 or shOCT4 + POU5F1) plasmids by site-directed mutagenesis and applied to the experiments. The FLAG-OCT4-6His portion was subcloned into the EcoRI and XbaI sites of the pLKO.1-TRC-shOCT4-FLAG-OCT4 plasmid15.The prokaryotic human His-OCT4 and His-SOX2 were generated by PCR and subcloned into the pET28a vector via EcoRI and SacI sites, and His-LSD1 (852 AA) was generated via NheI and XhoI sites. Lentiviral vector was constructed targeting the 3’-UTR of human POU5F1 mRNA to knock down the endogenous OCT4 while expressing the FLAG-tagged exogenous OCT4 WT (referred to as “shOCT4 + FLAG-POU5F1-WT”) was described previously16. 1 mg whole cell lysates (293 T and NCCIT) were incubated with 50 μl anti-FLAG M2 magnetic beads or 50 μl Pierce protein A/G agarose over 8 h at 4℃ respectively. 1 mg of whole cell lysates  was incubated with 100 μl of Ni–NTA agarose resin over 2 h at 4℃ respectively. Immunoblotting and Coomassie Brilliant Blue staining were performed after SDS-PAGE. In most cases, cropped immunoblot images were presented in the main figures, with their uncropped or partly-cropped images being shown in the Supplementary Information.Immunoprecipitation and immunoblotting were conducted as described previously15. Briefly, anti-OCT4 and anti-LSD1 antibodies were applied for immunofluorescence staining and their co-localization in NCCIT cells was determined by confocal microscopy (Carl Zeiss LSM-510).Immunofluorescence staining was conducted as described previouslyEMSAs were carried out using a LightShift chemiluminescent EMSA kit  as instructed by the manufacturer. Briefly, a 20 μl reaction mixture containing 1.5 μg purified recombinant His-OCT4 (WT and variants), 0.5 μg purified recombinant His-SOX2, 50 nM 5’-biotin end-labeled dsDNA probes  and 1 μl Poly (dl·dC) in 1 × Loading buffer was incubated for 30 min at 25℃. The sequences of the probes were as follows:NANOG-SORE probe,TTTTGCATTACAATGGCCTTGGTGAGACTGGTAGACG-3’.Sense:5’-biotin-GTCTGGGTTACTCTGCAGCTACAnti-sense:5’-CGTCTACCAGTCTCACCAAGGCCATTGTAATGCAAAAGTAGCTGCAGAGTAACCCAGAC-3’.OCT4 SORE probe,AGATGCATGACAAAGGTGCCGTGATGGTTCTGTCC-3’.Sense:5’-biotin-CCGTCTTCTTGGCAGACAGCAGAGAnti-sense:5’-GGACAGAACCATCACGGCACCTTTGTCATGCATCTCTCTGCTGTCTGCCAAGAAGACGG-3’.OCT4 PORE probe,ATTTGAAATGCAAATTTGACTGGGCACCCTGTATCTT-3’.Sense:5’-biotin-TATACTAAGCAATTCTTCATTGAnti-sense:5’-AAGATACAGGGTGCCCAGTCAAATTTGCATTTCAAATCAATGAAGAATTGCTTAGTATA-3’.OCT4 MORE probe,ATGCATATGCAAATAACCTGAGGTCTTCTGAGATAAATATA-3’.Sense:5’-biotin-TGTGAAATACCCTGCCTCAnti-sense:5’-TATATTTATCTCAGAAGACCTCAGGTTATTTGCATATGCATGAGGCAGGGTATTTCACA-3’.15. Firstly, total RNA was extracted by RNAiso Plus . Secondly, cDNA was synthesized using PrimeScript RT reagent kit with gDNA eraser  according to the manufacturer’s instructions. Thirdly, qRT-PCR was performed using the iTaq Universal SYBR Green Supermix  in an ABI 7500 Real-Time PCR instrument. All the PCR amplifications were performed in triplicates and repeated in three independent experiments. The relative quantities of mRNAs were normalized by the mRNA levels of the housekeeping gene PBGD (HMBS). The sequences of all RT-PCR primers were listed in Table Quantitative real time PCR analysis and primers used for lineage marker quantitation were as described previouslyThe pGL6-TA vector  was inserted with three tandem repeats of one of the OCT4 binding motifs  via XhoI and HindIII sites. The resulting constructs together with WT OCT4 or OCT4 variant plasmids were co-transfected into HeLa cells, and the luciferase mRNA expression levels driven by the OCT4 binding motifs were determined by qRT-PCR and normalized by the GAPDH mRNA levels. The sequences of the three tandem repeats of OCT4 binding motifs were as follows:TTTTGCATTACAATGTTTTGCATTACAATG3SORE, 5’-TTTTGCATTACAATGATTTGAAATGCAAATATTTGAAATGCAAAT3PORE, 5’-ATTTGAAATGCAAATATGCATATGCAAATATGCATATGCAAAT3MORE, 5’-ATGCATATGCAAATATTTGCATATTTGCAT3MONO, 5’-ATTTGCAT15. After sonication, the 15 ml E. coli lysate was centrifuged at 12,000 g for 10 min at 4℃ and the supernatants were mixed with 1 ml of 50% (w/v) slurry of Ni–NTA Agarose resin/beads incubated at 4℃ on a rotary shaker for 2 h. The mixture was then centrifuged at 800 g for 2 min and the supernatant was discarded. The saving beads were washed with lysis buffer containing 50 mM imidazole and the proteins binding on Ni–NTA beads were used for subsequent in vitro PTMs assay (prokaryotic proteins eluted with the same buffer containing 200 mM imidazole for EMSA experiments).The His-tagged human OCT4 (WT and variants) and SOX2 constructs were transformed into Rosseta E. coli strains and the purification procedures were as described previously16. An aliquot (5 μg) of purified recombinant His-OCT4 protein binding to the Ni–NTA beads (100 μl) was incubated with 1 mg of the whole lysate derived from NCCIT, H1 or U87 cells at 30℃ for 1 h in 1 ml PMA buffer  respectively. 2 μg purified recombinant His-LSD1 protein were added in the demethylation array. The His-OCT4 conjugated beads were sedimented by centrifugation at 800 g for 2 min, washed three times with ice-cold PMA washing buffer , eluted and denatured in SDS–PAGE sample loading buffer by heating at 100 ℃ for 5 min. Pooled samples (10–20 μg) were loaded and separated by SDS–PAGE and stained with Coomassie brilliant blue R250. After de-staining, the OCT4 bands (with a molecular weight of 45 kDa) were excised and analyzed by mass spectrometry.The procedures were largely similar as described previously48. The peptides were extracted with acetonitrile containing 0.1% formic acid and vacuum dried. Proteolytic peptides were reconstituted with mobile phase A (2% acetonitrile containing 0.1% formic acid) and then separated on an on-line C18 column . Mass spectrometry analysis was carried out on an LTQ-Orbitrap Velos mass spectrometer  operated in data dependent scan mode. Survey scan (m/z 375–1300) was performed at a resolution of 60,000 followed by MS2 scans to fragment the 50 most abundant precursors with collision induced dissociation. The activation time was set at 30 ms, the isolation width was 1.5 amu, the normalized activation energy was 35%, and the activation q was 0.25. Mass spectrometry raw file was searched by Proteome Discovery version 1.3 using MASCOT search engine with percolator against the human ref-sequence protein database (updated on 07–04–2013). Phosphorylation of Ser/Thr and Tyr, acetylation of Lys, mono-methylation and di-methylation of Lys and Arg, and di-glycine modification of Lys (ubiquitination) were used as variable modifications. A filter of 90% peptide confidence was applied according to the Peptide-Prophet and Protein-Prophet parsimony algorithms. Fragment assignment of each modified peptide was subject to manual inspection and validation using the original tandem mass spectra acquired in profile mode using Xcalibur software16.OCT4 samples were subjected to overnight digestion with trypsin or chymotrypsin as described by Liu et al.The band intensities of all the immunoblots were quantified by the ImageJ software and presented as means ± SD of triplicate measurements in one experiment representative of three similar ones. All RT-PCR quantitative data were presented as means ± SD of three independent experiments. The statistical significance of normally distributed data was evaluated using the two-tailed unpaired Student's test, and differences were considered significant at *P < 0.05 and **P < 0.01.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.


