Elaboration of the epigenetic regulation of chromatin is a long-standing aim in molecular and cellular biology. Hence, there is a great demand for the development of in vitro methods to reconstitute chromatin that can be used directly for biochemical assays. The widely used wheat germ cell-free protein expression method provides broad applications to investigate the function and structure of eukaryotic proteins. Such advantages, including high translation efficiency, flexibility, and possible automatization, are beneficial for achieving native-like chromatin substrates for in vitro studies.Drosophila and human chromatins can be reconstituted in the course of the in vitro translation of core histones by the addition of chromatin assembly factors, circular plasmid, and topoisomerase I in an ATP-dependent manner. Drosophila chromatin assembly was performed in 4 h at 26 °C, in the presence of premixed mRNAs encoding the core histones, dAcf1/dISWI chromatin remodeling complex, and nucleosome assembly protein, dNAP1. Similarly, the human chromatin was assembled by co-expressing the human core histones with Drosophila chromatin remodeling factor, dISWI, and chromatin chaperone, dNLP, for 6 h at 26 °C. The presence of reconstituted chromatin was monitored by DNA supercoiling assay, also the regular spacing of nucleosomes was assessed by Micrococcal nuclease assay. Furthermore, Drosophila linker histone H1-containing chromatin was reconstituted, affirming that the in vitro assembled chromatin is suitable for downstream applications.We describe a novel, single-step in vitro chromatin assembly method by using the wheat germ cell-free protein synthesis. We demonstrated that both Drosophila and human chromatins, possibly in native-like form, by using a wheat germ cell-free protein expression. Although both chromatins were reconstituted successfully, there were unexpected differences with respect to the required ratio of histone-coding mRNAs and the reaction time. Overall, our new in vitro chromatin reconstitution method will aid to characterize the unrevealed structure, function, and regulation of chromatin dynamics.The method described in this study allows the assembly of Supplementary information accompanies this paper at 10.1186/s12896-020-00655-6. Chromatin enables high-level compaction of the enormous genomic DNA into a few microns of nuclei in every eukaryote. The histone octamer, that is composed of four core histones, H2A, H2B, H3, and H4, is tightly wrapped around by approximately 147 base pair of DNA in a left-handed superhelix , 6, repaThe first detailed nucleosome structure was described by determining the X-ray crystallography of the refolded, purified, and in vitro assembled nucleosome on human α-satellite DNA . Since tXenopus laevis oocytes extract, and the assembly of nucleosome was determined to be ATP-dependent [Drosophila melanogaster embryo cell-free system, and it was shown that unknown factors in the embryo facilitated the formation of chromatin [Drosophila embryo were defined, including ATP-utilizing assembly factor (dAcf1 and dISWI), and nucleosome assembly protein-1 (dNAP1), that together help to assemble the regularly spaced chromatin, and became one of the standard methods [Besides the salt dialysis chromatin reconstitution technique, ATP-dependent chromatin assembly methods have been applied in a vast range of chromatin research, which utilizes a set of chromatin assembly factors –26. The ependent . Then, Bhromatin . Later,  methods , 26, 28. methods .Drosophila and human core histones, ATP-utilizing assembly factor, nucleosome assembly protein, and histone chaperone. For achieving the reconstitution of both Drosophila and human chromatins in co-expression manner, each set of core histones was translated along with the appropriate pairs of Drosophila chromatin assembly factors. Furthermore, the ratio of the mRNAs of core histones and incubation time were optimized in both Drosophila and human chromatin assembly systems. Finally, the reconstituted chromatins were evaluated by supercoiling and Micrococcal nuclease (MNase) assays. Overall, we describe a single-step in vitro chromatin assembly method, which may serve as a new, viable protocol for chromatin research and epigenetic studies.In this study, we sought to develop a novel chromatin reconstitution method by using a well-established wheat germ cell-free protein synthesis , 31 and Drosophila core histones DmH2A, DmH2B, DmH3, and DmH4, and chromatin assembly factors, dAcf1, dISWI, and dNAP1 were cloned into the pEU-E01-MCS expression vector from the original pET plasmids [Drosophila nucleoplasmin, dNLP, coding gene was amplified from the cDNA library of adult Drosophila whole body, then cloned into the pEU-E01-MCS vector. The human core histones were amplified from the human cDNA library and cloned into a pEU-E01-LICNot expression vector [Drosophila core histones  by MNase assay  ratio  and nucleosome assembly protein (dNAP1). Consistent with the previous studies, the Drosophila chromatin in our system was completed within 4 h. While, the combination of human core histones, Drosophila nucleoplasmin (dNLP), and dISWI was used in the human chromatin assembly, and the optimum reaction time was determined to be 6 h. Preeminently histone H3s and H4s, each of which differ only one amino acid between Drosophila and human , cloning (Toyobo), in vitro transcription (Promega), protein expression, including wheat germ extract  and SUB-AMIX translation buffer (CellFree Sciences), and creatine kinase  were purchased. Reagents used in chromatin assembly reaction, including pBluescript II SK plasmid DNA (Addgene), SUB-AMIX solution (CellFree Sciences), creatine kinase , and Topoisomerase I (Takara Bio Inc.) were also purchased. For gel analyses,100 bp DNA molecular weight marker (Toyobo) and protein size marker (Bio-Rad Laboratories) were purchased.Drosophila were prepared by using FastGeneTM kit (Nippon Genetics). cDNA libraries were synthesized by using 100 ng total RNA and with random 6-mer primer with PrimeScript II (Takara Bio Inc) by following the manufacturer protocol.Total RNA extracts of human culture Jurkat cell line  were a kind gift of Ryotah Uehara (Hokkaido University) and total RNA extracts of adult Drosophila core histones, DmH2A (UniProt ID: P84051), DmH2B (UniProt ID: P02283), DmH3 (UniProt ID: P02299), and DmH4 (UniProt ID: P84040), linker histone H1 (UniProt ID: P02255) and Drosophila chromatin assembly factors dAcf1 (UniProt ID: Q9V9T4), dISWI (UniProt ID: Q24368), and dNAP1 (UniProt ID: Q9W1G7) were a kind gift from Prof. Kadonaga . Genes encoding human core histones HsH2A (UniProt ID: Q6FI13), HsH2B (UniProt ID: P62807), HsH3.1 (UniProt ID: P68431), and HsH4 (UniProt ID: P62805), and Drosophila chromatin assembly factors dNLP (UniProt ID: Q27415) were amplified from human and Drosophila cDNA libraries, respectively. Genes of Drosophila core histones and chromatin assembly factors were cloned into a pEU-E01-MCS expression vector (CellFree Sciences) by PIPE-cloning [6-tag by PCR mediated deletion [Genes encoding -cloning . The pEUdeletion  with theTriticum aestivum genome was obtained from the NCBI on April 2014. Fixed modification of carbamidomethyl at cysteine and dynamic modifications of oxidation at methionine were used for the database search. The abundance of each protein was estimated by the Sequest score calculated by the Proteome Discoverer software.The sample preparation and proteomic analysis were performed by following the previous study . BrieflyDrosophila and human histone proteins, 10% stain-free SDS-PAGE (Bio-Rad Laboratories) was used for chromatin assembly factors and proteins were visualized by the gel documentation system (Bio-Rad Laboratories). For DmH1, a sample was run on 18% SDS-PAGE followed by Coomassie Blue staining. Expected molecular weights of in vitro translated proteins: Drosophila core histone DmH2A 13.4 kDa, DmH2B 13.7 kDa, DmH3 15.6 kDa, DmH4 11.4 kDa; human core histone HsH2A 14.1 kDa, HsH2B 14.0 kDa, HsH3.1 15.4 kDa, HsH4 11.4 kDa; Drosophila chromatin assembly factors, dAcf1 170.4 kDa, dISWI 118.9 kDa, dNAP1 42.8 kDa, dNLP 17.0 kDa  after in vitro transcription, precipitated and resuspend in 10.0 μl of RNase-free water (Nippon Gene). The ratio of appropriate amount core histone mRNAs was determined by testing the ratio of DmH2A/DmH2B and DmH3/DmH4 coding mRNAs from 1:0.5 to 1:2 in total of approximately 2.0 μg/reaction, then a stock mRNA mixture was made, that was used directly for chromatin assembly reaction. The reaction was performed by mixing 1.0 μl of Drosophila core histone mRNA mixture, 0.4 μl dAcf1, 0.4 μl dISWI, 0.4 μl dNAP1 mRNAs, 2.5 μg of supercoiled pBSK plasmid, 2 U of Topoisomerase I, 5.0 μl wheat germ extract, and 0.4 μg of creatine kinase and supplemented to 10.7 μl final volume with 1x SUB-AMIX solution. The mixture was gently underlaid to 103.0 μl of 1x SUB-AMIX solution in a 96-well plate to form bilayer. The reaction was carried out at the