Termination of the RNA polymerase III (Pol III)-mediated transcription requires the conversion of an elongation complex (EC) to a pre-termination complex (PTC) on poly-deoxythymidine (dT)-containing non-template strand, a mechanism distinct from Pol I and Pol II. Here, our in vitro transcription elongation assay showed that 5-7 dT-containing DNA template led to transcription termination of Pol III, but not Pol I or Pol II. We assembled human Pol III PTC on a 7 dT-containing DNA template and determined the structure at 3.6 Å resolution. The structure reveals that poly-dT are trapped in a narrow exit tunnel formed by RPC2. A hydrophobic gate of the exit tunnel separates the bases of two connected deoxythymidines and may prevent translocation of the non-template strand. The fork loop 2 stabilizes both template and non-template strands around the transcription fork, and may further prevent strand translocation. Our study shows that the Pol III-specific exit tunnel and FL2 allow for efficient translocation of non-poly-dT sequence during transcription elongation but trap poly-dT to promote DNA retention of Pol III, revealing molecular mechanism of poly-dT-dependent transcription termination of Pol III. Termination of eukaryotic RNA polymerase III (Pol III)-mediated transcription occurs when the polymerase reaches a stretch of four or more deoxythymidine nucleotides (poly-dT) on the non-template strand. Here, the authors present the 3.6 Å cryo-EM structure of a human Pol III pre-termination complex (PTC) that was assembled on a 7 dT-containing DNA template and discuss the mechanism of poly-dT-dependent transcription termination of Pol III. Dysregulation of transcription termination leads to a severe defect in gene expression and causes genetic diseases2.The eukaryotic transcription mediated by RNA polymerases requires tight control throughout transcription initiation, elongation, and termination. As an obligatory step of transcription, termination is critical for proper maturation and release of transcripts, recycle of polymerases, and clearance of template DNA for subsequent transcription6. Despite the well characterized mechanism of prokaryotic transcription termination, it remains incompletely understood how transcription is properly terminated in eukaryotes7. Among the three mammalian RNA polymerases , Pol I and Pol II require multipartite cis-regulatory elements and trans-acting factors to terminate the transcription7. By contrast, termination of Pol III-mediated transcription occurs when the polymerase reaches a stretch of more than three deoxythymidine nucleotides (poly-dT) on the non-template strand (strandNT), suggesting a Pol III-specific termination mechanism13.Distinct from the relatively conserved catalytic mechanism, the process of transcription termination seems to be more diversified among RNA polymerases across species12, a key process of transcription termination. It has been reported that poly-dT in non-template strand initiates the termination of transcription-independent of other cis-regulatory elements or trans-acting factors. The average lengths of the poly-dT in canonical Pol III termination sites vary across species, with an average of 5–7 dTs in Schizosaccharomyces pombe (S. pombe), 6–9 dT in Saccharomyces cerevisiae (S. cerevisiae), and 4–5 dT in vertebrates. This unique termination mechanism is beneficial to the functions of Pol III, which transcribes a large amount of short essential transcripts15 and requires high efficiency during the transition from termination to reinitiation17.In Pol III-mediated transcript, the productive elongation complex (EC) is converted into a transcriptionally active but metastable pre-termination complex (PTC)23. Previous studies reported the structures of yeast and human Pol III and the mechanisms of transcription initiation and elongation29. The structures revealed that the catalytic core of Pol III is similar to that of Pol I and Pol II, consistent with its conserved function in transcription elongation19. The three RNA polymerases also contain distantly related stalk subunits, A14/43, Rpb4/7, and C17/25 in yeast Pol I, Pol II, and Pol III, respectively. In addition, Pol I contains TFIIF-like heterodimer A49/34.5 and Pol III contains TFIIF-like heterodimer C37/53 and TFIIE-like heterotrimer C82/34/3118. These peripheral subunits play a regulatory role in transcription initiation and termination by directly interacting nucleic acids and/or involving in protein–protein interaction network19. The termination-reinitiation subcomplex (RPC4-RPC5-RPC10) of Pol III plays a critical role in regulating transcription termination32. Previous studies also showed that termination-defect mutations were mapped to residues 300–325 and 455–521 of the yeast RPC2 subunit. Despite these studies, it remains largely unknown how such a stretch of poly-dT on strandNT could lead to effective transcription termination on Pol III but not on Pol I or Pol II.Human Pol III consists of 17 subunits that are organized into a catalytic core , a stalk module (RPC8-9), peripheral trimer  and a dimer module (RPC4-5)21, we reconstituted a mimetic PTC complex and determined the cryo-electron microscopy (EM) structure at 3.6 Å resolution. The structure indicates that poly-dT in strandNT is trapped in a non-template strand exit tunnel (termed exit tunnel below). Translocation of this poly-dT is unfavorable due to the conformational restrain by the exit tunnel and the stabilization of the transcription fork. Our study provides structural insights into the transition from transcription elongation to pre-termination and reveals the molecular mechanism of poly-dT-dependent transcription termination of Pol III.Following our recent structural study of the human Pol III EC complex21  substrates to a final concentration of 1.25 mM each. As expected, Pol II could read-through all the TS1 templates without early termination  is paired with the template strand, undergoes a sharp kink between T+2 and T+1, is trapped within the exit tunnel , and makes a slight kink and flanks out of the exit tunnel (T−3 to T−4)  near the active site Fig. . The temNT G+8 to C+3 4) Figs. .Fig. 2Bi22, consistent with the previous findings that PTC is transcriptionally active12, albeit exhibiting the decreased elongation rate35. Structural comparison of the PTC and EC reveals a more compact core of PTC than that of EC and substantial conformational differences outside the core complex, suggesting the following modular reorganization during the transition from EC to PTC  on one side, the fork loop 1 (FL1) on the other side, and a part of the protrusion on the bottom  faces toward the hydrophobic surface of the protrusion whereas the acidic side of poly-dT (multiple phosphate groups) faces toward the positively charged surface of the lobe  of FL2 is in parallel with the phosphate backbone around T+2 and guides the strandNT to run toward the exit tunnel. Residue R463 stacks with the base of T+2 of the template strand. Residue K461 stacks with the base of T+2 of the strandNT. Residue F459 under the T+2 supports the ribose groups of T+2 and T+1. The strandNT makes a sharp kink over residue F459 by ~90° with the upstream region running toward the exit tunnel  Fig. . The FL2el Figs. . Thus, t37  is located proximal to and makes contact with the 5′ terminus of poly-dT  of C37 (RPC5 counterpart), which was termed a hotspot in decreased termination mutants in vivo38.In yeast, the termination-reinitiation subcomplex C53-C37-C11  is believed to regulate Pol III transcription termination. The Pol III devoid of this subcomplex has no decrease in transcript output but requires more dTs in termination, indicative of a deficient termination22  of RPC10 in the PTC structure binds the polymerase core and may support binding of RPC4-RPC5 to the core enzyme  with the polymerase.It is known that RPC10 is essential for the termination but is independent of its RNA cleavage activityyme Fig. . The C-rPTC Fig. . PreviouΔT scaffold). Pol I and Pol II could read-through 5S rDNA scaffold  in the non-template strand (Supplementary Table 40 and yeast Pol II EC (PDB: 5C4J)37, which both contain visible nucleotides in equivalent strandNT tunnels. Compared to Pol III EC/PTC structures, the Pol I EC structure40 reveals a nearly closed tunnel  through the interaction between the counterparts of the FL1 and a loop of the lobe 42 and our recently reported Pol II preinitiation complex structure (PDB: 7EG7)33 also reveal similar conformation of FL1 to that in the yeast Pol II EC. In agreement with the structural difference, sequence alignment of FL1  In Pol III-mediated transcript elongation, regular strandNT has no specific contact with the exit tunnel and easily passes through the open exit tunnel, permitting an efficient strand translocation. (II) The poly-dT of the strandNT enters the exit tunnel and tends to generate specific intermolecular contacts and induce closure of the exit tunnel, which in turn stabilizes and traps the strandNT. (III) The methyl groups of the poly-dT face toward the partially closed hydrophobic gate, possibly generating hydrophobic repulsion and a higher energy barrier to prevent the translocation of the strandNT. (IV) The FL2 stabilizes the transcription fork and may prevent the translocation of both non-template and template strands. As a result, the Pol III-mediated transcription elongation tends to be paused at the poly-dT region and the DNA retention of Pol III may correlate with the number of tandem deoxythymidines  may facilitate the EC to PTC conversion and/or efficiency of termination in cells.We assembled the memetic PTC in an in vitro system using DNA–RNA hybrid in the presence of poly-dT in the strand13. Despite considerable changes in the exit tunnel and associated regions, the formation of PTC unlikely affects the polymerase stability. The core region of PTC remains largely unchanged when compared to the cryo-EM structure of the human Pol III EC, with a root-mean-square deviation (RMSD) of 0.74 Å for 1881 Cα atoms  sequence in the nascent RNA.It has been proposed that the generated rU:dA hybrid at Pol III active site is unstable and the RNA product tends to dissociate from the PTC, possibly facilitating the release of RNA and transcription terminationS. cerevisiae C11 mutant. The resulted core polymerase has no