CDK12 aberrations were found in many other malignancies and have the potential to be used as biomarkers for therapeutic intervention. Moreover, the inhibition of CDK12 emerges as a promising strategy for treatment in several types of cancers. In this review, we summarize mechanisms that CDK12 utilizes for the regulation of gene expression and discuss how the perturbation of CDK12-sensitive genes contributes to the disruption of cell cycle progression and the onset of genome instability. Furthermore, we describe tumour-suppressive and oncogenic functions of CDK12 and its potential as a biomarker and inhibition target in anti-tumour treatments.Cyclin-dependent kinase 12 (CDK12) phosphorylates the C-terminal domain of RNA polymerase II and is needed for the optimal transcription elongation and translation of a subset of human protein-coding genes. The kinase has a pleiotropic effect on the maintenance of genome stability, and its inactivation in prostate and ovarian tumours results in focal tandem duplications, a CDK12-unique genome instability phenotype.  Drosophila (CDK12 is among few recurrently mutated genes in ovarian carcinoma (Cyclin-dependent kinase 12 (CDK12) was discovered as a candidate transcription and splicing machinery component by the Jonathon Pines lab in 2001  is a 1490-amino-acid-long, ∼160-kDa protein consisting of a centrally located kinase domain and intrinsically disordered regions with N-terminal arginine/serine-rich (RS) and central and C-terminal proline-rich (PR) motifs  and is i tissues ,14, indiRNAPII directs the transcription of protein-coding genes in a process consisting of initiation, promoter-proximal pausing, elongation and termination . It contSaccharomyces cerevisiae  and BRD4  antibodies , and by revisiae ,28, Lsk1es pombe ,30, CDK1 elegans  and Drososophila ,33, CDK1and BRD4 ] on at lin vitro kinase assays showed that CDK12 can phosphorylate either Ser2 and Ser5 (in vitro (c-MYC and c-FOS genes (Earlier studies in human cells indicated that longer term (days) CDK12 depletion leads to a modest decrease ,5,11 or and Ser5 ,8,11 or and Ser5 . These ein vitro ,35. Intein vitro , while iOS genes ,37.Recent studies have used short (hours) CDK12-selective inhibition in cells  and P-CTDrosophila transcriptome has a gene-specific rather than global role, in which its targets include NRF2-dependent genes mediating oxidative stress response  rather than affecting global transcription ,45–48. Tresponse . CDK12 aal genes .et al. found CDK12 at promoters and gene bodies of protein-coding genes, largely overlapping with RNAPII occupancies  and other RNA-binding proteins ,35,37,46ocessing . Interesocessing . WhetherSRSF1)  , glial-sIV genes  and 3′ end c-FOS ,37. Howend c-FOS ,45,46. AS. cerevisiae  of mRNAs  Figure . In collS. cerevisiae provided the very first hint that a CycK-associated kinase(s) can regulate cell cycle progression , predominantly those involved in the HR repair pathway  in the tumour background must exist, and might even be essential for tumour onset and/or survival. Notably, CDK12 mutant prostate cancer tumours exhibit synthetic dependence on recurrent gains in several genes involved in the regulation of the cell cycle and DNA replication such as MCM7,CCND1 or RAD9A  leading to tumorigenesis. Interestingly, very recent analyses have revealed the presence of focal tandem duplications in many other cancers with a low incidence (<2%) of CDK12 inactivation  results in the loss of catalytic activity and tumour-suppressive function of the kinase. The mutations of CDK12 in high-grade serous ovarian cancer (HGSOC) are mostly homozygous, indicating that they are driver mutations of a tumour suppressor  at locus Ch17q12 (HER2-positive (amplified) breast cancer  or c-MYC enhancers as a secondary genetic event of CDK12 inactivation  have a non-functional HR pathway due to aberrations in either  pathway . CDK12 w pathway , consistse genes ,115. CDKHR genes , indicatR repair  and sensR repair ,83. In ato PARPi ,109. Dinhibitors . Likewishibitors . In metahibitors .BRCA1/2 mutations  carry aberrations in  pathway ,119,120.ifferent  and dispifferent . This inifferent ,121. Indutations . This suThe MYC family of transcription factors is deregulated in >50% of human cancers. In normal cells, their expression is tightly regulated; in tumours, they are often amplified and overexpressed, and MYC-driven transcription programmes are central drivers of the disease. MYC proteins are considered directly ‘undruggable’ , but theHER2-positive breast cancers are treated with anti-HER2 monoclonal antibodies such as trastuzumab (Herceptin), but >50% of patients develop resistance  regulates various steps of cell cycle progression ,78,81 prCDK12 and focal tandem duplications have increased gene fusion, fusion-induced neoantigen load and T-cell infiltration, suggesting that this new subgroup of mCRPC could also benefit from the treatment -deficient tumours with high neoantigen load and T-cell infiltration is highly sensitive to the therapy ,131. Remreatment . This wareatment  and recereatment . As the  tumours ,134 and  tumours , the CDK therapy .BRCA1-deficient HGSOC cells to metabolic inhibitors . Another important question is whether the inhibition of premature termination by CDK12 is a regulated or merely a passive process. Given the occupancy of CDK12 on gene promoters, its potential role in promoter-proximal pause release and re-initiation remains to be determined. Broader questions of high interest include determining how exactly CDK12-directed gene expression is integrated into the regulation of DNA replication and cell cycle progression as well as determining the mechanism of genesis of focal tandem duplications.Answers to these questions will move us forward in understanding the cellular functions of CDK12. They will likely create fresh avenues towards clinical applications, such as finding new synthetic lethal interactions and its use as a biomarker for treatments of various malignancies.