indicated time  extraction (pH 8.0) followed by ethanol precipitation, and the purified DNA was resuspended in HD buffer  containing a trace amount of Ribonuclease a (Macherey-Nagel GmbH & Co.). Supercoiled plasmid DNAs were separated by 0.8% TBE agarose gel in 0.5x TBE buffer, visualized with ethidium bromide (Nippon Gene), and analyzed by gel documentation system (Bio-Rad Laboratories).2) to 28.0 μl of total reaction mixture followed by 5, 10, 20, 30, 40 min incubation at 25 °C. The final concentration of MNase for Drosophila chromatin was 14 U/μl and for human chromatin was 10 U/μl. For the DmH1-containing chromatin, 13 U/μl MNase was used. The assay was halted by EGTA, and proteins were eliminated by phenol-chloroform-IAA extraction followed by ethanol precipitation. The purified DNA was resuspended in HD Buffer  containing a trace amount of Ribonuclease A and analyzed by 2.0% TAE agarose gel in 1x TAE buffer. Gels containing Drosophila DNA were stained with GelRed  and human DNA was stained with ethidium bromide. The average mononucleosomal DNA length measurement was performed by the gel documentation system (Bio-Rad Laboratories).MNase assay was performed by the addition of Micrococcal Nuclease (Bio-Rad Laboratories) and MNase buffer (20 mM Tris-HCl (pH 8.0), 5 mM NaCl, 2.5 mM CaClAdditional file 1: Supplementary Table S1. Protein list of mass spectrometry analysis of the wheat germ extract. Results of proteins determined in the wheat germ cell-free extract (WEPRO7240H). Seven hundred forty-four proteins were identified in the total of 61,984 protein sequences listed in the FASTA file. The score indicated the enrichment of proteins calculated by the Proteome Discoverer.Additional file 2: Supplementary Figure S1. Full scans of gel images shown in Fig. a A full gel image of determination of the appropriate ratio of DmH2A/DmH2B and DmH3/DmH4 mRNAs in the Drosophila chromatin reconstitution reaction shown in Fig. b Supercoiling assay of assembled Drosophila chromatin under the indicated reaction time 0, 1, 4, and 6 h, where 0 indicated the reconstitution reaction in the absence of mRNAs encoding histones shown in Fig. Supplementary Figure S2. Full scans of gel images shown in Fig. a A full gel image of DNA supercoiling assay to optimize the ratio of HsH2A/HsH2B and HsH3.1/HsH4 mRNAs in the chromatin reconstitution reaction for 4 h shown in Fig. b Supercoiling assay of assembled human chromatin. Incubation time indicated with 0, 1, 4, 6, and 8 h, where 0 indicated the reconstitution reaction in the absence of mRNAs encoding histones, which is shown in Fig. Supplementary Figure S3. Sequence alignment of Drosophila and human histones. Amino acid sequence alignment of a DmH3 and HsH3.1, b DmH4 and HsH4, c DmH2A and HsH3.1, d DmH2B and HsH2B are shown, respectively. Asterisks and light purple-highlighted amino acids showed the identical amino acid sequence between two species. Supplementary Figure S4. Time course of MNase digestion for the reconstituted Drosophila chromatosome. Assembled DmH1-incorporated chromatin was digested by 10 U/μl MNase for 0, 5, 10,20, 30, 40 min, then run on 2.0% agarose gel and visualized by ethidium bromide. Detected bands corresponding to mono-, di-, and tri-nucleosomes were indicated. NRL of reconstituted chromatosome was estimated to be 174.6 ± 2.1 bp.


S. pombe. Using a combination of biochemical, molecular, and genetic approaches, the authors show that Hap2–Ino80 destabilizes H3 nucleosomes on centromere DNA through transcription-coupled histone H3 turnover, driving the replacement of resident H3 nucleosomes with CENP-A nucleosomes.In this study, Singh et al. set out to define mechanisms for H3:CENP-A exchange at centromeric regions in  Cnp1 chromatin. All subunits of the Ino80 complex were enriched, including the auxiliary subunit Hap2. Chromatin association of Hap2 is Ies4-dependent. In addition to a role in maintenance of CENP-ACnp1 chromatin integrity at endogenous centromeres, Hap2 is required for de novo assembly of CENP-ACnp1 chromatin on naïve centromere DNA and promotes H3 turnover on centromere regions and other loci prone to CENP-ACnp1 deposition. Prior to CENP-ACnp1 chromatin assembly, Hap2 facilitates transcription from centromere DNA. These analyses suggest that Hap2–Ino80 destabilizes H3 nucleosomes on centromere DNA through transcription-coupled histone H3 turnover, driving the replacement of resident H3 nucleosomes with CENP-ACnp1 nucleosomes. These inherent properties define centromere DNA by directing a program that mediates CENP-ACnp1 assembly on appropriate sequences.Centromeres are maintained epigenetically by the presence of CENP-A, an evolutionarily conserved histone H3 variant, which directs kinetochore assembly and hence centromere function. To identify factors that promote assembly of CENP-A chromatin, we affinity-selected solubilized fission yeast CENP-A Drosophila centromeres, respectively  in place of canonical H3 to direct kinetochore assembly on such repetitive elements. Moreover, it is known that following deletion of an endogenous centromere, CENP-A incorporation and neocentromeres can arise at novel noncentromeric DNA locations  with and/or transcription from CENP-A-associated DNA so that noncoding transcription has become an apparent integral feature of centromeres . RNAPII osophila , and S. revisiae  centromerevisiae . Disruptrevisiae . Moreoverevisiae . The cenromoters . In additurnover . Such obw CENP-A . HoweverS. pombe, Hrp1 (ortholog of chromo-helicase DNA-binding protein 1 [CHD1]) is enriched at centromeres and is required to maintain normal CENP-ACnp1 levels at centromeres  as an auxiliary subunit of Ino80C that is required for the conversion of H3 chromatin to CENP-ACnp1 chromatin on naïve centromere DNA. We show that loss of Hap2 function reduces transcription and histone H3 turnover on centromere DNA. Our findings indicate that Hap2–Ino80 is required to promote transcription-associated chromatin remodelling events that drive H3 nucleosome eviction and the assembly of CENP-ACnp1 nucleosomes in their place.Here we used affinity selection of CENP-ACnp1 chromatin, GFP-tagged CENP-ACnp1 chromatin was affinity-purified from micrococcal nuclease (MNase)-solubilized chromatin extracts (Cnp1 domain of cen2 (cc2) compared with the 18 copies of flanking outer repeat (dg) sequences . In addition, SDS-PAGE analysis showed core histones to be prevalent in this affinity-selected GFP-CENP-ACnp1 material . Label-free quantitative mass spectrometry detected strong enrichment of all known subunits of both the inner and outer kinetochore complexes . As our procedure also showed association of the chaperones Scm3 (HJURP) and Sim3 (NASP) that mediate CENP-ACnp1 deposition we reasoned that other enriched, but non-centromere-specific, proteins might be involved in the incorporation of CENP-ACnp1 into centromeric chromatin.To identify proteins involved in the assembly of CENP-Aextracts A. QuantiCnp1 preparations . Affinity selection of Hap2-GFP followed by proteomic analysis revealed that all subunits of Ino80C, but not subunits of other remodelling complexes, are enriched with Hap2-GFP . IP-western analysis confirmed that Hap2-GFP associates with immunoprecipitated HA-tagged Ino80 (Ino80-HA) and vice-versa, independently of DNA/chromatin association . Furthermore, affinity selection of Ino80-HA enriched all known Ino80C subunits along with Hap2 . We conclude that Hap2 is a noncanonical subunit of the fission yeast Ino80 complex and that all Ino80C subunits are enriched in CENP-ACnp1 chromatin.It was notable that all subunits of the Ino80 chromatin remodeling complex (Ino80C) were enriched in our affinity-selected GFP-CENP-Aarations C. Also earations . To confCnp1 domain of centromeres (cc2), the flanking pericentromeric outer repeats (dg) and on the highly expressed act1+ gene (cen2-cc2) is inserted at the ura4 locus on a chromosome arm (ura4:cc2), it is assembled in H3 instead of CENP-ACnp1 chromatin  allows analysis across the resulting unique ectopic copy of cen2 central domain DNA (ura4:cc2) in the absence CENP-ACnp1 and kinetochore proteins , while Hap2 chromatin association is lost in the absence of Ies4 but remains unaffected in iec1Δ, ies2Δ and arp5Δ cells . In budding yeast, different subunits of Ino80 have been shown to have broad interactions around NFRs and +1 nucleosomes  and good growth on counter-selective 5-FOA plates . Consistent with this observation, no significant change was detected in the levels of the heterochromatin H3K9me2 mark or dg transcripts produced by the underlying outer repeats in hap2Δ relative wild-type cells . Reduced silencing in hap2Δ cells, indicated by reduced growth on counter-selective FOA plates, suggested a defect in CENP-ACnp1 chromatin integrity . qChIP analysis detected significantly lower levels of CENP-ACnp1 and a reciprocal increase in H3 levels across the central domain of centromeres in hap2Δ relative to wild-type cells . We conclude that loss of Hap2 specifically