decrease in transcript output but requires more dT (8–9 dTs) to terminate transcription30. Comparison of our human Pol III EC and PTC structure suggests that RPC4-RPC5 and N-ribbon of RPC10 directly interact with the RPC2 lobe. By stabilizing the RPC2 lobe, RPC4-5 may indirectly facilitate trapping poly-dT effectively. In the PTC map, we have not observed any additional density of the RPC5 C-terminal domain that is specific to mammals. This indicates that the human-specific C-terminal domain of RPC5 may have no direct role in trapping of poly-dT although we can’t fully exclude this possibility. During the transition from EC to PTC, the unchanged position of this subcomplex to RPC2 lobe indicates that the “termination-reinitiation” subcomplex may play a regulatory role in the formation of the non-template exit tunnel. Depletion of the “termination-reinitiation” subcomplex may destabilize the lobe and impair the termination gate formation. Thus, more tandem deoxythymidines are required for termination. An N-terminal loop adjacent to the hotspot of RPC5 is referred to as the “termination-reinitiation” loop and may play an important role during the transition from termination to reinitiation, the mechanism of which requires further investigation.The yeast “termination-reinitiation” subcomplex C53-C37-C11  can be dissociated from Pol III during purification from an 44. Mammalian Pol II was isolated from pig (S. scrofa) thymus as indicated in our recent studies34. Four residue differences  exist between S. scrofa and H. sapiens Pol II34. Protein purification of human Pol III was performed as previously indicated21.Human Pol I was purified from transient-transfected HEK Expi293F cells as indicated in our recent report2, 2 mM DTT, and 1.25 mM NTPs . Polymerase was first assembled with dsDNA–RNA hybrid on ice for 30 min. 4× assay buffer and 4× NTP solution were added to the polymerase-scaffold complex to initialize the reaction. Transcription assays were performed at 37 °C for 30 min and stopped by a 2× quenching buffer  in a 1:1 ratio. The RNA products were analyzed on 10% acrylamide-urea gels. About 4 μl sample was loaded to each lane. The gels were run with a power of 15 W for 70 min in 0.5 × TBE buffer. RNA signal was detected by scanning the fluorescence of the 5′-FAM label on the RNA primer.RNA extension assays were performed to test the termination activity of polymerases on human 5S rDNA and variants based on scaffold used for structural investigation in this study. A short 5′-FAM-labeled RNA primer 5′-FAM-GUGCUGACU was used for fluorescence detection in all assays. The DNA sequences used for RNA extension assays are listed in Supplementary Table 21. Given that PTC is transcriptionally active, to capture the pretermination state, we designed an elongation scaffold that contains 12 mismatched nucleotides and seven deoxythymidines in the non-template strand. Template DNA: 5′-GAAACAGCGGCTCTAGTCTGTCCAGCACCCGTAGCACCCGGTATTCCCAGGCGG-3′, non-template DNA: 5′-CCGCCTGGGAATACCGGGTGCTTACGATTTTTTTCAGACTAGAGCCGCTGTTTC-3′, and RNA: 5′-CCGGGUGCUG-3′. The Pol III PTC complex was incubated with fresh prepared DNA–RNA scaffold on ice for 30 min at a 1:2 molar ratio. The sample was dialyzed in buffer containing 20 mM HEPES, pH 8.0, 150 mM NaCl, 2 mM MgCl2, and 2 mM DTT at 4 °C for 12 h using Slide-a-lyzer mini dialysis pins .The PTC was assembled essentially similar to our previously reported reconstitution of human Pol III EC2 mixture for 15 s using a Gatan 950 Solarus plasma cleaning system with a power of 15 W. The negatively stained grids were loaded onto a Thermo Fisher Scientific Talos L120C microscope equipped with a Ceta CCD camera and operating at 120 kV at a nominal magnification of 92,000x, corresponding to a pixel size of 1.58 Å on the specimen.For negative staining EM grids preparation, 5 μL of Pol III PTC complex sample were applied onto glow-discharged copper grids supported by a continuous thin layer of carbon film for 60 s before negatively stained by 2% (w/v) uranyl formate solution at room temperature. The grids were prepared in the Ar/O2/O2 mixture for 30 s using a Gatan 950 Solarus plasma cleaning system with a power of 5 W. The ø 55/20 mm blotting paper (TED PELLA) is used for plunge freezing.For cryo-EM grids preparation, 3 μL of the sample at a concentration of 0.7 mg/mL dialyzed PTC complexes were applied to freshly glow-discharged Quantifoil R 1.2/1.3 holey carbon grids. After incubation of 5 s at a temperature of 4 °C and a humidity of 100 %, the grids were blotted for 8.5 s in a Thermo Fisher Scientific Vitrobot Mark IV and plunge-frozen in liquid ethane at liquid nitrogen temperature. The grids were prepared in the H45 with a nominal magnification of 130,000x in the EFTEM mode, which yielded a super-resolution pixel size of 0.527 Å on the image plane, and with a defocus ranged from −1.3 to −2.3 μm. Each micrograph stack was dose-fractionated to 36 frames with a total electron dose of ~50 e−/Å2 and a total exposure time of 8.2 s. About 3125 micrographs from a total of 3400 micrographs were selected for further processing.The cryo-EM grids of PTC were loaded onto a Thermo Fisher Scientific Titan Krios transmission electron microscope equipped with a Gatan GIF Quantum energy filter (slit width 20 eV) and operating at 300 kV for data collection. All the cryo-EM images were automatically recorded by a post-GIF Gatan K2 Summit direct electron detector in the super-resolution counting mode using Serial-EM45 and binned twofold to a calibrated pixel size of 1.054 Å/pix. The defocus values were estimated by Gctf46 from summed images without dose weighting. Other procedures of cryo-EM data processing were performed within RELION v3.0 and 3.147 and cryoSPARC v248 using the dose-weighted micrographs.For cryo-EM data, drift and beam-induced motion correction were applied on the super-resolution movie stacks using MotionCor2A subset of ~10,000 particles were auto-picked free of reference and subjected to reference-free 2D classification. Some of the resulting 2D class averages were low-pass filtered to 20 Å and used as templates for automatic particle picking of the whole dataset in RELION v3.0 resulting in an initial set of 1,771,522 particles for several rounds of 2D and 3D classification. About 848,562 particles were selected from good classes for further 3D classification. After two rounds of 3D classification, 148,433 particles were subjected to a masked 3D classification for the whole particle without alignment. Two classes showed distinct features of single-stranded DNA between Protrusion and lobe were subjected to 3D homogeneous refinement in cryoSPARC, respectively. One class  yielded a reconstruction (PTC) with a nominal global resolution of 3.6 Å. This map shows the continuous density of single-stranded DNA, including termination signal residues, extended from the unwinding site (transcription fork) to the junction position, while RPC10 is an outside funnel. The rest of the class  was reconstructed using the same strategies and yielded a reconstruction (EC) with a nominal global resolution of 3.6 Å. In this map, there is no apparent density of single-stranded DNA in the same position compared with that in PTC. Interestingly, there is a stronger signal belonging to the RPC10 C-ribbon inside the funnel. To improve the map quality in the core region of PTC, a local mask 3D classification and a masked local refinement were performed. This refinement excludes the stalk module and trimer module due to intrinsic mobility. About 131,442 particles in one of the selected three classes were selected and subjected to 3D auto-refinement, CTF refinement, Bayesian polishing, and post-processing in RELION v3.1 yielding a reconstruction of the core region (PTC core) at 3.3 Å resolution.https://3dfsc.salk.edu)49. All the visualization and evaluation of the 3D volume map and mask creation were performed within UCSF Chimera50. The local resolution variations were calculated in cryoSPARC v2 for PTC and EC and in RELION v3.1 for PTC core.All of the reported resolutions were based on the gold-standard Fourier shell correlation (FSC) = 0.143 criterion. The FSC curves were corrected for the effects of a soft mask with high-resolution noise substitution. All cryo-EM maps were sharpened by applying a negative B-factor estimated in cryoSPARC or RELION. Directional FSC curves and map anisotropy were assessed using the 3DFSC  and UCSF ChimeraX54.The structure of human Pol III EC (PDB: 7DU2) was used as starting structural template, which was docked into PTC core map by rigid-body fitting by UCSF Chimera. The model was then manually adjusted in COOTFurther information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Reporting summary


The ubiquitous host protein, CCCTC-binding factor (CTCF), is an essential regulator of cellular transcription and functions to maintain epigenetic boundaries, stabilise chromatin loops and regulate splicing of alternative exons. We have previously demonstrated that CTCF binds to the E2 open reading frame (ORF) of human papillomavirus (HPV) 18 and functions to repress viral oncogene expression in undifferentiated keratinocytes by co-ordinating an epigenetically repressed chromatin loop within HPV episomes. Keratinocyte differentiation disrupts CTCF-dependent chromatin looping of HPV18 episomes promoting induction of enhanced viral oncogene expression. To further characterise CTCF function in HPV transcription control we utilised direct, long-read Nanopore RNA-sequencing which provides information on the structure and abundance of full-length transcripts. Nanopore analysis of primary human keratinocytes containing HPV18 episomes before and after synchronous differentiation allowed quantification of viral transcript species, including the identification of low abundance novel transcripts. Comparison of transcripts produced in wild type HPV18 genome-containing cells to those identified in CTCF-binding deficient genome-containing cells identifies CTCF as a key regulator of differentiation-dependent late promoter activation, required for efficient E1^E4 and L1 protein expression. Furthermore, our data show that CTCF binding at the E2 ORF promotes usage of the downstream weak splice donor (SD) sites SD3165 and SD3284, to the dominant E4 splice acceptor site at nucleotide 3434. These findings