Regulation of the differentiated identity requires active and continued supervision. Inability to maintain the differentiated state is a hallmark of aging and aging-related disease. To maintain cellular identity, a network of nuclear regulators is devoted to silencing previous and non-relevant gene programs. This network involves transcription factors, epigenetic regulators, and the localization of silent genes to heterochromatin. Together, identity supervisors mold and maintain the unique nuclear environment of the differentiated cell. This review describes recent discoveries regarding mechanisms and regulators that supervise the differentiated identity and protect from de-differentiation, tumorigenesis, and attenuate forced somatic cell reprograming. The review focuses on mechanisms involved in H3K9me3-decorated heterochromatin and the importance of nuclear lamins in cell identity. We outline how the biophysical properties of these factors are involved in self-compartmentalization of heterochromatin and cell identity. Finally, we discuss the relevance of these regulators to aging and age-related disease. How differentiated cells maintain their identity is a fundamental question that researchers have been grappling with since the early days of modern biology. The “textbook view” was that the differentiated identity of cells is determined by sequential steps, and the term “terminal differentiation” was coined as a sign of an irreversible process . It What maintains differentiated cell identity? Conard Waddington once compared differentiation to balls rolling downhill, where the balls (i.e. the differentiating cells) are funneled into specific valleys of irreversible fates , 5. A laThe nucleus plays a central role in identity regulation. Sir J. Gurdon, who conducted nuclear transfer experiments as early as 1962, described the generation of adult frogs by transplanting a single nucleus from a somatic cell into a fertilized egg whose nucleus was removed . Later oIn addition to sequence-specific transcription factors, the regulation of large-scale chromatin and nuclear organization is emerging as crucial for regulation of cell identity. Recent findings, presented below, suggest that cell identity is maintained at the level of high-order chromatin and nuclear organization involving both genetic and epigenetic players as well as biophysical forces that safeguard cell identity. The changes in cell identity supervision during aging and the relevance of these perturbations to aged-related disease are discussed.From a molecular perspective, the differentiated identity is maintained by two parallel mechanisms: one is the machinery that enables the differentiated cell to express its unique gene signature (a topic outside this review), and the second is the machinery that actively silences previous fates and irrelevant gene programs. While the silencing of gene expression also involves post-transcriptional mechanisms, such as RNA decay and protein degradation –21, the Traditionally, the genome is divided into two entities: euchromatin, which is characterized by an accessible and loose chromatin structure that is highly transcribed and expressed, and heterochromatin (HC), which is dense, compact in structure, and less accessible and, therefore, genes within HC are rarely expressed , 23. HC In contrast, fHC has a condensed structure that can change its organization under specific developmental conditions. This flexible form of HC harbors silent genes, and may include regulatory elements, such as lineage and cell-type specific enhancers , 27. A rHC is characterized by specific posttranscriptional modifications and, specifically, by histone tail methylations, including histone 3 Lys 9 di- and tri-methylations (H3K9me2/3). These methylations are catalyzed by evolutionarily conserved histone H3K9 methylases (HMT) the SET-domain-containing family of methyltransferases . Once meOur understanding of the role of H3K9me3 in maintaining the differentiated identity has benefited from experiments of somatic cell reprogramming into iPSs. Reprogramming is an inefficient process, both when achieved by the expression of reprogramming factors (OSKM), and by somatic cell nuclear transfer (SCNT) , 39, 40.suv39h1/2, resulted in increased chromosomal instability and high risk for tumor development. Extensive loss of H3K9me3 across large chromatin regions has been observed in cancer cell lines and cells undergoing epithelial-to-mesenchymal transition [Moreover, during cell specification, marking of chromatin regions by H3K9me3 prevents the expression of irrelevant genes. For example, T helper cells differentiate to either Th1 or Th2 cells, where the expression of Th1-related genes is silenced by H3K9me3 methylation and HP1 binding in Th2 cells. Genetic loss of H3K9me3 methylase (SUv3-9H1) or HP1 resulted in inappropriate expression of Th1 genes in Th2 cells . Similaransition –47. Thesansition . Thus, HDoes H3K9me3-marked HC protect against cell fate conversion (trans-differentiation)? A seminal study by Becker et al. sheds light on this question. As a starting point, heterochromatin was isolated based on its biophysical properties, independent of histone-tail modifications . SpecifiTo directly study the role of srHC/H3K9me-bound proteins in cell conversion, the Zaret laboratory shRNA-eliminated fifty srHC-associated factors upon direct fibroblast conversion to hepatocytes  and the SUMO-conjugating enzyme UBC9 as such factors . CAF1 isC. elegans shed light on the nuclear organization of H3K9me2,3-marked heterochromatin. H3K9 methylations are required In C. elegans to anchor and silence repeat-rich heterochromatin at the nuclear periphery. Only double null mutants of met-2 and set-25 (DKO), which catalyze the methylations of H3K9, were unable to anchor repeat-containing sequences to the nuclear periphery [Recent studies in eriphery . Experimeriphery , 59. ThuC. elegans genome codes for several proteins that are “readers” of H3K9me2, 3  [C. elegans lamin protein (LMN1), and that it binds to mono-, di- and tri-methylated H3K9. When muscle differentiation was forced in the developing larva, the majority of cec-4 mutants failed to fully commit to muscle fate, likely due to the inability to silence other fate programs  mimicking human Emery-Dreifuss muscular dystrophy (EDMD) in a C. elegans model [The , HPL-2) . Using ans model , thus sumet-1 or mes-4  in the cec-4 mutant background resulted in displacement of an artificial repeat reporter from heterochromatin, similar to that observed upon loss of MRG-1.This demonstrates that the positioning of heterochromatin in differentiated cells involves the activity of MRG-1 and H3K36 methylases in euchromatin regions. Interestingly, the shRNA-mediated loss of dMES-4 in differentiated enterocytes (ECs) in the Drosophila midgut resulted in a loss of the differentiated identity of mature enterocytes, a decline in the expression of EC genes, and the ectopic expression of the intestinal stem cell marker Delta on the surface of EC-like cells [In line with these observations, Cabianca et al. studied differentiated intestinal cells and discovered a second indirect machinery safeguarding heterochromatin, in addition to CEC-4-dependent anchoring . A screeke cells .In humans, NSD2, the ortholog of MES-4, has a tumor-suppressive function and is deleted or mutated in Wolf-Hirschhorn syndrome, which involves a cranio-facial phenotype, intellectual disability, and high incidence of leukemias (T-ALL). NSD2 loss is also characteristic of pediatric B cell lymphomas –67. TakeThe formation of repressive chromatin and the regulation of gene expression are tightly connected to the nuclear periphery and to nuclear lamins. Nuclear lamins are type-V intermediate filaments that generate a network in the nucleus, which, in most cases, is dense and associated with heterochromatin below the nuclear membrane, and is sparser in the nucleus interior, where euchromatin dominates , 69. OneLMNA gene and are generated by alternative splicing. Type B lamins are encoded by the lamin B1 and lamin B2 genes. The C. elegans genome contains a single lamin gene and the Drosophila genome encodes a single type A lamin, termed lamC, and a single type B lamin, lamDm0 [Type A lamins (lamin A and C) are encoded by the , lamDm0 . Nuclear, lamDm0 , 75. TypDrosophila LamDm0 to bacterial DNA adenine