affects silencing through loss of CENP-ACnp1 and gain of H3 within the central domain of centromeres, thus Hap2 is required to maintain CENP-ACnp1 chromatin integrity at endogenous centromeres.Central CENP-Apressive , 1995. Dilencing . To testpe cells F,G, conshap2Δ cells exhibited a complete failure to establish functional centromeres, similar to clr4Δ cells that lack heterochromatin, while ies2Δ displayed increased establishment, iec1Δ and ies4Δ were less competent in establishing functional centromeres on pHcc2 . In S. pombe, de novo CENP-ACnp1 chromatin establishment on circular plasmid-based minichromosomes requires a block of heterochromatin in close proximity to central domain DNA  so that the plasmid-borne cc2 is the only copy of this element. qChIP revealed that CENP-ACnp1 was not assembled over cc2 carried by pHcc2 in hap2Δ cells . We conclude that Hap2 is critical for the de novo establishment of CENP-ACnp1 chromatin on naïve central domain centromere DNA.Many factors are known to assist CENP-A maintenance but little is known about the factors required for the de novo establishment of CENP-A chromatin. De novo establishment of functional centromeres can occur following the introduction of naked centromere DNA into cells . We firsmain DNA . Loss of2Δ cells C. Recipr2Δ cells D. qChIP 2Δ cells E. Interepe cells E. This sassembly . In contromosome F, bottomura4 locus on chromosome 3 (ura4:cc2), remains assembled in H3 nucleosomes and exhibits a high rate of histone H3 turnover  where it mediates the turnover of +1 nucleosomes  . Compared with wild-type cells, a significant decrease in the level of H3 turnover was evident on ectopic ura4:cc2 centromere DNA in hap2Δ cells  and over highly transcribed genes (act1+ and spd1+). Ino80 may mediate nucleosome turnover through eviction of H2A.Z . We conclude that Hap2 is required to ensure H3 nucleosome instability on centromeric sequences by mediating a high frequency of H3 turnover, which may consequently allow the incorporation of CENP-ACnp1 in place of H3.Central domain DNA inserted at a noncentromeric location on the arm of a chromosome, such as the turnover . The inhvailable . In S. cleosomes . The reqe (RITE)  to measuonitored A. Import2Δ cells B. Similaof H2A.Z . HoweverCnp1 results in low levels of promiscuous CENP-ACnp1 incorporation at noncentromeric locations , while loss of Hap2 significantly decreased CENP-ACnp1 incorporation within these islands . Interestingly, qChIP also revealed reduced histone H3 turnover within these noncentromeric CENP-ACnp1 islands in hap2Δ cells, whereas histone H3 turnover remained unchanged within the heterochromatic outer repeats, the act1+ gene, or the repressed meiosis-specific mei4+ gene . In contrast, the levels of total and elongating RNAPIIS2P associated with ectopic central domain DNA remained unchanged in hap2Δ relative to wild-type cells . Consistent with reduced transcriptional initiation, lower levels of central domain transcripts were produced from ectopic ura4:cc2 in hap2Δ cells . We conclude that Hap2 is required for efficient transcription from central domain promoters and that this facilitates transcription-coupled histone exchange, thereby providing an opportunity for replacement of H3 with CENP-ACnp1 to establish CENP-ACnp1 chromatin domains and assemble functional kinetochores.Regions upstream of TSS within the central domain of activity . To deteactivity D, inset.Cnp1 chromatin. In addition to kinetochore proteins, all Ino80C subunits, including the small auxiliary subunit, Hap2, were found to be significantly enriched in solubilized CENP-ACnp1 chromatin. Hap2 and the Arp5 Ino80C subunit exhibit similar chromosomal distributions and Hap2 chromatin association depends on the Ies4 Ino80C subunit. Hap2 was found to promote CENP-ACnp1 chromatin integrity at centromeres and to be required for the de novo establishment of CENP-ACnp1 chromatin on introduced naïve centromere DNA. The requirement for Hap2 in ensuring high histone H3 turnover on endogenous centromere DNA, ectopically located centromere DNA, and noncentromeric CENP-ACnp1 islands indicates that Hap2–Ino80C drives H3 nucleosome turnover at these locations. The loss of CENP-ACnp1 incorporation from NCIS islands in the absence of Hap2 underscores the role for Hap2–Ino80C-mediated H3 turnover in stimulating CENP-ACnp1 incorporation. Strikingly, Hap2 is required for efficient transcription specifically from central domain promoters. We propose a mechanism in which Hap2–Ino80C drives the inherent instability of H3 nucleosomes on centromeric DNA via transcription-coupled nucleosome turnover, providing the opportunity for incorporation of CENP-ACnp1 in place of histone H3, when CENP-ACnp1 is available  expected to increase the levels of stalled RNAPIIS2P on central domain chromatin, promote CENP-A incorporation . This indicates that, although lower levels of initiation and transcription take place within ectopic central domain chromatin in the absence of Hap2, elongating RNAPII is retained across the domain. We interpret these observations to indicate that Hap2–Ino80C is required to release elongating RNAPII that becomes trapped or stalled in central domain chromatin. We suggest that resident H3 nucleosomes are evicted by Hap2–Ino80C as part of the process involved in removing this stalled RNAPII. Thus, centromere DNA may be programmed to be pervasively transcribed and stall RNAPII over a relatively large 10-kb region in order to recruit Hap2–Ino80C and trigger H3 turnover throughout this extensive domain, thereby providing the opportunity for CENP-A incorporation. Stalling may result from collisions between converging RNAPII complexes or other obstacles such as the replication origin recognition complex (ORC) which is bound to the many AT-rich tracts present within central domain regions  suggests that Hap2–Ino80C does not mediate H2A.ZPht1 eviction from central domain chromatin and that it must affect some other aspect of centromere promoter function, perhaps through its known nucleosome sliding activity. Thus, Ino80C may be required to slide nucleosomes away from central domain promoters so that they are efficiently transcribed and the resulting transcriptional properties of this domain mediate H3 nucleosome turnover.Histone turnover rates are generally higher for nucleosomes close to promoters . The +1 Cnp1 competes with histone H3 for incorporation into centromeric chromatin and CENP-ACnp1 incorporation is promiscuous, replacing H3 when the opportunity arises , plasmids , and primers  used in this study.Additional methods are described in the Supplemental Material and Supplemental Tables S3–S5. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the Proteomics Identifications (PRIDE)  both coupled online to an Ultimate 3000 RSLCnano system . For details of the separation, mass spectrometer parameters, and data analysis, see the  (PRIDE)  partner Cells transformed with minichromosomes were plated on PMG-uracil-adenine plates and incubated for 5–10 d at 32°C until medium-sized colonies had grown. Colonies were replica-plated to PMG low-adenine (10 µg/mL) plates to determine the frequency of establishment of centromere function. These indicator plates allow minichromosome loss (red colony) or retention  to be determined. In the absence of centromere establishment, minichromosomes behave as episomes that are rapidly lost. Minichromosomes occasionally integrate giving a false-positive white phenotype. To assess the frequency of such integration events and to confirm establishment of centromere segregation function, colonies giving the white/pale-pink phenotype upon replica plating were restreaked to single colonies on low-adenine plates. Sectored colonies are indicative of segregation function with low levels of minichromosome loss, whereas pure white colonies are indicative of integration into endogenous chromosomes and the establishment frequency was adjusted accordingly.hap2Δ strains and grown on minimal medium.LacZ assays were performed as described . PlasmidSupplemental Material. The accession number of the sequencing data reported for Hap2-GFP and Arp5-3HA is GEO GSE141524 and for CENP-ACnp1 (HA and T7 tag), GEO GSE136305.ChIP-seq and ChIP-nexus were prepared essentially as described . For det