demonstrate that in the HPV life cycle both early and late virus transcription programmes are facilitated by recruitment of CTCF to the E2 ORF. Oncogenic human papillomavirus (HPV) infection is the cause of a subset of epithelial cancers of the uterine cervix, other anogenital areas and the oropharynx. HPV infection is established in the basal cells of epithelia where a restricted programme of viral gene expression is required for replication and maintenance of the viral episome. Completion of the HPV life cycle is dependent on the maturation (differentiation) of infected cells which induces enhanced viral gene expression and induction of capsid production. We previously reported that the host cell transcriptional regulator, CTCF, is hijacked by HPV to control viral gene expression. In this study, we use long-read mRNA sequencing to quantitatively map the variety and abundance of HPV transcripts produced in early and late stages of the HPV life cycle and to dissect the function of CTCF in controlling HPV gene expression and transcript processing. Human papillomaviruses (HPVs) are a family of small, double-stranded DNA viruses that infect cutaneous and mucosal epithelia. Most HPV types cause benign epithelial hyperproliferation, which is usually resolved by host immune activation. However, persistent infection with a subset of HPV types e.g., HPV16 and 18) is the cause of epithelial tumours including cervical and other anogenital cancers, and carcinoma of the oropharyngeal tract  A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Paul Francis LambertAssociate EditorPLOS PathogensKarl MüngerSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064***********************Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: Ferguson et al. describe a long-read RNA-seq analysis of HPV transcripts using cells that maintain wt HPV 18 genomes in comparison to those with mutated CTCF sites in E2. Previous studies by this group showed that that loss of CTCF binding at this sE2 site results in increased early transcripts, altered splicing patterns and changes in chromatin factors. In the present study the authors use long-read RNA seq to extend the analysis to late transcripts upon differentiation. Mutation of the CTCF site reduces late E1^E4 transcripts by 50% with slightly greater decreases in protein levels. The most significant effect of mutating CTCF site is alteration in splice site utilization along with substantially reduced levels of H4Ac histone modifications.This study provides useful information that extends the authors previous findings on altered splice site usage and epigenetic modifications of the HPV early region with the CTCF mutant genome. The manuscript could benefit from a more mechanistic analysis of how loss of CTCF binding leads to altered splice site choice and changes in histone modifications.Reviewer #2: This manuscript hones in on the understanding of HPV early and late gene regulation. The group has previously identified that the genome-organising protein CTCF is involved in forming a chromatin loop that regulates HPV gene expression. The group uses Nanopore sequencing to understanding HPV early and late transcriptional regulation that appears to be regulated by the presence of a CTCF binding site in the E2 ORF. CTCF is emerging as a new player in regulating alternative splicing via co-transcriptional, genomic and epigenetic mechanisms. Even more recently CTCF has been shown to be involved in regulating splicing in different viral genomes. This is a timely study and uses long-read sequencing to quantify the contribution of CTCF in alternative splicing regulation and transcriptional control in HPV18. Figure 3 is particularly impressive and the authors should be commended. However, there are some deficiencies in results, data analysed and mechanistic understanding that need to be addressed or expanded.Reviewer #3: Manuscript by Joanna Parish, et al. describes the chromatin insulator CTCF regulates HPV18 transcript splicing and differentiation-dependent late gene expression. The authors in this report first found a CTCF binding site in the N-terminal transacting active domain of E2 and then compared viral gene expression in HFKs transfected with recirculated wt HPV18 or �CTCF-HPV18 DNA initially by RNA-seq and then by Nanopore-seq (long reads sequencing) analyses using mRNAs extracted from the transfected HFKs. The authors found that the cells with �CTCF-HPV18 DNA transfection exhibited a much high level of viral early gene expression from P102 promoter by RNA-seq analysis . However, the data from Nanopore-seq did not show the similar profile with the enhanced expression of viral early genes in HFKs transfected with �CTCF-HPV18 DNA . Although most viral splicing profiles and promoter usage were confirmative and the observation of CTCF binding to the E2 region to regulate viral genome expression is attractive, the data provided in this report are not conclusive, and more confirmation experiments are needed to draw a definite conclusion. The data in RNA-seq and Nanopore-seq appear to be compiled from only a single sample each in wt and �CTCF-HPV18 genome transfection. To avoid sample bias and outliers and batch effects in RNA-seq and Nanopore (long reads sequencing), minimal three RNA samples in high quality in each group are needed for statistical analysis. Like any other methods, the authors should also provide the perimeters or cutoff values used to count the real splice-junction reads and their long reads. Most importantly, introduction of point mutations to disrupt the CTCF binding site in the N-terminal transacting domain of E2 should not affect E2 protein translation and E2 function. Thus, functional E2 data from the �CTCF-HPV18 DNA-transfected HFKs should be provided to ensure that CTCF, not the E2, regulates viral promoter activities.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: 1). CTCF was shown to interact by looping with YY1 in the URR. Does mutation of the YYI site in the LCR recapitulate mutation of the CTCF site in E2 on late splicing effects? YY1 levels decrease upon differentiation so it is unclear how loss of CTCF looping affects late events. Could the effects on late splice site choice be mediated by altered levels of an early gene product that is affected by CTCF YY1 looping?2). Is the 50% reduction in E1^E4 transcripts in the CTCF mutant the result of altered differentiation dependent replication or changes in cell cycle? Southern analysis should be performed to look at genome levels upon differentiation with the CTCF mutant genomes. Does the previously reported increase in E6/E7 transcription upon CTCF site mutation affect induction of late functions? Is reduced E1^E4 responsible for altered splicing patterns? Is there a difference in how CTCF affects splice site choice in the early region as opposed to late?3). The reduction in H4Ac is interesting but the effects only reduce late promoter activity by 50%. Is there an increase in repressive histone marks or other modified histones on the late or early regions?Reviewer #2: 1. Nearly one-third of the abstract is reporting previously published results. The focus should be more on the current study, the results and the implications/significance.2. More details should be provided of the E2 ORF CTCF binding site. This should be given a precise location (other HPV genomic features are) rather than a generic location. A sequence and co-ordinates should be provided as well as an alignment with the CTCF core consensus motif.3. ChIP sequencing nicely identified differences in binding to WT vs CTCFmutant HPV in infected keratinocytes. However, no changes in host CTCF genome-wide binding were mentioned. These should be analysed and described (at least in Supplementary data).4. In Figure 1B what differences in host gene expression was observed? If there were changes in gene expression in HPV genome due to the deletion of the CTCF sites, then changes in host gene expression would also be expected. Please detail.5. Examples of differentiation-dependent host cell gene expression in HPV-infected keratinocytes were provided (IVL and EMC1) but this was limited at best – cherry picking at worst. More genes need to be examined to create an overall picture. This could be in Supplementary.6. Line 315: ‘Viral host fusion transcripts’ – Is there evidence, if any, that an absence of CTCF binding site in HPV genome could affect HPV viral integration? Loss of a CTCF site could affect episomal folding and/or association with the host genome and/or viral integration. Please comment in Discussion.7. Why was H4 acetylation examined as opposed to other histone marks? This is not made clear.8. The confirmation of differentially expressed HPV proteins by Western blot is a bit limited  and only E1^E4 is shown. This reviewer would have more confidence in CTCF’s role in regulating late viral gene expression if more HPV proteins are examined. If possible, a viral protein that is predicted to remain unchanged (based on Nanopore sequencing) should also be included. E6 and E7 should be examined.9. Figure 7A: The authors had previously shown CTCF expression increase upon HPV transduction. However, this has not been yet shown for differentiation. For completeness, total CTCF protein levels should also be examined.10. A mechanism by which CTCF regulates alternative splicing is via the ‘roadblock model’ by pausing RNAPII elongation. Is RNAPII enriched at the HPV CTCF site during keratinocyte differentiation?11. Are cohesin subunits eg RAD21 enriched at the HPV CTCF site and enforcing the CTCF-mediated chromatin loop?12. By the authors own review (Ref 10), HPV episomal genome can be methylated. Is there any evidence that the E2 ORF CTCF binding site or other CTCF sites can be impacted by methylation. This is important as this could account for lack of CTCF binding during the HPV viral cycle. The authors should conduct COBRA or clonal bisulfite sequencing of the E2 ORF and other possible CTCF sites.Reviewer #3: Fig. 1.Lines 271-274 and Fig. 1A. Please show the details of the mutated sequences in the mapped CTCF binding site (nt 2960-3020) which is positioned at the N-terminal domain of E2. Hope the mutated sequences in the CTCF binding site are neutral  and most importantly, will not inactivate the functional transactivation domain of E2, with