methylase, which methylates adenine in GmATC sequences [In the nucleus, genomic regions associated with lamins are organized in a distinct manner and form lamin-associated domains (LADs), mainly at the nuclear periphery, where chromatin is in proximity to the nuclear lamina –80. LADsequences –84, thusequences , 85. In equences ).Similar to HC, LADs can be categorized as constitutive LADs (cLADs) and facultative LADs (fLADs) . cLADs aC. elegans, the human PRR14 protein that binds HP1 may be involved in anchoring chromatin to LADs in a phosphorylation-dependent mode. PRR14 has separate HP1-binding and lamin-binding domains and likely links H3K9me2/3 HP1-bound and enriched HC to LADs , together with genetic experiments, suggests the existence of multiple interactions that are only partly dependent on H3K9me . InteresThe regions between LADs, termed inter-LADs (iLADs), are highly transcribed, are associated with active and elongating Pol-II, and are in the vicinity of transcription factories and splicing speckles. The borders of LADs and iLADs are highly enriched with H3K27me3, which is a histone mark associated with fHC and polycomb repression , 77. EarDrosophila cells identified significant binding (~ 20%) of two NPC proteins Nup93 and Nup107 within LADs [A recent study in hin LADs . Nup93-bThe above observations suggest a general role for H3K9 methylations and lamins in the tethering of genomic regions to the nuclear periphery. Elimination of all lamins from mouse embryonic stem cells revealed, however, that they are dispensable for lamina-associated domain organization in cells . While tAmong tissue-specific anchoring proteins, there are nuclear envelope proteins associated with inner nuclear membrane (INM), as well as transmembrane proteins collectively termed NET , suggesting a chromatin-related nutrient-sensing machinery during differentiation .The organization of LamA/C in the nucleus interior is likely different from its tight peripheral association with the nuclear lamina. In the nucleus interior, it is observed microscopically like a “veil” and is easily extracted . This maIn many somatic cells, both types of lamins are co-expressed, with each generating distinct networks that do not mix . During Drosophila tissues. For example, loss of LamDm0 in female-derived fat body cells, led to detachment of testis -specific gene clusters from the nuclear lamina and to their ectopic expression [Drosophila enterocytes (ECs) [Drosophila midgut is differential; in intestinal stem cells (ISCs), the dominant lamin is the B-type lamin, LamDm0. Upon differentiation, LamDm0 levels decline and the level of LamC increases and is the dominant lamin in ECs. The switch in lamin gene expression is directly regulated by the HES-related transcription factor Hey, which binds to enhancers in the lamin genes, repressing the expression of stem-cell lamin, LamDm0, and enhancing the expression of the differentiated LamC [The active role of lamins in supervising the identity of stem cells and differentiated cells was studied in several pression . The actes (ECs) . The disted LamC . GeneticAging is intimately linked to loss of cell identity and is associated with age-related diseases, including increased susceptibility to infection . It is cIn the nucleus, aging is associated with changes in the epigenome, the function of nuclear pores, and large-scale re-organization of the nucleus affecting nuclear lamins and intranuclear organelles , 113. ThDrosophila, where depletion of LamDm0 from the fat body of young adult larvae resulted in decreased HC levels and re-expression of TEs [wash-deficient Drosophila cells exhibit a wrinkled nuclear morphology and disruptions of intranuclear organelles similar to those observed in laminopathic cells. Wash interacts directly with the type B lamin, LamDm0, and is required for cHC integrity. Loss of wash results in increased chromatin accessibility and changes in the distribution of repressive histone marks.These changes are likely due to a decline in the activity and levels of identity supervisors, leading to aging chromatin that is more homogeneous . Among tn of TEs . Similarn of TEs . Moreoven of TEs , 122. NuHow HC is maintained in the context of aging is the subject of extensive studies. At the level of the HC-associated histone tail modification, normal aging correlates with a reduction in H3K9me levels in HC, increase in H3K9me3 outside HC and redistribution HP1 . StudiesFor example, differentiating mesenchymal stem cells (MSCs) derived from Werner syndrome (WRN) patients, a pre-mature aging syndrome, exhibit epigenomic aging phenotypes , includiDrosophila adult midgut and immune tissues. In midgut aged enterocytes, the level of Hey protein decreases, leading to a decline in the level of LamC and, as a result, to the ectopic expression of stem cell genes, including LamDm0, and to subsequent silencing of the EC signature. Indeed, the phenotype observed upon acute loss of Hey in young ECs is highly similar to that observed in aged ECs. Remarkably, expression of Hey, or to a limited extent, of LamC, in aged ECs restores lamin organization and suppressed aging phenotypes [The active role of lamins in supervising cell identity in the context of aging was recently investigated in both enotypes , 128.Drosophila, factors secreted from the fat body (homolog of vertebrate liver) elicit systemic inflammation, as well as hyperplasia in the midgut [Another characteristic of aging is immune senescence, which is associated with activation of immune responses in the absence of a pathogenic challenge . In agine midgut , 131. Ine midgut , 133.The regulation of cell identity by lamins is also conserved in humans. The ability of cells to divide in vitro is limited by a cellular aging process called replicative senescence. Replicative senescence is associated with changes in chromatin organization, cell cycle arrest, and an increase in metabolic activity and cytokine production .Remarkably, loss of lamin B1 in proliferating fibroblasts induced cellular senescence and was accompanied by changes that are highly similar to cells undergoing senescence, including large-scale changes in the chromatin landscape and in gene expression –137. Intlamin A gene (C1824T) that leads to a splice variant of lamin A, which is permanently farnesylated, generating a protein called Progerin, which is the cause of the Hutchinson–Gilford progeria (HGPS), a premature aging syndrome [Numerous mutations in lamin genes and nuclear lamina proteins are associated with a group of diseases collectively termed laminopathies, which affect the muscular, skeletal, adipose, and neuronal tissues, as well as the heart and skin , 140. A syndrome , 142.progerin splice variant are also sporadically present in physiological wild-type aged cells [Interestingly, this single-nucleotide mutation and the ed cells , 144.Analyzing the changes in histone tail modification patterns in HGPS fibroblasts revealed global and rapid reduction of H3K9me3, specifically in the vicinity of the nuclear lamina, along with a decrease in HP1 that binds to H3K9me3 , 145. MoThese data experimentally substantiated the idea introduced by Goetzman and Foisner that laminopathies originate at the level of somatic stem cells, such as MSCs, which give rise to bone, muscle and cartilage, but are manifested upon differentiation and over time . Indeed,Ikagami et al.  recentlyAlong this line, the Foisner group generated an endothelium-specific HGPS mouse model with selective endothelial Progerin expression. These transgenic mice exhibited deregulated activity of a major cardiac transcription factor termed mechanoresponsive myocardin-related transcription factor-A (MRTFA) and developed myocardial and perivascular fibrosis, left ventricular hypertrophy, and premature death .De-regulated and elevated levels of inflammatory markers were observed in a progeria mouse model , whose pMolecular mechanisms were described that regulate HC-dependent silencing involved in the regulation of cell identity. Chromatin partitioning is, however, also determined by the self-assembly of proteins, the biophysical properties of macro-molecules, such as viscous chromatin, and the nucleoplasm environment. These forces determine intra-nuclear domains, nuclear bodies, and the segregation of chromatin loops, including euchromatin and HC. They govern gene regulatory