Proper regulation of the chromatin landscape is essential for maintaining eukaryotic cell identity and diverse cellular processes. The importance of the epigenome comes, in part, from the ability to influence gene expression through patterns in DNA methylation, histone tail modification, and chromatin architecture. Decades of research have associated this process of chromatin regulation and gene expression with human diseased states. With the goal of understanding how chromatin dysregulation contributes to disease, as well as preventing or reversing this type of dysregulation, a multidisciplinary effort has been launched to control the epigenome. Chemicals that alter the epigenome have been used in labs and in clinics since the 1970s, but more recently there has been a shift in this effort towards manipulating the chromatin landscape in a locus-specific manner. This review will provide an overview of chromatin biology to set the stage for the type of control being discussed, evaluate the recent technological advances made in controlling specific regions of chromatin, and consider the translational applications of these works. Differential regulation of the chromatin landscape (composed of DNA wrapped around histone linkers or octamers) allows for genetically identical eukaryotic cells to perform specific, discrete functions . This isChromatin compaction has been identified as a physical determinant of DNA’s accessibility to transcription-initiating proteins, and these processes are highly regulated through deposition of histone protein variants, ATP-dependent nucleosome remodeling, methylation of DNA, and posttranslational modifications of histone tails . These iProtruding from nucleosomes are N-terminal histone tails, capable of being post- translationally chemically modified, and DNA bases can also be modified at cytosine (C) and adenine (A) nucleotides ,10,11,12The Allis group coined the phrase “the histone code” to describe the observation that distinct histone marks function sequentially or in combination to initiate distinct downstream changes ,13. ChanEukaryotic DNA methylation is most often observed as 5-methylcytosine in the context of a cytosine–guanine (CpG) dinucleotide and is strongly associated with gene repression, though there are some notable exceptions ,22. The 3) [3 is observed in the gene body of transcriptionally active genes, whereas H3K9me3 is linked with gene repression when concentrated near transcriptional start sites (TSS) [3, has been used for recruitment to induce targeted control of chromatin . Methyl groups are transferred from the methyl donor, adenosyl-methionine. KMTs are split into two families based on the presence (or lack) of a SET domain . Mammali3) . The rem3) , whereas3)  utilize 3) ,57,58,593) . For exaes (TSS) ,61. For + [Lysine residues can be acetylated, which typically results in a less condensed chromatin state coupled with an increase in transcriptional activity ,63. Hist+ ,65,67. F+ . BRD-con+ ,68,69 in3 is deposited, followed by hypoacetylation of the gene body [3) results in the deposition of DNA methylation, an epigenetically stable and transcriptionally repressive modification [Models that describe the close interplay between specific chromatin environments and their associated transcriptional activities are based on two characteristics: the physical accessibility of the DNA due to the level of chromatin compaction and the recruitment of transcriptional machinery by chromatin modifications. At the eukaryotic promoter, RNA polymerase II (Pol II) binds to DNA, in coordination with other general transcription factors, and forms the preinitiation complex (PIC) . The chrene body . Lastly,fication . While tfication .For decades, researchers have sought to understand the mechanisms by which the chromatin regulatory machinery functions to control higher-ordered genomes. Understanding the function and transcriptional effects induced by various DNA and histone writers, readers, and erasers are a major goal for basic scientists and translational scientists. There are several ways in which scientists have worked towards understanding and controlling these pathways by modulating the activity of a chromatin modifying enzymes, either by inhibiting their catalytic domains ,77,78, cBy directly binding and inhibiting the catalytic activity of chromatin modifying enzymes, effects on transcription and translation have been inferred. For example, the first two DNMT inhibitors (DNMTi) synthesized, Azacytidine (5-azacytidine) and Decitabine (5-aza-2′-deoxycytidine), are classified as “nucleoside” inhibitors. They mimic the structure of cytidine, are incorporated into the DNA, and form irreversible interactions with DNMTs . SeveralAnother means by which scientists have studied and controlled the chromatin regulatory machinery is through targeted degradation with Proteolysis Targeted Chimeras (PROTACs). These bifunctional molecules bind a protein of interest with one portion of the PROTAC, while the other end binds an E3 ubiquitin ligase protein . ThroughDrosophila are replaced with histone genes incapable of having certain post-translational modifications. In a seminal paper, McKay et al., mutated the H3K27, H3K36, or H4K20 residue from lysine (K) to Alanine (A), which is incapable of being methylated or acetylated and observed that H3K36 is required for viability and H3K27 is essential for maintenance of cellular identity [Lastly, by blocking the chromatin-based target, the function of specific chromatin marks and enzymes can also be inferred. For example, DNMTis have been designed and synthesized in the “non-nucleoside” category. For instance, Procainamide binds CpG regions of DNA to block DNMT-based activity . These tIn the above examples, global chromatin is being impacted, either through inhibiting or degrading the chromatin modifying machinery or by preventing a DNA or histone modification from being deposited. The disadvantage of chemically inhibiting or degrading chromatin regulatory machinery in the whole cell or organism is that many of the existing chemicals bind off-target proteins. Especially in instances where a conserved domain is being targeting , specificity is difficult to achieve ,86,87. TWhile a lot can be learned through global perturbation of chromatin, site-specific technologies offer an opportunity to examine chromatin regulation in the context of a more physiologic setting without gross changes to the cellular environment. To recruit chromatin effectors to a specific gene, technologies have been developed and utilized to genetically modify a gene-of-interest, insert a Gal4-bind arrays, and recruit Gal4-fused chromatin modifiers ,90,91,922-His2 zinc-finger domain has been the most common type of zinc-finger used for these editing platforms. Each zinc-finger consists of roughly 30 amino acids (aa) to form a ββα conformation, with a α-helix and the adjacent turn designed to bind 3 bps of DNA. The optimized systems often use six zinc finger domains strung together, targeting 18 bps. This 18 bp length provides specificity within 68 million bases [VEGF-A locus with a gene-specific zinc finger fused to an HMT. They demonstrated increased H3K9 methylation as well as targeted gene repression [ZFNs were the first of these technologies to be developed for editing the mammalian genome a. The Cyon bases . Zinc fion bases ,100,101.RHOXF2 with a TALE-TET1 fusion. Upon recruitment of this fusion protein, CpG methylation levels decreased and transcriptional activity increased [Transcription Activator-Like Effector (TALE) proteins are found natively in bacteria and contain a series of 33-35 amino acid repeat domains that recognize a single DNA base pair a. Similancreased .The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system provides a technique for mammalian genome editing. Cas9 is the most widely used CRISPR protein for epigenomic engineering. CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) recruit Cas9 to a complementary sequence of DNA and sequence-specific nuclease cleavage is introduced. The CRISPR-Cas system was first discovered in prokaryotes as an adaptive immunity to foreign viruses and plasmid DNA. To apply the CRISPR/Cas9 system to mammalian genome manipulation, a single-chain chimeric RNA was created by fusing crRNA and tracrRNA, termed single guide RNA (sgRNA) . This sgVEGFA and NTF3. In this work they also created a multiple sgRNA targeting system with which a set of sgRNAs can be co-expressed to target the same gene at several places showing that the activation level of the targeted genes was elevated when compared to using only one sgRNA. A variety of different mammalian genes were activated by other groups by directly fusing activating factors, including VP64 [Shortly after Qi et al. published the design of the deactivated Cas9 (dCas9), several labs began to engineer activating protein domains to dCas9 to increase endogenous gene expression. One of the first works published was the dCas9-VP64 fusion from Meader et al. VP64 is a tetrameric repeat of herpes simplex VP16′s minimal activation domain . Meader ing VP64 ,110,111,ing VP64 , VP192 [ing VP64 , p65 AD ing VP64 ,115, Teting VP64 ,117,118,ing VP64   enhancer region with sgRNAs and dCas9-activator fusion proteins, multiple downstream hemoglobin genes were significantly overexpressed simultaneously [3 deposition, decreasing chromatin accessibility [In addition to the promoter region of genes, different publications have shown the ability to target dCas9 fusions to non-promoter regulatory elements as a means to control the chromatin environment. Hilton et al. expanded the targeting region to core enhancers (CE), distal regulatory regions (DRR), proximal enhancers (PE), and distal enhancers (DE). Both dCas9-p300 core and dCas9-VP64 were used in their experiments targeting aneously . Thakoresibility . Kearns sibility . As moresibility . HoweverAfter the development of dCas9 direct fusion technologies, researchers developed dCas9-based indirect effectors. The incorporation of SunTag into the dCas9 system has significantly increased the number of epigenetic modulators being recruited. The SunTag is a repeating peptide array, which can recruit a single chain variable fragment (scFv) antibody fusion. Tenenbaum et al. tested the recruitments of 4, 5, 10, or 24 scFv-VP64 fusions and successfully promoted the regulatory effect by increasing the number of antibody fusions . They coASCL1 than placing the addition loops downstream of the sgRNA stemloops. This improvement likely occurred because either the positioning or the degradation protection of the MS2 protein was improved. In the same study, it was demonstrated that engaging different recruitment strategies (dCas9-VP64 and MCP-P65) was more powerful than using the same activator in both dCas9 and MCP fusions. By utilizing this MS2 stemloop sgRNA, MCP-TET1-CD can activate gene expression by DNA demethylation [Not only have groups focused on the dCas9 fusion protein engineering, but also on the scaffolding sgRNAs (scRNAs). By engineering the sgRNA with additional stem loops, the recruitment power of the sgRNAs can be increased. Zalatan et al. created three scRNA systems and compared the recruitment efficacy between them. They engineered MS2 stemloops to recruit MS2-capping proteins (MCP), PP7 stemloops to recruit PP7-coat proteins (PCP), and Com stemloops to recruit Com proteins. By fusing MCP, PCP, and Com proteins to VP64, transcriptional activation through stemloop-based recruitment was observed. Furthermore, they developed a two-stemloop version with identical stemloops in the scRNA and the activation was improved . Furtherhylation . With thhylation .While researchers were focusing on controlling gene expression by utilizing synthetic devices, several labs began developing chemically responsive gene expression control constructs. By introducing small molecules into biological systems, gene expression can be controlled in a temporal manner. The Conklin lab took advantage of doxycycline Dox; a and iPSApart from the two chemically dependent systems mentioned above, there is another approach, chemical inducers of proximity (CIP), being applied to both study and control chromatin biology. This system utilizes a class of bifunctional molecules, which can recruit two protein fusions together and initiate a new biological response. The first CIP was a homodimer of FK506 (FK1012) . FK506 iASCL expression at a level comparable to the full-length dCas9-VP64 (57-fold overexpression compared to untreated cells). The Qi lab also developed a highly intricate dCas9 and CIP system. They utilized both ABA and GA to create dCas9-based synthetic gene circuits. They can recruit activating or repressive machinery in a chemically dependent manner by creating dCas9-AB1/Pyl1-fusions and dCas9-GAI/GID1 fusions. The ABA- and GA- inducible recruitments were reversible and orthogonal [With the development of CRISPR/Cas9, many labs have incorporated CIPs and dCas9 fusion proteins to control chromatin structure and gene expression. The Crabtree lab developed a dCas9-based rapamycin, FKBP, and FRB system. By creating ms2-FKBP and FRB-chromatin regulator fusions , they achieved reversible control of the chromatin environment of endogenous mammalian genes and a change in transcription activities . Zetschethogonal . Anotherthogonal . A rapamthogonal  and Stanthogonal .Optogenetic systems have an advantage in precise spatial and temporal controls. Polstein and Gersbach engineered a light-activated dCas9 effector (LACE) system to induce the transcription of endogenous genes by illuminating blue light . They uttrans-splicing strategy (PTS) and designed dCas9-InN  and InC-effector. They tested dCas9-InN and InC-VP64 as a direct fusion, which led to transcriptional activation of targeted genes (IL1RN and NTF3). Next, they expanded the effector into engineered InC-JQ1x4  and InC-UNC3866 (synthetic peptide) and recruited endogenous BRD4 and PRC1 complexes, respectively [Similar to the split dCas9 protein technology, the Muir lab used another split intein-mediated protein ectively .CXCR4, MYOD1, etc.) in multiple cell lines, and CEM87, which is composed of FK506 and iBet762 (a BRD inhibitor), was determined to be the best-in-class CEM. With different concentrations of CEM treatment, graded activation of targeted genes can be achieved. CEM can also provide reversible gene activation by washing out the molecule. To determine the off-target effects, ChIP-sequencing (ChIP-seq) and RNA-sequencing (RNA-seq) were conducted. With expression of the targeting sgRNA and addition of CEM87, no off-target effects were observed, which is indicative of the specificity of CEM technology.Recruiting endogenous chromatin modifying enzymes has a great advantage over exogenous engagement in the simplicity of the technology. With the development of dCas9 approaches to regulate chromatin structure, research groups have expanded the application, utilizing chemical endogenous recruiting warheads. We have developed a bifunctional molecule named chemical epigenetic modifier (CEM), which has two functional components, FK506 and a molecule interacting with cellular epigenetic machinery  Figure b. We incFXN) gene and were conjugated to JQ1 (BRD4 ligands). By recruiting BRD4 to FXN, JQ1 can engage the transcription elongation machinery P-TEFb and facilitate transcription elongation. Activation of FXN was observed in lymphoblastoid cell lines, and no off-target effects were seen with RNA-seq and ChIP-seq. With treatment of Syn-TEF to overexpress FXN, they induced iPSCs to differentiate into cardiomyocytes that expressed cardiac-specific markers and were observed to beat rhythmically in culture. Finally, they examined the utility of Syn-TEF in vivo by establishing an immunocompromised mouse model bearing a luciferase reporter fused with FXN, and upon Syn-TEF treatment luciferase expression was stimulated. More ATF technologies can be found in the review written by Heiderscheit et al. [Transcription factors (TFs) are diverse signaling proteins that play important roles in gene expression and cell differentiation in a dynamic manner. During cellular development, transcription factors interpret signals and initiate programmed transcriptional changes. Artificial transcription factors (ATFs) have been designed and customized to reprogram somatic cells into induced pluripotent stem cells (iPSCs) . Repurpot et al. .Discoveries over the past few decades have demonstrated the importance of chromatin regulatory pathways and have implicated their dysregulation in several human diseases . TechnolWith the understanding that changes in chromatin can contribute to disease initiation, growth, and/or therapeutic resistance, small molecules have been developed to study epigenome modulators and regulate their activity . The ideIn some cases, groups have been able to specify a tumor suppressor or oncogene for a given disease, in which the chromatin environment is altered at the gene locus, thereby affecting the transcriptional activity . These eStaphylococcus aureus dCas9) [sa-Cas9 proteins lacking non-essential domains  ,219, smas dCas9)  and deacs dCas9) ,222), CRs dCas9) , moleculs dCas9) , and sa-ini-Cas9 ). The smini-Cas9 .Development of new ways to tether proteins and proteins associated with RNA to chromatin has opened up the ability to modulate specific endogenous genes by an assortment of chromatin modifying activities. Using gene-specific targeting technologies in conjunction with chromatin regulators has allowed for directed epigenomic engineering. Furthermore, bringing temporal and spatial control with these technologies has allowed finer governing of chromatin state at specific times and in subsets of cell populations. These technologies have been used to study the mechanisms of chromatin regulation in new ways and across a diverse set of genes. Modern research is also examining the ability of these approaches to be used to treat human diseases, yet most of this is still in the preclinical setting. The overexpression of synthetic effectors is required in most of the technologies summarized in this review, having undesirable impacts to the epigenome. While epigenomic engineering has been empowered by technology, it is likely to be driven further by techniques that are yet to be developed. Expect the future to hold a wider assortment of chromatin regulatory activities to be tethered to chromatin as only a subset of all the wide array of chromatin regulatory enzymes have been tested by these approaches. Additionally, new methods to anchor chromatin-modifying enzymes to target genes will lead to even more effective and specific chromatin modulation than current techniques.