normal E2 protein production.Lines 280-285 and Fig. 1B. The increased splicing of 233^416 was most likely resulted from increased expression of delta CTCF genome because total viral reads were all increased across the entire virus genome! How did the authors confirm the same amount of recirculated wt and mt HPV18 DNA being transfected into the HFKs? Some quantitative verification data on input viral genome copies in transfected cells are needed at the beginning of the study.Fig. 2C. How many repeats do you have? Is this just one sample data or one representative of three qualified Nanopore-seq? Plus, all RNA-seq raw data should be submitted to NCBI Geo before manuscript submission.Fig. 3.Lines 327-336 and Fig. 3 transcriptome summary. Additional methods should be applied to verify “novel transcripts” identified from this single sample study.Lines 337-339. Not sure about E2^E4L from the reported study (reference #38) but should be verified too in this report.Fig. 3. HPV18 transcripts of wt vs CTCF mt in HFKs under undifferentiated and differentiated conditions: As the reads are so low (many are less than 5 reads per million) in so called “new transcripts”, how did we know these splice-junction reads were not background noise reads. What were the bioinformatics perimeters and thresholds used in the analyses? Where are the verification data on these new transcripts? Obviously, the late transcripts are so low due to poor HFK differentiation under methylcellulose condition where the RNA was prepared for RNA-seq or Nanopore-seq.Plus, all Nanopore-seq raw data should be submitted to NCBI Geo to obtain a Geo Accession number for manuscript inclusion and submission.Fig. 4 and Fig. 5. Because the RNA 5’ cap prevents linker ligation, RNA-seq and Nanopore-seq can’t be used to precisely map a transcription start site. What the authors’ reads counts in the two figures are the reads counts downstream of a real TSS.Fig. 4B. Since only one or two reads were identified at each nt position in the virus genome, it would be better to delete those reads or state clearly in text that the biological functions of these rare TSSs are minimal.Fig. 4D. If possible, please verify the promoter P3000 TSS by 5’ RACE.Line 410-411 and Fig. 5. Need to be careful on the statement only until the authors ensure a functional E2 expression from �CTCF-HPV18 virus and the HFKs receiving the same amount of wt and mt HPV18 DNA. What are reads-scales in the Fig. 5A and 5B? To this reviewer, both the P102 and the P811 promoters appear more active from the �CTCF-HPV18 virus than wt HPV18 virus in differentiated HFKs, despite that the authors claim only the P102, but not the P811, being more active in the differentiated HFKs. The difference in Fisher’s test must be showing more transactivation in �CTCF-HPV18 virus than wt HPV18.Fig. 6 and Fig. 7.Fig. 6. Please make the annotated genome positions in scale to relative H4Ac positions. Where is your Fig. 6B (lines 24-426)?Line 429. Not sure this statement is correct on reduction of P811 promoter activation from �CTCF-HPV18 virus in differentiated HFKs .Fig. 7D and 7E. Now we can see the sample variations when three biological samples were examined. Thus, the same principle should be applied to all RNA-seq and Nanopore-seq for the data shown in Figs. 1-5.**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: (No Response)Reviewer #2: 1. Line 134: splicing2. Include antibody dilutions or amounts used in each application3. Include amplicon size in Table 14. It is not obvious how images  were scored or analysed. Was it done by image analysis or by a researcher blinded to the experimental arm?5. A scale and 0- Z(?) x-co-ordinates should be provided on each HPV plot. At a suggestion, the CTCF site should be indicated with a black triangle or similar on Figure 1B & 6.6. Figure 1 ‘wild type’ is used, but HPV18 is used thereafter. Please be consistent with usage in all Figures and text.7. Figure 1 Legend: ‘Next generation sequencing data were IGV.’ The sentence isn’t complete?8. Figure 2B – number and label the exons. No mention of EMC1 isoform(?) numbers 1, 2 & 3 are mentioned in the legend.9. Lines 513-514 – ‘….in virus unable to bind CTCF’. Please rectify.Reviewer #3: IntroductionLines 97-103. In addition to CTCF, YY1 also interacts with HPV E7.Line 105. It would be better to describe this promoter as P102 instead of P105 for HPV 18 E6/E7 and make it consistent in the entire manuscript. In fact, the main TSS in raft cultures starts from the P102, not much so from the P105.Lines 119-123. The full HPV18 transcriptome has been summarized in a JVI article  and host SRSF3 and hnRNPA1 regulation on HPV18 RNA splicing has been described in the same paper.DiscussionPlease make the promoter P102 as a name consistently in the entire manuscript, including figures and text.Lines 469-483. These spliced transcripts are rare and should not be emphasized too much. In your best estimate, they are less than 10 reads per million both in undifferentiated and differentiated conditions !Lines 484-509. Because the RNA-seq and Nanopore-seq lack qualified biological sample repeats and there was no data on functional E2 data from �CTCF-HPV18 virus, this paragraph is not relevant.**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, Data Requirements:http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here on PLOS Biology: Reproducibility:https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocolsTo enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at   12 Oct 2021AttachmentPLoS Path response to reviewers.docxSubmitted filename: Click here for additional data file. 13 Oct 2021Dear Professor Parish,We are pleased to inform you that your manuscript 'The chromatin insulator CTCF regulates HPV18 transcript splicing and differentiation-dependent late gene expression' has been provisionally accepted for publication in PLOS Pathogens.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.Best regards,Paul Francis LambertAssociate EditorPLOS PathogensKarl MüngerSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064***********************************************************Reviewer Comments : 29 Oct 2021Dear Professor Parish,We are delighted to inform you that your manuscript, "The chromatin insulator CTCF regulates HPV18 transcript splicing and differentiation-dependent late gene expression," has been formally accepted for publication in PLOS Pathogens.We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles  are generated on a different schedule and may not be made available as quickly.Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens. Best regards,Kasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064


Eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1) encodes the 4EBP1 protein, a negative regulator of mRNA translation and a substrate of the mechanistic target of rapamycin (mTOR), whose function and relevance in cancer is still under debate. Here, we analyzed EIF4EBP1 expression in different glioma patient cohorts and investigated its mode of transcriptional regulation in glioblastoma cells. We verified that EIF4EBP1 mRNA is overexpressed in malignant gliomas, including isocitrate dehydrogenase (IDH)-wildtype glioblastomas, relative to non-neoplastic brain tissue in multiple publically available datasets. Our analyses revealed that EIF4EBP1 overexpression in malignant gliomas is neither due to gene amplification nor to altered DNA methylation, but rather results from aberrant transcriptional activation by distinct transcription factors. We found seven transcription factor candidates co-expressed with EIF4EBP1 in gliomas and bound to the EIF4EBP1 promoter, as revealed by chromatin immunoprecipitation (ChIP)-sequencing data. We investigated the ability of these candidates to activate the EIF4EBP1 promoter using luciferase reporter assays, which supported four transcription factors as candidate EIF4EBP1 regulators, namely MYBL2, ETS1, HIF-1A, and E2F6. Finally, by employing transient knock-down experiments to repress either of these transcription factors, we identified MYBL2 and ETS1 as the relevant transcriptional drivers of enhanced EIF4EBP1 expression in malignant glioma cells. Taken together, our findings confirm enhanced expression of EIF4EBP1 in malignant gliomas relative to non-neoplastic brain tissue and characterize the underlying molecular pathomechanisms. EIF4EBP1) encodes 4EBP1, a substrate of the nutrient-responsive hub mechanistic target of rapamycin complex 1 (mTORC1). Upon nutrient deprivation, 4EBP1 gets activated ), pcDNA2O and 1 µl of forward and reverse primers . PPIA, GusB, and β-actin were used as housekeepers. For primer sequences, see Table RNA was extracted using the RNeasy Plus Mini Kit . The extraction was performed according to the protocol provided by the manufacturer. Isolated RNA was retro-transcribed to cDNA using 1 μg of RNA per reaction with either the QuantiTect Reverse Transcription Kit  or the High-Capacity cDNA Reverse Transcription Kit  according to the manufacturer’s protocol. Real-time PCR was performed in triplicates using 1 µl cDNA and 9 µl master mix consisting of 5 µl SYBR Green PCR Mix , 3 µl Hg for 15 min at 4 °C and supernatants were collected. Protein concentration was quantified using the PierceTM BCA Protein Assay Kit  according to the manufacturer’s protocol. Twenty micrograms of total protein were loaded either on a 12% polyacrylamide-SDS gel or on a NativePAGE™ 4–12%, Bis-Tris Gels  and transferred to a 0.2 µm nitrocellulose membrane . Membranes were blocked with 5% bovine serum albumin (BSA)  TBS-Tween  and probed with primary antibodies  supplemented with proteinase inhibitor cocktail  and phosphatase inhibitor . Cell lysates were centrifuged at 14,000 × t-test was used to compare differences between control and experimental groups. Results were considered as being statistically significant at p < 0.05. Statistical tests were calculated with GraphPad Prism version 7.04.All experiments were carried out in three biological replicates. Data are represented as mean +/− standard deviation (SD). A one- or two-sided Student’s Original Data FileOriginal Data FileSupplementary legendsSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Tables