regions, such as super-enhancers, and accessibility of the transcriptional machinery and repressive complexes –160. ForAt the heart of this “self-organization” phenomenon is a process termed phase-separation and formation of localized condensates . One typWhile the above explanations for the establishment and spreading of HP1-dependent HC are attractive, this view was recently challenged . Erdel eDrosophila and Ciona, and TLE/Grg1-4 in vertebrates, are co-repressors that interact with multiple transcription factors, including Hey and other HES-related TFs. Gro/TLE regulates “transcriptional memory” cell fate and identity [Another recent example of LLPS-dependent transcriptional repression involves the developmental co-repressor Groucho (Gro)/TLE 142 . Gro in identity , 170. Gridentity . Treen eidentity . Thus, iCollectively, these pioneering studies suggest that, once determined by genetic factors, the biophysical properties of these proteins and the local nuclear environment self-generate chromatin and nuclear compartmentalization that safeguard cell identity.As postulated by Blau and Baltimore , regulatFinally, observations that epigenomic aging pathways are conserved and that aging phenotypes can be suppressed by re-expression of identity supervisors, are promising –177. Ind


Nuclear Speckles (NS) are phase-separated condensates of protein and RNA whose components dynamically coordinate RNA transcription, splicing, transport and DNA repair. NS, probed largely by imaging studies, remained historically well known as Interchromatin Granule Clusters, and biochemical properties, especially their association with Chromatin have been largely unexplored. In this study, we tested whether NS exhibit any stable association with chromatin and show that limited DNAse-1 nicking of chromatin leads to the collapse of NS into isotropic distribution or aggregates of constituent proteins without affecting other nuclear structures. Further biochemical probing revealed that NS proteins were tightly associated with chromatin, extractable only by high-salt treatment just like histone proteins. NS were also co-released with solubilised mono-dinucleosomal chromatin fraction following the MNase digestion of chromatin. We propose a model that NS-chromatin constitutes a “putative stable association” whose coupling might be subject to the combined regulation from both chromatin and NS changes.Abbreviations: NS: Nuclear speckles; DSB: double strand breaks; PTM: posttranslational modifications; DDR: DNA damage repair; RBP-RNA binding proteins; TAD: topologically associated domains; LCR: low complexity regions; IDR: intrinsically disordered regions. In this context, phase-separated condensates refer to the membrane-less organelles or aggregates of macromolecules (proteins or RNAs) that exhibit liquid droplet like behavior. These condensates are dynamic and the assembly/disassembly processes are highly regulated. The low-complexity regions (LCRs) or intrinsically disordered regions (IDRs) of certain RNA binding proteins promote multivalent weak interactions, that lead to aggregation/separation of such proteins into different ‘phases’ ,2. TheseDNA damage response (DDR) has not been well characterized so far. Moreover, DDR, being a global cellular response, is expected to elicit a DNA-repair centric rewiring of transcriptional modulation via RNA splicing changes following the remodeling of NS. However, strong experimental support in favor of such a model is still not available. We surmise that NS remodeling during DDR is associated with specific RNA splicing changes possibly involving the dynamic association of NS with nuclear chromatin, a model that has not been well tested so far. This posits a direct query on whether NS exhibit stable anchorage on cellular chromatin at all, which is the main focus of the current study. Historically, NS (interchromatin particles) were thought to be assembled in ‘chromatin-free’ regions of the nucleus and were detected by electron microscopic analysis as ‘clouds of RNA’  for 1 hour at room temperature. Immuno-detection of the proteins was performed using the following antibodies Anti-Phosphoepitope SR proteins Antibody, clone 1H4  , Anti-SR protein family Antibody, clone 16H3  and anti-SRRM1 protein , Stat3 , HDAC1 , HnRNP1A , and Histone H3  as these proteins show enrichment in different fractions.For MNase treatment the purified nuclei were incubated with MNase  1 U at 37°C for 30 minutes in a Ca2+ containing buffer . Post treatment, the supernatant was collected and used for DNA purification and Western blotting. Differential salt elution was also performed on MNase treated nuclear pellet as previously described.Nuclei were isolated as previously described and 50ul was stored as the nuclear fraction. The purified nuclei were then suspended in chromatin elution buffer . Such an analysis revealed that genotoxic treatment led to statically significant drop in the number of NS and increase in the speckle particle volume per nucleus in the population of cells as well as purified nuclei. Interestingly, these changes were observed to be reversible as reflected by the return of NS number and volume to normal distribution following 6 hours of cellular recovery (Sup 1a and 1b) suggesting that NS exhibit reversibly dynamic changes in morphology as well as the particle number following genotoxic stress. It appears that any stress in the genome leads to changes in NS organization leading to their particle coalescence into larger particles thereby leading to a drop in the average number of particles per cell as pointed out by previous studies as well.In the current study, we set out to explore the characteristics of NS changes following sublethal doses of double-strand breaks (DSBs) induced by Etoposide treatment in mammalian cells. As a comparison, we also performed Actinomycin D-mediated transcriptional inhibition as another case of genotoxic stress treatment. Transcriptional inhibition and osmotic stress have already been reported to change the NS morphology in several previous studies ,27,28. ISuch chromatin-level changes leading to dynamic alterations in NS prompted us to address whether the NS show any direct association with chromatin. This query was also prompted by the recent findings in the field that NS serve as regulatory hubs for inducible gene expression changes that appear highly tunable to cellular needs . Motivatversus the same in whole cells. Moreover, the NS dynamic changes observed during genotoxic stress treatments (Etoposide and Actinomycin D treatment) in whole-cell imaging were well recapitulated in the images retrieved with purified nuclei as well  interacting with topological associated domains (TADs) . HoweverIn order to test the ‘NS-chromatin’ association hypothesis, we designed a minimally disruptive biochemical strategy : We askevice-versa, limited DNase 1 nicking clearly resulted in large-scale disruption of NS particle phase-separation in the nuclei . We, therefore, believe that the conditions chosen for DNase 1 treatment were mild enough to induce limited nicking in the genome. The role of poly A RNAs and ncRNAs has been well documented in the maintenance of NS integrity , so we uIn order to ascertain that the limited DNAse 1 nicking condition employed in our experiments did not lead to any discernible changes in the nuclear substructures, we probed for Nucleolar morphology using Nucleolin and NPM (B23) as markers, inner nuclear membrane using Emerin marker and the nucleoplasm marker distribution profile using HNRNPA1 protein marker.Nucleoli are highly dynamic and responsive phase-separated condensates, exhibiting stress-dependent changes in their morphology . The DNaInterestingly, inner nuclear membrane morphology was completely intact in DNase 1 treated nuclei, which was indistinguishable from that of control untreated nuclei (Sup. 1j). HNRNP nucleoplasm distribution, marked by HNRNPA1 protein staining was also unaltered by DNase 1 treatment (Sup. 1j). We also tested for NS behavior upon DNase 1 treatment using SON and SRRM1 proteins as additional NS markers, both of which also exhibited dispersal and aggregation upon DNase 1 treatment to varying extent, which was somewhat different from Sc35 staining. Such differences of NS specific markers could arise from their intrinsic differences in protein association with NS