In Drosophila, the function of grainyhead (grh) gene is essential during developmental processes such as epithelial differentiation, tracheal tube formation, maintenance of wing and hair polarity, and epidermal barrier wound repair. Three main mammalian orthologs of grh: Grainyhead-like 1-3  are highly conserved in terms of their gene structures and functions. GRHL proteins are essentially associated with the development and maintenance of the epithelial phenotype across diverse physiological conditions such as epidermal differentiation and craniofacial development as well as pathological functions including hearing impairment and neural tube defects. More importantly, through direct chromatin binding and induction of epigenetic alterations, GRHL factors function as potent suppressors of oncogenic cellular dedifferentiation program – epithelial-mesenchymal transition and its associated tumor-promoting phenotypes such as tumor cell migration and invasion. On the contrary, GRHL factors also induce pro-tumorigenic effects such as increased migration and anchorage-independent growth in certain tumor types. Furthermore, investigations focusing on the epithelial-specific activation of grh and GRHL factors have revealed that these factors potentially act as a pioneer factor in establishing a cell-type/cell-state specific accessible chromatin landscape that is exclusive for epithelial gene transcription. In this review, we highlight the essential roles of grh and GRHL factors during embryogenesis and pathogenesis, with a special focus on its emerging pioneering function.Among the transcription factors that are conserved across phylogeny, the  Originally identified in Drosophila (previously known as Elf-1 or NTF-1) as an embryonic lethal locus, grh mutant Drosophila embryos show immature cuticle development, patchy tracheal network and the most notable ‘granular’ head skeleton aberration  in Drosophila. Recent critical reviews have discussed the role of LSF/CP2 subfamily members  in various aspects of development and human diseases including cancer . Additionally, we focus on human GRHL2 as an important determinant of the epithelial phenotype during development and as a gatekeeper of epithelial differentiation in several human cancers. Finally, we also discuss in detail the novel pioneering role of grainyhead and Grainyhead-like proteins in contouring the chromatin landscape during embryonic development and cancer progression.The erration . The LSFerration . Therefoerration . These tg cancer . In thisgrh gene in Drosophila and Caenorhabditis elegans exists as multiple orthologs in the vertebrates, which are denoted as grainyhead-like (grhl) genes. These genes remain evolutionarily conserved from insects to humans. In addition, a varying number of splice variants generated from alternative splicing events and alternative transcriptional initiation sites further highlight the underlying complexity and their gene regulatory networks operating during development and disease progression (grainyhead exist in humans: Grainyhead-like 1 (GRHL1), Grainyhead-like 2 (GRHL2) and Grainyhead-like 3 (GRHL3). Each protein contains three annotated functional domains: a transcriptional activation domain (TAD) at the N-terminus; a central DNA-binding domain (DBD) structurally similar to the equivalent of p53, and a dimerization domain (DD) at the C-terminus with unique ubiquitin-like folds. The TAD is the least conserved domain between Drosophila and mammalian orthologs, which could be partially due to the presence of an isoleucine-rich segment that has poor conservation across phylogeny  and en (GTGAGCCGGCGAAACCGGTT), whereas the binding motif on Ubx and ftz promoters is (T/C)NAAC(C/T)GGT(T/C) (Cdh1 (ACCAGTCAAACCAGTAAT) and the promoter of Cldn4 (ACCAGAGATACTGGTCAA) are strikingly similar to the human GRHL2 binding motif on the intron 2 of the CDH1 (GCACCAGCCAAACCAGTTAAT) and the promoter of CLDN4 (GGACCAGAGATACTGGTCAAAG) , with different levels of variability for some target genes . In DrosGGT(T/C) . In mammTGGTCAG) . The invTGGTCAG) . A recenTGGTCAG) .In metazoans, two major cell types form the basis of organ development: epithelium and mesenchyme. Epithelial cells are generated first during the embryonic development, while mesenchymal cells are derived from the pre-existing epithelial cells through a process called epithelial-to-mesenchymal transition (EMT) . EpithelThe recurrence of EMT is also observed during the early development of the nervous system to generate neural crest cells. During the embryonic process termed neurulation, epithelial neural plate  bends, invaginates and fuses along the dorsal midline to form a cylindrical structure called the neural tube. Subsequent closure of the neural tube in anterior and posterior directions guides formation of the future brain and spinal cord . MeanwhiDrosophila, the formation of protective exoskeleton called cuticle during the early stages of larval development serves many important functions during the adult life including the protection against water loss and the maintenance of structural framework for locomotion. grh remains to be essential for the development of the epidermal barrier and the repair of barriers after wounding. The embryonically lethal larval cuticles of grh mutants are multilayered and grossly inflated structures, generating the “blimp” phenotype that are functionally weaker when compared to wild-type cuticles , one of the two key enzymes contributing for the formation and hardening of larval and adult cuticles. This results in the defective phosphorylation of grh by ERK which is required for wound-dependent regeneration of the epidermal barrier  have identified the direct regulation of Grh on major targets including stit, msn, cora, sinu, and fas3  and mice deficient of Grhl1 show an abnormal desmosome phenotype in the interfollicular epidermis, ultimately delaying the initial skin coat growth and poor hair anchoring to the follicle , and an epithelial-specific member of small guanosine triphosphatase Rab25, which are crucial for epithelial differentiation  signaling pathway (Grhl3 have digit fusion (syndactyly) due to abnormal adhesion of the periderm covering the developing digits  ectoderm prior to neural tube closure. The fate choice between neural and surface ectoderm is highly regulated through signaling interplay of Wnt, FGF and BMP activity . At the a bifida . Single-n humans . Therefogrhl3/Grhl3 in zebrafish and mouse embryos do not affect any stages of neural crest cell development and activity  and Claudin 4 (Cldn4), are significantly reduced in the surface ectoderm leading to neural tube defects  expression and demonstrated the presence of a positive feedback loop between GRHL2 and AR to promote prostate cancer growth  functionally dictate the cellular dedifferentiation and differentiation status respectively and determine the nature of cellular behavior. Although EMT/MET processes occur spontaneously during fundamental events such as gastrulation, neural crest dissemination and organogenesis, execution of EMT/MET is also observed during wound healing, fibrosis and cancer . Overcomgression . In briegression . The expgression , ovariangression , bladdergression , and sargression . On the gression , ovariangression  cancers gression .high/CD24low mesenchymal subpopulation cells of breast cancer cells and restores the anoikis sensitivity by altering intracellular H2O2 ROS levels  and/or polycomb repressive complex 2 (PRC2) to generate a repressive chromatin around epithelial genes . GRHL2 aEvidences have shown that GRHL members are the gatekeepers of early phenotype transition. Our assessment on a heterogeneous ovarian cancer cell line panel revealed the presence of intermediate cellular phenotypes that dually expressed epithelial and mesenchymal markers . ConsequDrosophila larvae carrying grh mutations have pronounced deformation in the chitinous head-skeleton morphology generating a granular head appearance  has revealed that GRHL2 expression is prevalent in cochlear duct lining cells, hair cells, and supporting cells of the inner ear  are often observed in genes that are suppressed at a particular cell state/type (On top of gene expression regulations through ate/type . A staggate/type . Alteratate/type . GRHL faUpkII) in mouse bladder epithelial cells is also regulated through Grhl3-mediated active H3K9ac mark enrichment on the UpkII promoter  that are essential for lumen expansion and barrier formation , keratin 1 (KRT1), filaggrin (FLG) and cyclin dependent kinase inhibitor 2A (INK4A) (TGM1 promoter to control the expression of transglutaminase (TGM1)  . During e (TGM1) , a Ca2+-envelope . GRHL3 fntiation . These sAbout two meters long, the double stranded DNA is condensed and packed into a typical eukaryotic interphase nucleus that only measures about six micrometers in diameter. This composite level of condensation starts primarily by wrapping the DNA around an octameric protein complex made of four core histones into a structure called nucleosome. Arrays of nucleosomes undergo further condensation into higher-order chromatin structures that functionally demarcate the chromatin boundaries into densely condensed heterochromatin and relatively less compressed euchromatin units. Although the high level of DNA packaging significantly deals with containing the genetic material in a miniscule space, DNA access to gene regulatory proteins during key cellular process such as transcription is greatly restricted. In this context, one crucial question is “How do TFs find their way to gene regulatory elements that are repressed or latent amidst these convoluted nucleosomal barriers in order to initiate transcription for diverse cellular processes?” A new set of regulatory proteins called ‘pioneer factors’ have been identified to accomplish this phenomenal task .cis-regulatory units within permissive heterochromatin, and subsequently prime the chromatin for additional factors to bind, prior to transcription initiation (Pioneer factors belong to a unique class of TFs that can recognize and bind specific itiation . The pioitiation . Compellcis-regulatory elements (such as enhancers) that recruit the binding of transcription factors, cofactors and collectively form a stable regulatory complex. Promoters are usually a minimal stretch of DNA sequences, located in proximity to the transcription start sites within a nucleosome-free chromatin landscape to enable easy access to the transcription machinery. In contrast, enhancers tend to be located far (either upstream or downstream) from the cognate promoters and enhance transcriptional outputs in a cell-type/state specific and spatiotemporal manner  to the primed pluripotent epiblast-like cells (EpiLCs), GRHL2 binds to latent enhancers (regions with low or no histone marks) and restores the epigenetic landscape toward an activation state  of key genes that control the epithelial state and cell adhesion . ImportaMARVELD3, ESRP1, GRHL1, RAB25, OVOL2 and MUC20 (PCDH1) and serine peptidase inhibitor 1 (SPINT1)  could be annotated using an automated ChromHMM algorithm . In ovarnd MUC20 . Upon rend MUC20 . However(SPINT1) . After p(SPINT1) . ChIP-se(SPINT1) . This in(SPINT1) . Indeed,(SPINT1) . This co(SPINT1) . These dDrosophila to mammals, and is a conserved feature of the three-dimensional chromatin architecture . The presence of TADs is evident across many species, ranging from itecture . These ditecture . Therefoitecture . The funitecture . In the pression . Using cial gene . FormatiERBB3 and PERP) is crucial for their expressions  model has showed that Grh functions as a transcriptional repressor to regulate genes essential for the development of epithelium and molting of old integument (ecdysis)  . This inGrhl genes were implicated in multiple human abnormalities such as hearing impairment, ectodermal dysplasia syndrome, and cleft palate formation, somatic mutations in GRHL genes in human cancer samples occur at a very low frequency  and potentially regulate their access along the epithelial gene loci during cellular differentiation/dedifferentiation. Of note, chromatin conformational changes during EMT/MET programs during development and cancer progression are starting to emerge recently . Since GVS and RH conceptualized and wrote the complete first version of the manuscript. All authors contributed to the manuscript revision, read, and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Importance of sperm-derived transcripts and chromatin imprints in organismal development is poorly investigated. Here using an integrative approach, we show that human sperm transcripts are equally important as oocyte. Sperm-specific and sperm-oocyte common transcripts carry distinct chromatin structures at their promoters correlating with corresponding transcript levels in sperm. Interestingly, sperm-specific H3K4me3 patterns at the lincRNA promoters are not maintained in the germ layers and somatic tissues. However, bivalent chromatin at the sperm-specific protein-coding gene promoters is maintained throughout the development. Sperm-specific transcripts reach their peak expression during zygotic genome activation, whereas sperm-oocyte common transcripts are present during early preimplantation development but decline at the onset of zygotic genome activation. Additionally, there is an inverse correlation between sperm-specific and sperm-oocyte lincRNAs throughout the development. Sperm-lincRNAs also show aberrant activation in tumors. Overall, our observations indicate that sperm transcripts carrying chromatin imprints may play an important role in human development and cancer.  •Sp-lincRNAs carry distinct chromatin structures correlating with transcript levels•Sp-lincRNAs are active during ZGA in preimplantation developmental stages•SpOc-lincRNAs are active in pre-ZGA and decline at the onset of ZGA•Sp-lincRNAs are silent post implantation but show aberrant cancer-specific activation Chromosome Organization; Endocrinology; Transcriptomics Maternal or oocyte-derived transcripts are known to regulate maternal to zygotic transition (MZT), which involves zygotic genome activation (ZGA) and maternal transcript degradation. These are the two important events necessary for subsequent preimplantation embryonic development . On the Cancer testis antigens (CTAs) are a group of testis-specific proteins that show predominant expression on cancer cells. This observation suggests a functional connection between testis-specific expression and cancer. Majority of the CTA genes are preferentially expressed in testicular cell types such as spermatogonia or spermatocytes. Hence, we were particularly interested in characterizing whether sperm-derived RNAs show any preferential expression in different tumor types, which would in turn pave the way for understanding the contribution of sperm-derived transcripts in tumor development and progression .At the outset, we analyzed the transcriptome profiles of sperm, oocyte, and preimplantation embryo stages (two cell to late blastocyst) in human and mouse  Figure A. We incOur analysis show that sperm and oocyte, from both mouse and human, harbor comparable number of transcripts, and in particular, mouse harbors nearly twice as many transcripts in sperm compared with oocyte B and 1C.-K27-)  A. In Sp-) (-K27- B. In mou) (-K27- C. In the) (-K27- . Mouse S) (-K27- D. We nexspecific A and 2E.specific  and S3. specific B–S1D. Th-K27-) showed immune defense-related functions such as immune response, neutrophil chemotaxis, and defense response  from sperm-derived chromatin clusters show highly temporal expression patterns with no expression in early preimplantation-stage embryos and specific activation between four- and eight-cell stages of embryos A. Like i-K27- and low-K4 sperm-derived chromatin clusters from four-cell stage onward. More importantly, SpOc PCGs from low-K4 group become super activated during preimplantation development , mesoderm (middle layer), and endoderm (innermost layer) A. Each iromoters A.Figure -K27- clusters maintain the same chromatin structure at their promoters in all the three germ layers and somatic tissues. Interestingly, sperm-derived chromatin clusters from SpOc-PCG promoters showed higher enrichment of H3K4me3 in all the three germ layers and their derived somatic tissues. However, sperm-derived bivalent domains from SpOc-PCG promoters maintained their bivalent chromatin structure in all the three germ layers but in the germ-layer-derived tissues only traces of bivalency were maintained , meiosis , and post-meiosis (round spermatid) stages of spermatogenesis . Sp-lincIn summary, tracing the expression dynamics from different spermatogenic cell types, gametes (matured sperm and oocyte), and preimplantation (zygote to late blastocyst) and post-implantation stages (germ layers and matured tissues) reveal that Sp and SpOc transcripts have opposing expression patterns throughout the human development C.cis- or trans-acting regulatory elements can control the transcription of genes , prostate (PRAD), testis (TCGT), and uterine or ovary -related cancers.Previous studies have shown that RNAs involved in organism development and having testis-specific expression are known to take part in cancer development and progression . To democlusters A; thus, clusters B. One coclusters . A detaiclusters A and S9AP4HA3-AS1, LINC01518, and AP001476.1), representing each of the three sperm-derived chromatin clusters, in human embryonic kidney cell line HEK-293 and cervical carcinoma cell line HeLa. We found that the Sp lincRNAs show higher expression in HeLa cells compared with the HEK293 cells  occurs, even though their expression is restricted to meiotic or pre-meiotic spermatogenic cell types. These observations indicate that low levels of transcription may persist during sperm maturation, and this notion is consistent with the data that sperm transcripts differs in caput and cauda and moreover, as discussed earlier, sperm RNA alterations in response to motility and capacitation  transcripts were identified based on the microarray analysis of RNA from the ejaculated sperm of nine individuals . The precitation . Thus, fThe next important question is whether genes encoding sperm-specific transcripts and/or the transcripts per se have any role in preimplantation development. Multiple lines of evidence suggest that sperm transcripts play an important role in early preimplantation development. For example, defective blastocyst embryos generated from dicer knockout germ cells can be rescued with sperm RNA . SimilarAnother interesting aspect of the current investigation is the temporal expression of genes that encode sperm-specific and sperm-oocyte transcripts during preimplantation development. Genes that encode sperm-specific transcripts carry distinct chromatin structures and show stage-specific expression across preimplantation stages. This exclusive stage-specific expression is not seen with genes that encode sperm-oocyte transcripts, rather the majority of these transcripts show expression during early preimplantation stages, primarily between two- and four-cell stages and start to decline or degrade during the onset of ZGA. Highly temporal expression of genes that encode sperm-specific transcripts in the preimplantation stages signifies their importance during early preimplantation development. Moreover, genes that encode sperm-specific and sperm-oocyte transcripts have crucial non-overlapping roles in preimplantation development. It is reasonable to assume that the sperm-inherited chromatin imprints together with the help of preimplantation development associated transcription factors may bring in the observed stage-specific expression . Indeed,-K27-, and bivalent (H3K4me3/H3K27me3) promoters, transcripts are enriched in sperm, and they express throughout preimplantation development, the germ layers, and in multiple somatic tissues. Thus, sperm-encoded information seems to take part in important developmental decisions.Strikingly, the expression of Sp-lincRNAs as well as their promoter epigenetic profiles were lost in the three germ layers and their derived somatic tissues. This again emphasizes the importance