The human genome encodes thousands of non-coding RNAs. Many of these terminate early and are then rapidly degraded, but how their transcription is restricted is poorly understood. In a screen for protein-coding gene transcriptional termination factors, we identified ZC3H4. Its depletion causes upregulation and extension of hundreds of unstable transcripts, particularly antisense RNAs and those transcribed from so-called super-enhancers. These loci are occupied by ZC3H4, suggesting that it directly functions in their transcription. Consistently, engineered tethering of ZC3H4 to reporter RNA promotes its degradation by the exosome. ZC3H4 is predominantly metazoan –interesting when considering its impact on enhancer RNAs that are less prominent in single-celled organisms. Finally, ZC3H4 loss causes a substantial reduction in cell proliferation, highlighting its overall importance. In summary, we identify ZC3H4 as playing an important role in restricting non-coding transcription in multicellular organisms. Most of the human genome can be transcribed by RNA polymerase II (Pol II). Among these transcripts are thousands of long non-coding RNAs, broadly classified as greater than ~200 nucleotides in length . They shTermination of protein-coding transcription is coupled to 3’ end processing of pre-mRNA via cleavage at the polyadenylation signal (PAS) . A PAS cThe Integrator complex aids termination of many non-coding transcripts, with the archetypal example being snRNAs . IntegraA variety of processes attenuate transcription at protein-coding genes . FrequenA less-studied termination pathway at some intragenic non-coding regions is controlled by WDR82 and its associated factors . In mammHere, we performed a proteomic screen for new termination factors by searching for proteins that bind to Pol II complexes in a manner that depends on PAS recognition by CPSF30. This uncovered ZC3H4, a metazoan zinc finger-containing factor without a characterised function in transcription. Because of the nature of our screen, we anticipated a role for ZC3H4 in 3’ end formation; however, its effects on this process are mild and apply to a small number of genes. Its main function is to restrict non-coding transcription, especially of PROMPT and eRNA transcripts, which can be extended by hundreds of kb when ZC3H4 is depleted. ZC3H4 interacts with WDR82, the depletion of which causes similar defects. Tethered function assays show that ZC3H4 recruitment is sufficient to restrict transcription and cause RNA degradation by the exosome. In sum, we reveal ZC3H4 as a hitherto unknown terminator of promoter-proximal transcription with particular relevance at non-coding loci.CPSF30 with a mini auxin-inducible degron (mAID)  . The int to work . CPSF30- to work . This re to work  demonstrCPSF30-mAID cells to homozygously tag the largest subunit of Pol II, Rpb1, with mini(m)-Turbo -Turbo . mTurbo m)-Turbo . This ocm)-Turbo .CPSF30-mAID:RPB1-mTurbo cells were exposed to biotin before western blotting with streptavidin horseradish peroxidase (HRP). This revealed multiple bands with a prominent one corresponding in size to Rpb1-mTurbo and indicating the biotinylation of its proximal proteome  , then deMYC, which is undetectable in control siRNA-treated cells, but abundant following ZC3H4 depletion. Loss of ZC3H4 also leads to the extension of this transcript by more than 100 kilobases. This is made clearer by comparing the loss of ZC3H4 to AID-mediated depletion of the catalytic exosome (DIS3)  . DIS3 dee (DIS3) . These dMYC SE where upregulation and extension of eRNAs is obvious  and SE  RNAs  RNAs . All werP1) RNAs .ZC3H4-DHFR cells grown with or without TMP for 4 hr. To additionally confirm their specificity to ZC3H4 (vs. Integrator), we also depleted the catalytic Integrator subunit utilising our previously engineered cell line in which INTS11 is tagged with a small molecule assisted shut-off module  alongside that of Pol II.RPL13 is shown as an example of recruitment of ZC3H4 upstream of the promoter, where its loss causes stabilisation and extension of the antisense transcript  or following its release was uncertain. Therefore, we purified chromatin-associated RNA . As mentDIS3-AID cells that were then treated or not with auxin to eliminate the catalytic exosome. RNA upstream and downstream of the MS2 hairpins was detected by qRT-PCR and their ratio plotted ), which negatively regulates transcription via promoter-proximal termination  suggests that elements of specificity are required to explain its mechanism. Most obviously, this could be sequences within DNA or RNA, to which ZC3H4 (and Su(s)) binds via ChIP and XRNAX, respectively see . While ontribute . Our eviBeyond transcriptional regulation, ZC3H4 occupancy of SEs is interesting. Other notable SE-associated factors (e.g. BRD4 and MED1) are much more generally implicated in Pol II transcription than ZC3H4 . MoreoveIn conclusion, we have uncovered ZC3H4 as a factor with a function in restricting transcription. Its most notable effects are at non-coding loci where transcriptional termination mechanisms are less understood than at protein-coding genes. Further dissection of ZC3H4 and its targeting should reveal additional important insights into how the unstable portion of the transcriptome is controlled. The non-overlapping effects of Integrator and ZC3H4 at protein-coding genes indicate the possibility that multiple factors may control gene output via premature transcriptional termination.HCT116 parental cells and engineered cell lines were tested negative for mycoplasma and cultured in Dulbecco modified eagle medium, supplemented with 10% foetal calf serum and penicillin streptomycin (Gibco). For RNAi, 6 or 24-well dishes were transfected with siRNA using Lipofectamine RNAiMax (Life Technologies) following the manufacturers’ guidelines. The transfection was repeated 24 hr later and, 48 hr after the second transfection, RNA was isolated. For MS2 assays, cells were seeded in 24-well dishes overnight, then transfected with 50 ng MS2hp-IRES-GFP and 100 ng of MS2-GFP, ZC3H4-MS2 or ZC3H4-GFP using JetPRIME (PolyPlus) for 24 hr. To deplete DIS3-AID or PNUTS-AID, auxin was used at a final concentration of 500 uM. To deplete ZC3H4-DHFR, cells were washed twice in PBS and grown in media with or without TMP (30 uM).CPSF30-mAID and CPSF30-mAID:RPB1-mTurbo cells were generated using CRISPR/Cas9-mediated homology-directed repair (HDR). CPSF30 and RPB1 homology arms and gRNA sequences are detailed in 2. Residual final wash buffer was used to resuspend the beads, which were then flash frozen in liquid nitrogen and sent for tandem mass spectrometry at the University of Bristol Proteomics Facility.10 cm dishes at ~80% confluency were labelled with 500 µM biotin for 10 min and the labelling reaction quenched immediately by washing cells in ice cold PBS. Cells were lysed in RIPA buffer  containing protease inhibitors  for 30 min on ice, then clarified via centrifugation. 350 uL of washed Streptavidin Sepharose High Performance slurry  was incubated with biotinylated or control lysates with inversion at room temperature for 1 hr. Samples were then washed twice with RIPA buffer, twice with Urea buffer , twice with 100 mM sodium carbonate, and once with 20 mM Tris HCl (pH 8), 2 mM CaCl<0.70 were classified as having a decreased interaction with RNA polymerase II following CPSF30 depletion. Finally, proteins with <5 peptides were discarded. Remaining candidates were plotted in First, contaminant proteins (e.g. keratin) or those that are known to be preferentially biotinylated in ligase assays (e.g. AHNAK) were excluded. Samples with an average abundance ratio 1 µg of total RNA (DNase treated) was reversed transcribed using random hexamers according to manufacturer’s instructions ; cDNA diluted to 50 uL. qPCR was performed using LUNA SYBR (NEB) on a Rotorgene (Qiagen). Fold changes were calculated using Qiagen’s software based on delta CT values. Graphs were plotted using Prism (GraphPad). Numbers underpinning qPCR-derived bar graphs are provided in CPSF30 , RNA Pol II , PNUTS , WDR82 , EXOSC10 , ZC3H4 , HA tag , GFP , TCF4/TCF7L2 . Uncropped western blots are provided in 2, 20 mM Tris HCl pH 7.5, 1% Triton X-100, 250 units Benzonase [Sigma]). Samples were then clarified through centrifugation , split in two and incubated with 25 ul of GFP TRAP magnetic agarose (Chromotek) for 1 hr with rotation at 4°C. Beads were washed 5x with lysis buffer and samples eluted by boiling in 2x SDS buffer  SDS (sodium dodecyl sulfate), 0.2% (w/v) bromophenol blue, 20% (v/v) glycerol, 200 mM β-mercaptoethanol) before analysis by western blotting.10 cm dishes were transfected , washed with PBS, and lysed for 30 min on ice in 1 mL lysis buffer  with a 10% sucrose cushion and RNA was isolated using Tri-reagent. Following DNase treatment, RNA was phenol chloroform-extracted and ethanol-precipitated. After assaying quality control using a Tapestation (Agilent), 1 μg RNA was rRNA-depleted using Ribo-Zero Gold rRNA removal kit (Illumina), then cleaned and purified using RNAClean XP Beads (Beckman Coulter). Libraries were prepared using TruSeq Stranded Total RNA Library Prep Kit (Illumina) and purified using Ampure XP beads (Beckman Coulter). A final Tapestation screen was used to determine cDNA fragment size and concentration before pooling and sequencing using Hiseq2500 (Illumina).Nuclei were extracted from 1 × 30 mm dish of cells per condition using hypotonic lysis buffer , and resuspended in 400 ul RIPA buffer . Sonication was then performed in a Bioruptor (30 s on/30 s off x10 on high setting) and debris pelleted (13000 rpm x 10 min). Supernatants were then incubated for 2 hr at 4°C with 40 ul of sheep anti-mouse dynabeads to which 4 ug of anti-Pol II  was pre-bound. Beads were washed 6x with RIPA buffer and then bound chromatin was eluted by 30 min incubation at room temperature with rotation (500 ul 0.1 M NaHCO3 +1% SDS). Cross-links were reversed overnight at 65°C with the addition of 20 ul 5M NaCl. Following phenol chloroform extraction and ethanol precipitation, chromatin was resuspended in 100 ul water of which 1 ul was used per qPCR reaction.Cells were cross-linked for 10 min at room temperature  and quenched for 5 mins (125 mM glycine). Cells were washed in PBS, pelleted  according to manufacturer’s instructions. 