versus chromatin. (Sup. 2a-f). All these observations, put together, suggest that limited DNase 1 treatment of purified nuclei showed selective disruption of NS morphology while the overall distribution of other nuclear substructures, namely nuclear membrane, nucleoli, chromatin and nucleoplasm were relatively intact. Poly A mRNAs are also known to regulate NS integrity ,39,40. IHistones and other tightly chromatin-bound proteins are extractable from chromatin scaffold only with high salt treatment . This prWe subjected purified nuclei to a regime of successively increasing salt washes as shown in the schematic . Nuclei One of the simplest biochemical tests of any chromatin-associated factor is its ability to corelease with the soluble fraction following MNase digestion of total nuclear chromatin. We performed this test on isolated nuclei incubated in MNase compatible buffer followed by initiating MNase digestion reaction. MNase is an endonuclease that digests chromatin selectively at the linker regions thereby releasing oligonucleosomes in the soluble chromatin fraction, where an increasing time of enzyme digestion leads to a relatively higher level of mono-dinucleosomes released in the soluble chromatin fraction recovered in the supernatant . MNase dTaken together, all these results point toward an interesting new model  of NS orNS, conventionally and historically, were regarded as interchromatin granules, whose subsequent cell biological characterization unveiled that they were functionally very important intranuclear bodies. Several structural and functional studies related to NS strongly posit that their association with chromatin was very dynamic and supposedly transient ,46 ChromWe believe that our current study is not only novel but also very significant in the field for the following reasons. The current study sets up a simple framework to start rethinking of NS as a novel biochemical entity that is ‘in-continuum’ with chromatin. We strongly believe that the evidence provided in the current study does justify this rethink. In fact, such a rethink does not contradict the existing models of NS as dynamic interchromatin granule structures. Our model  only ten31. G4s are non-canonical four-stranded DNA structures comprising of repetitive guanines (G) forming stable stacked tetrad structures [31. Interestingly, the G4s have been reported in highly active, GC rich euchromatin. The GC rich regions have been implicated for NS formation in earlier studies as well. Jabbari et al. reported that genomic sequence can dictate nuclear body formation, with high GC density positively correlating with NS [The candidates that look attractive in mediating the association between NS and chromatin are possibly RNA (coding or noncoding) associated with chromatin, the histone proteins, even the post-translationally modified histones or some tightly bound adapter proteins. Recent studies have hinted toward possible ‘stable’ NS-chromatin interactions mediated via specialized chromatin structure G-quadruplexes (G4s)ructures . These sructures . Komurko with NS . The ide with NS . Recentl with NS . Direct  with NS . In lighvis-à-vis different states of chromatin  and establish the nature of communication between the changes in NS-proteome versus multiple chromatin functional states. We believe that this aspect of research has been the missing puzzle in the mystery of NS biochemistry.It will be very interesting to systematically probe the association dynamics of NS Click here for additional data file.


Additionally, PRKCA-AS1 was further determined to bind with promoter of PRKCA via 5ʹ terminal and interact with DNMT1 via 3ʹ terminal. Taken together, our results illuminated a novel regulatory mechanism of DNA methylation on regulating PRKCA transcription through lncRNA PRKCA-AS1, and shed light on the molecular pathogenesis of RHD occurrence.In the present study, mitral valve tissues from three mitral stenosis patients with RHD by valve replacement and two healthy donors were harvested and conducted DNA methylation signature on PRKCA by MeDIP-qPCR. The presence of hypomethylated CpG islands at promoter and 5ʹ terminal of PRKCA was observed in RHD accompanied with highly expressed PRKCA and down-regulated antisense long non-coding RNA (lncRNA) PRKCA-AS1 compared to health control. Furthermore, the enrichments of DNMT1/3A/3B on PRKCA were detected by ChIP-qPCR assay  Rheumatic heart disease (RHD) is an autoimmune inflammatory disease responding to group A streptococcal infection, which results in mitral valve damage and hemodynamic changes. RHD remains a health burden in developing countries . Althougde novo methylation in zygotic and embryonic development [DNA methylation as the most extensively epigenetic study is regulated by DNA methyltransferases (DNMTs) family, and controls gene expression without DNA sequence changes. In general, DNA methylation negatively governs transcription activity via impacting with polarity of promoter coordinated with multiple transcription factors and epigenetic regulators . The modelopment . Neverthin vivo and identified protein kinase C alpha (PRKCA) as one of the candidate genes whose DNA methylation is associated with the pathogenesis of RHD [Increasing evidences have reported that abnormal DNA methylation plays an important role in RHD development and progression ,6. Previs of RHD . HoweverIn this study, we take PRKCA for example, and mainly focuses on the regulatory roles of DNMTs in PRKCA in RHD. Our results may provide a common research model of studying a single gene on DNA methylation, and advance the understanding on the epigenetic regulation for RHD pathogenesis.Three RHD patients  who received mitral valve replacement in Yantai Yuhuangding Hospital from April 2016 to May 2018 were enrolled in this study as the following criteria: I) Diagnosis of mitral valve stenosis; II) left ventricular ejection fraction volume >50% and left ventricular end-diastolic diameter <55 mm; III) Antistreptolysin O > 500 U, positive C reactive protein; IV) no history of cardiomyopathy, congenital heart disease, liver disease or renal disease. Healthy mitral valve tissues were obtained from two remains donation by traffic accidents. The average age of participants is 66.85 ± 7.33 year. All the participants and their families understood and signed the informed consent. The documents of signed informed consent and Ethics Committee of Yantai Yuhuangding Hospital were all provided to approve this study. The freshly isolated human tissues were washed by saline and stored in −80°C for the subsequent experiments.6 cells were transfected with plasmids using Lipofectamine 3000  for 24 h. The conditions of small molecular drugs or recombined proteins treatment were listed in Table S1.Human cardiomyocyte-like AC16 cells  and fetaThe levels of biochemical indicators in the serum were determined, including interleukin (IL)-1β  and IL-4  in serum according to the manufacturer’s instructions.6 cells was extracted by phenol chloroform. For ChIP assay [6 cells were fixed with 1% formaldehyde, quenched with 0.125 M glycine at room temperature and then lysed in 500 μl of lysis buffer (10 mM Tris-HCl (pH 8.0), 10 mM NaCl, 0.2% NP40 and 100 U/ml protease inhibitor cocktail) on ice for 30 min, and collected supernatant after 14,000 rpm centrifugation. For RIP assay [6 cells were added 1 ml nuclear isolation buffer , 20 mM MgCl2, 4% Triton X-100) on ice for 20 min for nucleus isolation. After 2500 g centrifugation for 15 min, pellet was resuspended by 1 ml RIP buffer , 5 mM EDTA, 0.5 mM DTT, 0.5% NP40, 100 U/ml RNAase inhibitor and protease inhibitor cocktail).For MeDIP assay , genomicIP assay , tissuesIP assay , tissuesDNA or RNA was mechanically sheared by Bioruptor Pico 60 KHz Sonicator . Ten percent of whole lysate was stored as input, whereas the rest of it was incubated with 1 μg primary antibodies of interest including 5-Methylcytosine , DNMT1  and SMAD2  at 4°C overnight. Additional 2-h pull down by protein A/G beads (Thermo Fisher Scientific) was performed at 4°C with gentle rotation. Beads were washed by the appropriated buffer for three times. Coprecipitated RNA was isolated by RNAiso . Coprecipitated DNA was purified by phenol chloroform methods. Primers designed to encompass approximately 150 bp around the target regions were used to detect the enrichment using qPCR.RNA was extracted using RNAiso reagent, quantified with a NanoDrop (Thermo Fisher Scientific). 100 ng RNA was conversed into cDNA using a reverse transcription kit . PRKCA and PRKCA-AS1 expressions were detected by qPCR.The DNA templates were detected using Fast Universal SYBR Green Real-time PCR Master Mix (Roche) under the following conditions: 95°C/2 min and 50 cycles of 95°C/5 s, 60°C/10 s, 72°C/ 30 s, and 72°C/10 min. GAPDH was used for the loading control. All primers used in this study are listed in Table S2.6 cells were added the 500 µl RIPA buffer (Thermo Fisher Scientific) with protease inhibitor cocktail , and quantified the concentration using BCA methods. Aliquots of proteins (40 µg) were added into the lanes of 10% SDS polyacrylamide gel, and the proteins were separated through electrophoresis and transferred onto nitrocellulose membranes. Subsequently, the membranes were congested with 5% nonfat dry milk in 0.01 M PBS buffer (pH 7.4) and 0.05% Tween-20 for 1 h at room temperature (RT). The blocked membranes were then incubated with primary antibodies of PRKCA , DNMT1 , DNMT3A , DNMT3B  and GAPDH  overnight at 4°C, followed by incubation with the appropriate secondary antibodies  for 30 min at RT. The expression was determined by enhanced chemiluminescence method using Amersham Imager 600 system  whereas the density of the immunoblots was measured with Quantity One 4.62 software .5x10The FISH probes designed for PRKCA labeled with R-Phycoerythrin (excitation wavelength 565 nm) and for PRKCA-AS1 labeled with FITC (excitation wavelength 495 nm) were purchased from G&P Medical Enterprises . Probes sequences can be found in Table S2. 40 μm mitral valve tissues sections or slides filled with AC16 cells washed by PBS were hypotension treatment in 0.075 M KCl at 37°C for 25 min, fixed by pure ethanol for 10 min and dropped on the glass slide, then aged at 56°C for 60 min. The slides were washed by PBS twice and dehydrated with the 70, 85, and 100% ethanol for 3 min in order, and naturally dried. 10 μl probes were added on and mounted immediately. Slides were next denatured 75°C for 5 min and 42°C for 16 h, then removed the cover glass and incubated in 2 × SSC for 5 min and 0.1% NP-40/2 × SSC for another 2 min at 46°C, followed by 70% ethanol at RT for 3 min and dried in dark. 15 μl DAPI were dropped and mounted again. After 10 min incubation in dark, the slides were observed under the fluorescence microscope with the appropriate filters.p-value less than 0.05 was considered to indicate a significant difference.All statistical analyses were conducted in SPSS 20 . For qPCR data, the 2^delta delta method was used to calculate the expression and protein enrichment on DNA. Student’s t-test was used to evaluate differences between groups. A in vivo and in vitro, and further found that the abnormal DNA methylation was ascribed to DNMT1 binding affinity. Antisense lncRNA PRKCA-AS1 was determined to connect and recruit DNMT1 on the promoter of PRKCA. Finally, we further investigated the effect of p38/MAPK pathway on PRKCA-AS1 expression in RHD.To investigate the epigenetic role of PRKCA in RHD, we characterized the DNA methylation pattern of PRKCA in RHD p = 0.021), where DNA methylation of promoter  and the first exon  of PRKCA was significantly lower in RHD compared to health control (p = 0.022) (p = 0.036) (p < 0.05) and exon  were strongly negatively correlated with PRKCA transcription  , which w control . The mRN= 0.022)  and prot= 0.036)  of PRKCAcription . Taken tde novo methylation) have been declared. Next, we investigated which DNMTs were responsible for regulating gene body methylation of PRKCA. Two human cardiomyocyte cell lines AC16 and RL-14 were exposed to 100 ng/mL TNF-α for mimicking the in vitro inflammatory model or direct DNMTs knockdown via RNA interference or 20 ng/mL 5-Azacytidine (DNMTs inhibitor) treatment as a positive control. IL-1β and IL-4 levels in medium were detected by ELISA to confirm the inflammatory effect of TNF-α in cardiomyocyte cells . We observed that PRKCA was up-regulated after TNF-α treatment  or DNMT1 deficiency  or 5-Azacytidine addition  both in AC16  or DNMT1 deficiency  or 5-Azacytidine treatment  compared to negative control and DNMT3A/B knockdown in AC16  and DNMT3A/B  as well as in TNF-α treated AC16  and RL-14 cells  compared to control  and first exon  of PRKCA in vivo and in vitro. As an antisense lncRNA transcribed from the second intron of PRKCA, the potential interaction between PRKCA-AS1 and genomic region of PRKCA was investigated by FISH. Multiple single-strand DNA probes spanning across PRKCA promoter and PRKCA-AS1 introns were designed for PRKCA-AS1 and PRKCA (in vivo (http://www.regulatory-genomics.org/tdf/basic-introduction/) [In general, DNMTs recognize unmethylated CpG loci and transfer methyl group for DNA methylation catalysis. Although DNMTs expression did not change, DNA methylation on PRKCA was weakened after TNF-α stimulation, suggesting an unknown epigenetic regulation coordinated with DNMT1 for DNA methylation on PRKCA. Previous studies have indicated that the antisense lncRNAs can link with DNA and transcription factors, and govern gene expression via chromatin organization ,16,17, w control . Certainnd PRKCA . Althoug indicatp = 0.037) and RL-14 cells  compared to TNF-α treatment  and first exon  of PRKCA were both elevated concurrent with higher DNMT1 occupancy  by PRKCA-AS1 over-expression  and fourth  exons deficiency resulted in the loss of DNMT1 recruitment  and fourth  exons deficiency of PRKCA-AS1 compared to wild type (p = 0.011) and the other truncations  and TGFB1  blocking could enhance PRKCA-AS1 transcription although all these pathways inhibition caused PRKCA reduction against the effect of TNF-α (p = 0.032) and TGFB1  compared to TNF-α   . The outin vitro environment of inflammatory reaction. We observe that both NF-κB and JAK/STAT signaling pathways are all activated, which indicates that the ectopic cytokine stimulators can mimic rheumatism to induce auto inflammation in myocardial cells.The pathogenesis of RHD is the result of valve damage caused by an abnormal immune response consisting of humoral and cellular components after exposure to group A streptococcal infection . The rolin vivo and in vitro. Moreover, we verify that transcription of PRKCA is up-regulated after DNMT1 knockdown, which suggests that the promoter and 5ʹ terminal methylated by DNMT1 contributes to regulating PRKCA transcription, and the other CpG loci on PRKCA showing no significant association with PRKCA transcription may not affected RNA polymerase. In RHD, previous studies reported a controversial phenotype of DNMTs expression [PRKCA has been associated with emotional memory formation, posttraumatic stress syndrome, cancer, and aging. PRKCA as a key regulator of cardiac contractility is reported to be implicated in heart failure risks and treatment outcomes . The suppression ,23. In tAntisense lncRNA regulates the adjacent target gene via RNA-DNA interaction has been determined previously. X-chromosome inactivation by lncRNA Xist is one of the most classic case ,25. BesiAlthough the sequences of PRKCA and PRKCA-AS1 are aligned but fail to find the identical sequence, the substantial interaction is confirmed by FISH assay, which implies that PRKCA-AS1 may recognize a certain DNA motif of PRKCA promoter through a complicate loop structure, and need to be clarified in future study. Moreover, FISH data shows the presence of PRKCA-AS1 transcript in nucleus and indicates that PRKCA-AS1 may bind multiple genomic regions via the similar DNA motif with PRKCA, which can be verified by high-throughput sequencing followed by pull down assay.Finally, p38/MAPK/TGF-β1/Smad signaling pathway is determined to be the reason behind aberrant DNA methylation of PRKCA. The activated p38/MAPK/TGF-β1/Smad is able to suppress PRKCA-AS1, thereby retard DNMT1 recruitment to PRKCA promoter, and modulate DNA methylation and transcriptional repression similarly with a previous regulation model . InteresOverall, our data suggests that PRKCA can be hypermethylated by PRKCA-AS1-meidated DNMT1 through p38/MAPK/TGF-β1/Smad signaling pathway against RHD, which advance the understanding of pathogenesis and progression of RHD, and provide the potential therapeutic targets for RHD.Click here for additional data file.