5 µg of RNA Pol II  or ZC3H4  were used for immunoprecipitation, Dynabeads α-mouse/α-rabbit (Life Technologies) were used for capture.2, 0.5% NP40), underlayered with 10% sucrose (w/v in HLB) on ice for 5 min, then spun at 500xg to isolate nuclei. Supernatant and sucrose were removed and nuclei resuspended in 100 μL of NUN1 , before being incubated with 1 mL NUN2  on ice for 15 min. Samples were spun at 13,000xg to pellet chromatin, this was dissolved in Trizol and RNA extracted.HCT116 cells were scraped into PBS, pelleted, incubated in hypotonic lysis buffer  for 10 min.g for 15 min, then the interphase carefully aspirated into fresh tubes. The interphase was washed thrice with Tris-SDS , before being dissolved in 1 mL Tris-SDS. 1 uL glycogen, 60 uL of 5M NaCl, and 1 mL isopropanol were added and samples precipitated at −20°C for 10 min, then pelleted at 18,000xg for 15 mins. Precipitated protein was washed with 70% ethanol, air dried, resuspended in 180 uL water and pellets dissolved on ice. DNA was removed via TurboDNase (ThermoFisher) treatment, before samples were repelleted, redissolved in RNase buffer  and RNA-digested with RNase A (NEB) and 1 uL of RNase T1 (Roche). 4x SDS loading buffer was added before gel electrophoresis and western blotting.We essentially followed the protocol of usegalaxy.org and usegalaxy.eu servers were used.All sequencing data were uploaded to the Galaxy web platform and processed as detailed below; Data newly generated in this paper (GSE163015); Pol II HEPG2 ChIP-seq (GSE32883); ZC3H4 HEPG2 ChIP-seq (GSE104247); DIS3-AID HCT116 RNA-seq (GSE120574); INTS1 RNAi Chromatin-associated RNA-seq (GSE150238). 4sU labelled RNA differential expression in HeLa cells depleted of INTS11 or ZC3H4 .<20 were removed from alignment files using SAMtools  and default settings. For each experiment, bedtools was used to establish common peaks from individual reps , creating a bed file of high confidence peaks. For ZC3H4, peaks called in the INPUT sample were subtracted via bedtools. All bed files were annotated and plotted in R using ChipSeeker .For ChIP heat maps, computematrix (DeepTools) was used to generate score files from ChIP bigwig files using an hg38 bed file; parameters used for each heat map are detailed in figure legends. Plots were redrawn in R. Transcription read-through analysis was calculated for each condition by comparing the first 1 kb downstream of the TES to a 500 bp region directly preceding the TES (PAS). A log2 ratio (depletion/control) was then applied to identify increased read-through.A bed file with the coordinates of SE locations from dbSUPER in HCT116 cells was used as a basis . All regSplit strand metagene plots were generated using RPKM normalised sense and antisense  bigwig coverage files separately with further graphical processing performed in R. For identifying ZC3H4 PROMPT regions, ncRNA genes were filtered from hg38 refgene gtf file to give protein-coding genes that were used with feature counts on siCont RNAseq , to gainFeatureCounts was used to count mapped reads over exons and differential expression was performed using DESeq2 .For PROMPT analysis, we used a catalogue of 961 PROMPTs generated by de novo assembly following acute DIS3 depletion . Due to To identify ZC3H4 homologue protein sequences, sequences from UniRef100  were searched using a profile HMM search: ‘hmmsearch’, part of HMMer V3.2.1 , with PAhttps://doi.org/10.5281/zenodo.4637127).Identified PTHR13119 domains were aligned using MAFFT v7.4 Sequences are provided in  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by James Manley as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Joan Steitz (Reviewer #3).Thank you for submitting your article "ZC3H4 restricts non-coding transcription in human cells" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:The manuscript by Estell et al. investigates the impact of the protein ZC3H4 on RNA levels in human cells. The authors first perform a screen aiming at the identification of factors involved in transcription termination, by identifying proteins in the vicinity of Pol II in a WT context compared to the rapid depletion of the Cpsf30 subunit of the cleavage and polyadenylation complex. The poorly characterised paraloguous proteins ZC3H4 and ZC3H6 display reduced signal upon Cpsf30 depletion. These proteins are then depleted, demonstrating that ZC3H4 depletion gives rise to clear alterations in RNA expression profiles. Based on this, and additional bioinformatics analysis, the authors focus on the impact of ZC3H4 on RNA levels at protein coding genes, PROMPTs and enhancers. It is concluded that ZC3H4 acts as a terminator of transcription for the latter two categories of transcription units. The observed effects are then compared to those of the Integrator complex, that also acts as an early transcription terminator at such loci, which leads to the suggestion that these proteins act on different substrates. Moreover, it is determined that ZC3H4 binds chromatin at regions corresponding to the targeted RNAs. Finally, using a reporter system, the authors further show that ZC3H4 targeting to a transcript reduces its expression level.The identification of a previously uncharacterized factor involved in transcription regulation is interesting. However, the reported results lack in-depth analysis. Most statements are quite general and would benefit from further analyses.1. ZC3H4 binds to Pol II in a CPSF30-dependent manner. Moreover, ZC3H4 appears to share similar functions with the Integrator complex. Therefore, the relationship between ZC3H4 and CPSF30 on the one hand and ZC3H4 and Integrator on the other would benefit from further elucidation and exploration. For example:i. Analyze the connection between ZC3H4 and pA sites e.g by measuring whether ZC3H4-sensitive transcripts are enriched for PASs.ii. What's the exact number of protein-coding genes demonstrating increased downstream transcription upon depletion of ZC3H4, and how are these distinguished from non-sensitive genes? What is the overlap between CPSF30 and ZC3H4-dependent protein-coding genes?iii. What are the genes included in the metagene analysis comparing the effects of CPSF30 and ZC3H4 in Figure 3B? Are genes that are read-through in the absence of either CPSF30 or ZC3H4 plotted in the Figure? Or is this a metaplot of the 1795 protein-coding genes demonstrating read-through upon CPSF30 depletion as in Figure 1C?iv. Comparison of INTS1 and/or ZC3H4-dependent protein-coding genes . Are there any characteristics  of the genes upregulated upon depletion of INTS1 or ZC3H4 or both?v. What is the overlap between the non-coding loci (PROMPTs and SEs) affected by depletion of INTS1 or ZC3H4?vi. Because the performed screen is related to transcription termination and pA site RNA processing, the described effects of ZC3H4 are predicted to be at the level of transcription termination. However, the possibility that ZC3H4 acts on transcription or RNA stability is not addressed. The usage of the DIS3 depleted samples only serves the purpose of mapping exosome sensitive locations.2. ZC3H4 versus ZC3H6: Providing a sequence alignment of ZC3H4 and ZC3H6  would yield a better appreciation of the similarity between the two proteins. Overall, this whole panel would be more fitting as a supplementary figure.3. Figure 4: The comparison of RNA-seq and chromatin-seq is not optimal. This is exemplified in panel E where the two controls display different profiles, leading to a strong effect of Ints1 depletion and no effect of ZC3H4 depletion.4. Figure 5: While affirming the phenotype also upon rapid depletion of ZC3H4 represents an important control, these data do not bring any new information and should probably be considered supplementary.5. Figure 6: Considering the weak affinity of the ZC3H4 antibody in ChIP, displaying an input track would help estimate the specificity of the signal. In the same vein: To ensure that the peaks presented in the IGV tracks are not background, one could normalize ChIP-seq data to input DNA and plot the tracks as the ratio of ChIP/Input.6. Figure 7: While the tethering assay nicely demonstrates that recruitment of ZC3H4 negatively regulates the reporter RNA, additional explanation or experiment is warranted. In their discussion of chromatin-associated RNA, it is unclear how the reporter RNA is found in the chromatin fraction? Do the authors mean chromatin formed on the plasmid or the host chromatin? If the latter, how does the reporter associate with the chromatin? Is detection of uncleaved RNA at the BGH poly A site a faithful readout of nascent RNA? ZC3H4 also affects read-through. Can other methods be used to quantify nascent RNA? The tethering experiment should be performed on at least one endogenous target of ZC3H4 (that was upregulated upon its depletion). Does ZC3H4 over-expression rescue the effect of its depletion or is this not sufficient?7. Clarification of Figures and Figure legends: Despite presenting interesting experiments, the figures require thorough revision to ensure clarity.a. Please specify the direction of all genes in the IGV browser tracks in a more visible manner.b. Adequately label all axes and scales . When referring to fold change, specify of what.c. When presenting normalized data, include a control in the graph even if its set to 1. .d. Figure 1D: Label CPSF30 green.e. Figure 2A should be supplementary to Figure 1.f. Figure 3B: How many genes are being plotted?g. 3E: Where is MYC SE on the track?h. Figure 5C and S4: Explain the 5´ and 3´ primer probes .i. Figure 7D: Both bars should be colored red.8. Discussion: Several paragraphs in the discussion feel disconnected to the findings presented in the manuscript and how they tie in with the current knowledge regarding transcription termination (eg. Lines 382-395). The authors should revise this section to present a stronger argument highlighting the importance of the findings presented in their manuscript. Essential revisions:The manuscript by Estell et al. investigates the impact of the protein ZC3H4 on RNA levels in human cells. The authors first perform a screen aiming at the identification of factors involved in transcription termination, by identifying proteins in the vicinity of Pol II in a WT context compared to the rapid depletion of the Cpsf30 subunit of the cleavage and polyadenylation complex. The poorly characterised paraloguous proteins ZC3H4 and ZC3H6 display reduced signal upon