We identified length-dependent and strand-specific mechanisms that account for CAG slip-out induced transcriptional arrest. We found substantial R-loop formation for the distal transcriptional pausing induced by template strand (TS) slip-out, but not non-template strand (NTS) slip-out. In contrast, Pol II backtracking was observed at the proximal transcriptional pausing sites induced by both NTS and TS slip-out blockage. Strikingly, we revealed that Rad26 and TFIIS can stimulate bypass of NTS CAG slip-out, but not TS slip-out induced distal pausing. Our biochemical results provide new insights into understanding the mechanism of CAG slip-out induced transcriptional pausing and functions of transcription factors in modulating transcription-coupled CAG repeat instability, which may pave the way for developing potential strategies for the treatment of repeat sequence associated human diseases.Transcription induced CAG repeat instability is associated with fatal neurological disorders. Genetic approaches found transcription-coupled nucleotide excision repair (TC-NER) factor CSB protein and TFIIS play critical roles in modulating the repeat stability. Here, we took advantage of an  Genomic instability of ubiquitous simple sequence repeats, such as trinucleotide repeat expansion and contraction, is associated with >50 genetic disorders in humans ,2. In paTrinucleotide DNA repeats have been shown to form slipped-strand, non-B-form secondary structures (slip-out), in which the complementary DNA strands display an out-of-register alignment . CAG repEscherichia coli  pathway to removes transcription-blocking DNA lesions . Intriguin vitro study results for the in vivo repeat instability?Although it has been proposed that the CAG slip-out induced Pol II arrest may trigger TC-NER ,40,41 anin vitro reconstituted system with purified proteins from budding yeast to address these questions. First, we showed that CAG slip-out has a length-dependent and strand-specific effect on Pol II transcription. The TS slip-out structures lead to both proximal and distal transcriptional pausing, whereas the NTS slip-out structures only cause proximal transcriptional pausing. We found that R-loop formation plays a specific role in distal pausing sites of TS slip-out, but not in the proximal pausing sites of the TS or NTS slip-out. In contrast, translocation blockage induced backtracking that correlates with both NTS and TS slip-out induced proximal pausing. Strikingly, we uncovered strand-specific roles of Rad26 and TFIIS in modulating Pol II bypass of CAG slip-out transcriptional barriers. Taken together, our biochemical data provide new mechanistic insights to understand how slip-out structures induce transcriptional arrest and the functions of Rad26/CSB and TFIIS in modulating CAG repeat induced transcriptional arrest and transcription-coupled CAG repeat instability.Here, we used an Saccharomyces cerevisiae 10-subunit Pol II was purified essentially as previously described  to 23°C and subsequent native PAGE purification. Annealing of the oligonucleotide strands resulted in 9-nt sticky ends for ligation to the downstream of the elongation complex. The DNA/RNA sequences used in this study are shown in Preparation of the DNA templates were performed essentially as previously described . Briefly2, 40 mM KCl, 5 mM DTT). The immobilized elongation complex was ligated to the downstream slip-out DNA and washed two times with EB buffer. Next, Rpb4/7 was added to a final concentration of 5 μM and incubated for 10 min at 23°C to generate 12-subunit elongation complex, followed by washing three times with EB buffer to remove excess Rpb4/7.Pol II elongation complex (EC10) was assembled essentially as previously described . First, Transcription reaction was started by adding 1 mM rNTPs  with an additional 3 mM dATP to support Rad26 ATPase activity. The concentration for Rad26 and TFIIS are 100 nM. Reactions were performed at 30°C and allowed to continue for the desired time point and then quenched by adding an equal volume of stop buffer . Samples were denatured for 15 min at 95°C in formamide loading buffer, and the extended RNA was separated from the unextended RNA by denaturing urea PAGE (6 M urea). The gels were visualized by phosphorimaging and quantified using Imagelab software (Bio-Rad). All bands above the ligation truncations  were used in quantification. Bands beneath the run-off bands but above the ligation truncation bands are grouped as ‘blockage bands’ for quantification. The percentage of bypass was calculated as intensity of run off bands/total intensity above the ligation truncation.N = 0) and short slip-out (CAG)2 repeats and obtain run-off transcript . In sharp contrast, we observed persistent strong pausing on template strand containing TS (CAG)20 and (CAG)40 slip-outs. This result recapitulates the effect of CAG repeat expansion seen in its associated patients (N = 0).We previously showed that B-form duplex and non-B form slip-out of CTG•CAG repeat can cause Pol II pausing in a very distinct manner . Strikint Figure  and C, wt Figure . The amot Figure  and C. Fion time  min. In patients . Run-off40 slip-outs, we observed over 90% of Pol II remain in persistent stalling induced by the TS (CAG)40 slip-out structure (at 10-minute time point), whereas we found most of Pol II slowly bypass NTS (CAG)40 slip-out induced pausing and only 28% of Pol II remain stalled after 10-minute incubation. The pattern of CAG slip-out induced transcriptional pausing/stalling is also strand-specific , and low concentrations of TFIIS was added to the system. We found that TFIIS shortens the transcripts corresponding to proximal stalling bands induced by TS slip-outs and almost all stalling bands induced by NTS slip-outs  identified transcription of CTG•CAG repeats induces R-loop formation 40 slip-out t Figure . The addt Figure . Strikint Figure .40 slip-out 40 slip-out template which recapitulates the effect of CAG repeat expansion seen in its associated patients patients ,49. Withpatients . The naspatients . Our finpatients ,54,55,59Transcription factors Rad26, TFIIS, and Spt4/5 have been shown to promote Pol II transcription elongation on naked DNA ,47,60 orImportantly and surprisingly, the effects of the transcription factors on slip-out structures are factor-specific and strand-specific. In this study, we showed that the effect of Rad26 and TFIIS on slip-out induced Pol II transcriptional arrest are strand-specific. Both Rad26 and TFIIS can promote Pol II bypass of NTS slip-out structures, but not TS slip-out structures. We recently reported that elongation factor Spt4/5 along with Elf1 strongly inhibit Pol II bypass of both TS and NTS slip-outs structures, which is in sharp contrast to their canonical roles in promoting Pol II elongation processivity in naked B-form DNA duplex or nucleosome substrates . These f70 fragility (breakage) and a modest 2-fold increase of contraction of (CAG)70 in budding yeast t Figure , they art Figure ,41,64,65t Figure . XPG exct Figure . Interest Figure ,25,59. Wgkab573_Supplemental_FileClick here for additional data file.