Cpsf30 depletion. These proteins are then depleted, demonstrating that ZC3H4 depletion gives rise to clear alterations in RNA expression profiles. Based on this, and additional bioinformatics analysis, the authors focus on the impact of ZC3H4 on RNA levels at protein coding genes, PROMPTs and enhancers. It is concluded that ZC3H4 acts as a terminator of transcription for the latter two categories of transcription units. The observed effects are then compared to those of the Integrator complex, that also acts as an early transcription terminator at such loci, which leads to the suggestion that these proteins act on different substrates. Moreover, it is determined that ZC3H4 binds chromatin at regions corresponding to the targeted RNAs. Finally, using a reporter system, the authors further show that ZC3H4 targeting to a transcript reduces its expression level.The identification of a previously uncharacterized factor involved in transcription regulation is interesting. However, the reported results lack in-depth analysis. Most statements are quite general and would benefit from further analyses.We thank all reviewers for taking the time to carefully read our paper and provide the constructive criticism that has helped us to revise it. Below, you will find our detailed responses to each point raised. We would like to point out that while we were revising our paper, another group published some complementary findings . They also identify ZC3H4 as a WDR82 interactor and find some defects in intragenic transcription. Our respective independent findings synergise and make a compelling case for the function of ZC3H4 in mammalian non-coding RNA transcription and metabolism.1. ZC3H4 binds to Pol II in a CPSF30-dependent manner. Moreover, ZC3H4 appears to share similar functions with the Integrator complex. Therefore, the relationship between ZC3H4 and CPSF30 on the one hand and ZC3H4 and Integrator on the other would benefit from further elucidation and exploration. For example:i. Analyze the connection between ZC3H4 and pA sites e.g by measuring whether ZC3H4-sensitive transcripts are enriched for PASs.Although we identified ZC3H4 via a screen involving CPSF30 depletion, we reported that it did not often affect transcription beyond pA sites of protein-coding genes. This suggests that its transcriptional functions are unlikely to universally require pA sites. As requested, we provide more analyses to support this claim that can be found in Figure 2—figure supplement 2F. We looked at this using PROMPTs since they are easily identified and many are sensitive to ZC3H4 depletion. We took a list of PROMPT transcripts that we previously annotated in HCT116 cells based on their acute sensitivity to exosome (DIS3) loss . Of this 961, 438 are upregulated by 2-fold or more following ZC3H4 RNAi. When we compared the pA density in the affected vs unaffected set we found no link between pA density and susceptibility to ZC3H4. In fact, affected PROMPTs tended to have a slightly lower density of pA sequences.ii. What's the exact number of protein-coding genes demonstrating increased downstream transcription upon depletion of ZC3H4, and how are these distinguished from non-sensitive genes? What is the overlap between CPSF30 and ZC3H4-dependent protein-coding genes?As we stated in the original manuscript, only a small number of protein-coding genes are affected by ZC3H4 compared to CPSF30 loss. As requested, we now present a bioinformatics analysis . To approximate read-through, we looked at a region 1kb downstream of the poly(A) signal and compared the read density to a region 500bp upstream. This was to maximise the number of genes in the dataset. From over 7000 genes, most show some read-through in the absence of CPSF30 and 4343 have a 2-fold or more increase. By contrast, only 583 have over a 2-fold increase upon ZC3H4 depletion. The majority overlap with those affected by CPSF30 (369/583). In general, the effects of ZC3H4 beyond pA sites are also smaller than for CPSF30. This is illustrated by the fact that the proportion of genes with larger fold effects Log2FC>2 is even lower than those with Log2FC>1 . Manual inspection of ZC3H4 effects also reveals some read-through examples as having low read coverage beyond the pA site, which may lead to the inclusion of some false positives. However, our analysis provides valuable unbiased support for the generality of CPSF30 effects versus those associated with ZC3H4 loss.We looked at characteristics of protein-coding genes that show evidence of read-through after ZC3H4 loss. There was no clear effect of length or intron density and only a slight tendency for affected genes to be expressed at lower levels. We have not included these analyses but can do so if necessary.iii. What are the genes included in the metagene analysis comparing the effects of CPSF30 and ZC3H4 in Figure 3B? Are genes that are read-through in the absence of either CPSF30 or ZC3H4 plotted in the Figure? Or is this a metaplot of the 1795 protein-coding genes demonstrating read-through upon CPSF30 depletion as in Figure 1C?Genes for these metaplots were not selected based on their sensitivity to ZC3H4 or CPSF30 and were intended to provide unbiased analysis. We selected expressed genes separated from any annotated neighbour by 3kb upstream and 5kb downstream to eliminate closely spaced/overlapping transcription units. The conclusion drawn from them  is supported by the broader analysis provided in response to the above point (ii) and our extensive manual inspection of the data.iv. Comparison of INTS1 and/or ZC3H4-dependent protein-coding genes . Are there any characteristics  of the genes upregulated upon depletion of INTS1 or ZC3H4 or both?We looked for enriched GO processes but found nothing striking/obvious in terms of over-represented functions that could link the effects of either factor to large sets of genes with a common function. We can provide tables for this if necessary. Of the other facets, we found that genes affected by INTS1 and ZC3H4 have lower steady-state expression under normal circumstances than those that are unaffected. This is interesting and supports the notion that there are mechanisms (i.e. Integrator and ZC3H4) that restrict their expression. This new data is in Figure 3E.v. What is the overlap between the non-coding loci (PROMPTs and SEs) affected by depletion of INTS1 or ZC3H4?The requested analyses are now provided in Figure 3—figure supplement 1C and D. We used our annotated list of 961 PROMPTs, described above for point i), and found that 438 show 2-fold or more upregulation in the absence of ZC3H4 while fewer (222) have this effect without INTS1. 153 of these 222 overlap with those affected by ZC3H4. We had noted in the original manuscript that ZC3H4 effects at PROMPTs were typically greater in magnitude than those associated with INTS1 RNAi. Our new bioinformatics analyses support this. For example, we find that only 26 PROMPTs have a change of 4-fold or more in the absence of INTS1, whereas the figure is 154 for those affected by ZC3H4 depletion. For SEs the effects of INTS1 are much less robust than those of ZC3H4 as can be seen by comparing the metaplots in Figures 2F and 3H.vi. Because the performed screen is related to transcription termination and pA site RNA processing, the described effects of ZC3H4 are predicted to be at the level of transcription termination. However, the possibility that ZC3H4 acts on transcription or RNA stability is not addressed. The usage of the DIS3 depleted samples only serves the purpose of mapping exosome sensitive locations.MYC and ITPRID2 PROMPT regions). This new experiment  shows increased Pol II occupancy upstream of both PROMPTs following rapid (4 hours) depletion of ZC3H4 and provides important additional support for a transcriptional effect. In addition, we looked at extended transcripts from both PROMPTs via the isolation of chromatin-associated RNA which is enriched in nascent transcripts. This confirmed the ChIP result by demonstrating substantially more (~30 fold) extended RNA following the rapid loss of ZC3H4 . It is important to note that ZC3H4 may additionally play roles in RNA stability alongside or as part of its function in controlling non-coding transcription. However, to our knowledge, it has never been recovered as an interactor of the exosome complex.We agree that ZC3H4 effects are predicted to be at the level of transcription (termination). In our view, this is indicated by the long extensions to non-coding RNA caused by its loss. The most credible reason for such distal RNA signal is aberrant transcription caused by ZC3H4 depletion. We say this for two main reasons. First, there is normally no Pol II over these regions indicating that they are not transcribed in unmodified cells. Second, there is no DIS3 effect at these extended regions  arguing against the presence of low level unstable RNA at these positions. To confirm this, we performed Pol II chromatin immunoprecipitation (ChIP) in the presence and absence of ZC3H4 and analysed two loci affected by its loss in our RNA-seq  would yield a better appreciation of the similarity between the two proteins. Overall, this whole panel would be more fitting as a supplementary figure.We have now provided the requested alignment and moved the updated figure concerning phylogenetic analyses to the supplement .3. Figure 4: The comparison of RNA-seq and chromatin-seq is not optimal. This is exemplified in panel E where the two controls display different profiles, leading to a strong effect of Ints1 depletion and no effect of ZC3H4 depletion.A reason for the two controls looking different is because chromatin-associated RNA (INTS1) is enriched in nascent transcripts whereas the nuclear RNA (ZC3H4) will contain an accumulation of completed mRNA transcripts – hence the higher control level of TM7SF2 versus in the chromatin. We would like to note that not all comparisons look like TM7SF2 with respect to the control profiles. Many examples are not like this because ZC3H4 and Integrator often act on genes that normally show low expression (as we discovered when addressing point 1 iv) above). To better illustrate this, we have provided more IGV snapshots in addition to retaining the TM7SF2 example . We have also provided new data in the revision to further demonstrate that ZC3H4 and Integrator depletion affect different mRNAs and that the ZC3H4 effect is rapid and at chromatin.To demonstrate that the limited overlap of ZC3H4 and Integrator targets is not due to our comparison of two slightly different sequencing methodologies, we compared recently published datasets that used 4sU to sequence nascent RNA in HeLa cells depleted of the catalytic Integrator subunit  or ZC3H4 . Similar to our data this revealed several hundred protein-coding transcripts upregulated by either depletion with very little overlap . This orthologous and independent approach, performed in a different cell line, provides strong support for the idea that ZC3H4 and Integrator affect different subsets of mRNAs.To test the rapidity, and confirm the selectivity, of the ZC3H4 effect in our system, we performed qRT-PCR on chromatin-associated RNA after depletion of ZC3H4-DHFR. We selected three transcripts  that are affected by ZC3H4 via our nuclear RNA-seq. All three are enhanced in the chromatin-associated fraction after just 4 hours of ZC3H4 depletion . The same three transcripts were unaffected by Integrator according to our chromatin-associated RNA-seq – a result that we confirmed by qRT-PCR of chromatin-associated RNA isolated from cells depleted of the catalytic Integrator subunit, INTS11. This experiment demonstrates that at least some transcripts are rapidly and specifically (versus Integrator) upregulated by ZC3H4 loss.In all, we feel that these new analyses and experiments more convincingly highlight some separate transcriptional functions of ZC3H4 and Integrator at different mRNA subsets.4. Figure 5: While affirming the phenotype also upon rapid depletion of ZC3H4 represents an important control, these data do not bring any new information and should probably be considered supplementary.We do agree with this assessment in the context of the original manuscript. However, as part of the revision process we have made more use of the ZC3H4-DHFR cells to address some of the important points raised by the reviewers. E.g. to show that ZC3H4 acts quickly on transcription of mRNA targets and, importantly, that the effects of its depletion are rapidly reversed when its levels are allowed to recover. We have incorporated some of these new data into an expanded version of Figure 5 .5. Figure 6: Considering the weak affinity of the ZC3H4 antibody in ChIP, displaying an input track would help estimate the specificity of the signal. In the same vein: To ensure that the peaks presented in the IGV tracks are not background, one could normalize ChIP-seq data to input DNA and plot the tracks as the ratio of ChIP/Input.As requested, we have included an input track for these experiments. This makes clear that the presented peaks are not background.6. Figure 7: While the tethering assay nicely demonstrates that recruitment of ZC3H4 negatively regulates the reporter RNA, additional explanation or experiment is warranted. In their discussion of chromatin-associated RNA, it is unclear how the reporter RNA is found in the chromatin fraction? Do the authors mean chromatin formed on the plasmid or the host chromatin? If the latter, how does the reporter associate with the chromatin? Is detection of uncleaved RNA at the BGH poly A site a faithful readout of nascent RNA? ZC3H4 also affects read-through. Can other methods be used to quantify nascent RNA?We apologise for the lack of clarity here and have provided some more explanation in the revision as requested. As mentioned, chromatin-associated RNA is enriched in nascent transcripts. Importantly, previous reports  demonstrated that this method also effectively enriches nascent RNAs transcribed from plasmids. This likely reflects the co-sedimentation of transcribed plasmids with chromatin during this isolation procedure and shows that this method is effective in enriching nascent RNA from exogenous DNA plasmids. Our analysis of RNAs uncleaved at the BGH poly(A) was designed to support this. Because poly(A) site cleavage is co-transcriptional, these transcripts are very likely nascent.The reviewers also put forward the reasonable opinion that ZC3H4 affects read-through at some protein-coding genes and may therefore influence the BGH pA site to account for some of the tethering effect. We have considered this carefully and are confident that this is not a confounding issue for the following reasons. In response to 1 ii), we show that ZC3H4 rarely affects read-through at pA sites and we have provided (see below) a new experiment that its depletion does not affect the BGH pA site in this reporter. One might posit a scenario in which tethering ZC3H4 to the reporter improves BGH pA processing, thereby accounting for the lower levels of this uncleaved species when ZC3H4-MS2 is used. However, this hypothetical is not supported by our evidence. Such enhanced pA processing would almost certainly result in more reporter mRNA whereas what we observe is much less (i.e. tethered ZC3H4 reduces the recovery of gene body amplicons). Finally, our exosome depletion experiment in this tethering system argues for an effect exerted at, or close to, its site of recruitment (i.e. the MS2 hairpins) and not at the downstream BGH pA. We say this because exosome depletion has a greater effect on the stability of RNA upstream of the MS2 hairpins rather than in the vicinity of the BGH pA.The tethering experiment should be performed on at least one endogenous target of ZC3H4 (that was upregulated upon its depletion).We apologise for the lack of clarity here and have provided some more explanation in the revision as requested. As mentioned, chromatin-associated RNA is enriched in nascent transcripts. Importantly, previous reports  demonstrated that this method also effectively enriches nascent RNAs transcribed from plasmids. This likely reflects the co-sedimentation of transcribed plasmids with chromatin during this isolation procedure and shows that this method is effective in enriching nascent RNA from exogenous DNA plasmids. Our analysis of RNAs uncleaved at the BGH poly(A) was designed to support this. Because poly(A) site cleavage is co-transcriptional, these transcripts are very likely nascent.The reviewers also put forward the reasonable opinion that ZC3H4 affects read-through at some protein-coding genes and may therefore influence the BGH pA site to account for some of the tethering effect. We have considered this carefully and are confident that this is not a confounding issue for the following reasons. In response to 1 ii), we show that ZC3H4 rarely affects read-through at pA sites and we have provided (see below) a new experiment that its depletion does not affect the BGH pA site in this reporter. One might posit a scenario in which tethering ZC3H4 to the reporter improves BGH pA processing, thereby accounting for the lower levels of this uncleaved species when ZC3H4-MS2 is used. However, this hypothetical is not supported by our evidence. Such enhanced pA processing would almost certainly result in more reporter mRNA whereas what we observe is much less (i.e. tethered ZC3H4 reduces the recovery of gene body amplicons). Finally, our exosome depletion experiment in this tethering system argues for an effect exerted at, or close to, its site of recruitment (i.e. the MS2 hairpins) and not at the downstream BGH pA. We say this because exosome depletion has a greater effect on the stability of RNA upstream of the MS2 hairpins rather than in the vicinity of the BGH pA.Does ZC3H4 over-expression rescue the effect of its depletion or is this not sufficient?ZC3H4-DHFR cell line because the degradation of ZC3H4-DHFR that is triggered by withdrawing TMP from media can be reversed by re-administering TMP. This is advantageous to over-expression because re-expression of ZC3H4-DHFR is achieved more quickly than typically required for protein over-expression by transfection. The effectiveness of this approach is shown by a new Western blot in Figure 4C. This re-expression is sufficient to rescue the level of every ZC3H4 target that we tested . By supporting our other depletions, the MS2 tethering result and the ChIP showing ZC3H4 occupancy of affected loci, this result provides additional evidence that ZC3H4 is responsible for the effects that we observe in its absence.We have performed the requested rescue using our 7. Clarification of Figures and Figure legends: Despite presenting interesting experiments, the figures require thorough revision to ensure clarity.a. Please specify the direction of all genes in the IGV browser tracks in a more visible manner.We have now included arrows on each browser track that are colour coded to match sense (blue) or antisense (red) directions.b. Adequately label all axes and scales . When referring to fold change, specify of what.We have now done this and provided some additional clarification in the legends.c. When presenting normalized data, include a control in the graph even if its set to 1. .We have now done this.d. Figure 1D: Label CPSF30 green.We have made this change.e. Figure 2A should be supplementary to Figure 1.In response to this comment (and another above), we have moved all of the material from the original figure 2 into the supplementary material .f. Figure 3B: How many genes are being plotted?As explained above, this is 1795 selected solely on the basis that they are expressed and separated from annotated transcription units . There was no selection for any perceived effect of ZC3H4 and/or CPSF30.g. 3E: Where is MYC SE on the track?As explained above, this is 1795 selected solely on the basis that they are expressed and separated from annotated transcription units . There was no selection for any perceived effect of ZC3H4 and/or CPSF30.h. Figure 5C and S4: Explain the 5´ and 3´ primer probes .Further information on their location (relative to the beginning of the PROMPT) are provided in the legend for Figure 4D.i. Figure 7D: Both bars should be colored red.We have made this change.8. Discussion: Several paragraphs in the discussion feel disconnected to the findings presented in the manuscript and how they tie in with the current knowledge regarding transcription termination (eg. Lines 382-395). The authors should revise this section to present a stronger argument highlighting the importance of the findings presented in their manuscript.We have made this change.