of the genes that encode sperm lincRNAs in fertilization and preimplantation development. On the other hand, genes that encode Sp-PCGs and SpOc transcripts (SpOc-lincRNAs and SpOc-PCGs) continue to express in all the three germ layers and the germ-layer-derived multiple tissues. Comparison of the histone profiles of sperm, germ layers, and mature tissues revealed that Sp-PCGs and SpOc-PCGs retain their sperm-specific bivalent chromatin in the germ layers and somatic tissues; however, the ratio of H3K4me3 and H3K27me3 is different from that in the sperm. These observations indicate that the sperm-derived bivalent chromatin imprints at the Sp-PCGs and SpOc-PCGs promoters are maintained during the germ layers formation and these sperm-inherited chromatin imprints may play an important role in crucial developmental decisions by regulating spatiotemporal expression of Sp-PCGs and SpOc-PCGs. Thus, our study found two sets of genes: one set, sperm-specific genes with high-K4 promoters, whose transcripts are enriched in sperm, activated during ZGA and transcriptionally silenced in the three germ layers and somatic tissues. In the other set of genes, comprising Sp-PCGs, SpOc-lincRNAs, and SpOc-PCGs with high-K4, low-K4, K4-K27-). Interestingly, the level of their deregulation in cancer matches with the sperm H3K4me3 levels at the promoters. For example, lincRNAs with high-K4 levels at their promoters showed more deregulation in cancer compared with the lincRNAs with low-K4, indicating that mechanisms that establish sperm-specific chromatin imprints are recapitulated in cancer. Their higher expression in tumors compared with the corresponding normal tissues indicate that they may behave as oncogenes. We have also tested the oncogenic properties of selected lncRNAs from high-K4, low-K4, and K4-K27- clusters in HeLa cell line by measuring their effect on crucial cancer cell hallmarks that define the oncogenic drivers such as cell proliferation, cell cycle progression, and apoptosis. Loss of function of these lncRNAs in HeLa cells using siRNAs resulted in decrease in cell proliferation, cell cycle progression, and apoptosis, indicating that these sperm lincRNAs harbor oncogenic properties. Thus, Sp-lincRNAs may serve as comprehensive resource for diagnostic/prognostic markers and potential therapeutic targets. SpOc-lincRNAs also showed deregulation in multiple cancers but to a lesser extent compared with the Sp-lincRNAs. Surprisingly, Sp-PCGs and SpOc-PCGs were mostly downregulated in cancers, and this in particular is consistent with their role in immune response regulation as immune response genes are mostly suppressed in many cancers.Previous studies have proposed a functional link between early embryonic gene expression program and cancer development and progression . In partIn sum, the abundance of RNA, in particular lincRNAs, in sperm necessitates further investigation on their importance in sperm maturation, fertilization, and preimplantation development and cancer. More importantly, our study laid a strong basis for further investigation on the functional role of sperm-inherited chromatin imprints in organismal development.Lack of chromatin data (ChIP-seq) for different stages of human preimplantation embryos limited our possibility to explore whether sperm-derived chromatin imprints are preserved during preimplantation development. Exploring this would have added an in-depth resolution to our analysis.kanduri.chandrasekhar@gu.se.Correspondence to lead contact Chandrasekhar Kanduri, E-mail: This study did not generate any new sequencing data. Analysis was performed using public datasets.https://dx.doi.org/10.17632/695c4zvr6d.1.The processed datasets generated during this study are available publicly at Mendeley Data repository with the following DOI All methods can be found in the accompanying


Among 760 tested genes linked to known neuronal pathways, knockdown of circZNF827 deregulates expression of numerous genes including nerve growth factor receptor (NGFR), which becomes transcriptionally upregulated to enhance NGF signaling. We identify a circZNF827-nucleated transcription-repressive complex containing hnRNP-K/L proteins and show that knockdown of these factors strongly augments NGFR regulation. Finally, we show that the ZNF827 protein is part of the mRNP complex, suggesting a functional co-evolution of a circRNA and the protein encoded by its linear pre-mRNA host.Circular RNAs are important for many cellular processes but their mechanisms of action remain poorly understood. Here, we map circRNA inventories of mouse embryonic stem cells, neuronal progenitor cells and differentiated neurons and identify hundreds of highly expressed circRNAs. By screening several candidate circRNAs for a potential function in neuronal differentiation, we find that  The mammalian non-coding transcriptome, which includes long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs), plays pivotal roles in biological decisions during differentiation and normal cell maintenance , leading to increased mRNA expression of their respective miRNA-targets , promotes the destruction of Mir7-1, which in turn upregulates ciRS-7 by a still unidentified mechanism , which becomes robustly upregulated upon circZNF827 knockdown. Mechanistically, our evidence suggests that circZNF827 is a necessary scaffold for a transcription-repressive complex containing its own host-encoded protein; ZNF827, together with hnRNP K and hnRNP L.Despite these intricate molecular interactions between circRNA, miRNA, and lncRNA, many important questions regarding neuronal differentiation and function remain unanswered. For example, it is largely unknown how the tightly controlled expression of circRNAs potentially affects neuronal development. Here, we present the circRNA inventory of mouse embryonic stem cells (mESC), neuronal progenitor cells (NPC) and differentiated glutamatergic neurons, which represents a well-established model for CNS-type neuronal differentiation . We repoto-be called circRNAs, we first performed standard rRNA depletion and subsequently either included or excluded RNase R treatment step prior to RNA-sequencing. Specifically, we used an established differentiation model for CNS-type glutamatergic neurons, based on E14 mouse embryonic stem cells (mESCs) that reportedly yields a purity of glutamatergic neurons of >90% , while stem cell pluripotency marker Nanog became significantly reduced upon differentiation (circular-to-linear ratio of identified circRNAs (find_circ), by comparing splice site usage in circular vs. linear splicing events  using RT-qPCR with amplicons across the backsplicing junction  by lentivirally delivered dishRNAs  (circZNF827 (and to a lesser extent circANKIB1), produced a significant and reproducible increase in neuronal marker expression upon differentiation   and subsCAMSAP1) . While tntiation . Importanockdown . The uprkinetics . This phcircZNF827. Indeed, knockdown of circZNF827 leads to a moderate but significant increased expression (1.5–2.5 fold) of RARB and RARG, while RARB remained constant   measured at steady-state, our results suggest that circZNF827 contributes to controlling the RARA receptor levels in order to keep neuronal differentiation in check.We next asked whether the retinoic acid receptors (RARs), which represent central nodes in relaying anti-proliferative differentiation cues during neuronal development , and areconstant . Since m signal) . We therrcZNF827 . Next, wnockdown . AlthougcircZNF827 levels are low. To test how circZNF827 knockdown affects other key factors of the neuronal transcriptome, we next performed Nanostring analyses using a neuro-differentiation/pathology panel of 760 genes with RNA purified from differentiated or non-differentiated L-AN-5 cells. 135 genes become differentially expressed  due to circZNF827 knockdown after differentiation , which plays a central role in regulating neuronal differentiation, death, survival, and neurite outgrowth (Phosphatase and tensin homolog (PTEN), STAT3 and NAD(P)H quinone dehydrogenase 1 (NQO1) were all significantly downregulated upon circZNF827 knockdown (2–4 fold), which reportedly also contributes positively to neuronal differentiation  at the level of transcription  vectors proved very efficient in depleting nuclear circRNA  and control RNAs  in vitro and subjected these to pull-down experiments using L-AN-5 cell lysates and streptavidin-coupled magnetic beads as previously described  bound strongly to any of the bait RNAs (circZNF827 compared to IgG controls (>100 fold), suggesting that these interactions can be recapitulated in L-AN-5 cells , proved unfeasible due to low expression levels of the ZNF827 gene in the K562 and HepG2 cells used by ENCODE. Using RBPmap , but these interactions remain unaffected by increased expression of circZNF827   , suggestcircZNF827 complex could facilitate transcriptional repression of NGFR by interacting with gene-regulatory regions, consistent with NGFR upregulation upon circZNF827 knockdown. To this end, publicly available ChIP-seq data (ENCODE consortium) in K562 and HepG2 cells demonstrate that hnRNP K indeed interacts with transcription regulatory regions  of the NGFR gene , and ZNF827 protein co-immunoprecipitated circZNF827 more efficiently than GAPDH mRNA (~19 fold more enriched)  . Finallyckground .circZNF827 represses NGFR transcription  by bridging a hnRNP K/L-contaning inhibitory complex with their genomic loci, facilitated by the ZNF827 protein.Taken together, our results are consistent with a model where circZNF827 nucleates the hnRNP-ZNF827 complex to potentially prepare/activate it for engagement with chromatin, or whether circZNF827 itself, is part of the chromatin-bound complex. To address this question, we first cloned a circZNF827 expression construct, which includes a sequence encoding 2 X MS2 hairpins (circZNF827-MS2). Next, we transfected these cells, HEK293 control cells (Empty) or HEK293-circZNF827-WT (lacking MS2 hairpins) with a plasmid encoding FLAG-tagged MS2 coat protein (FLAG-MS2cp) and subsequently performed anti-FLAG ChIP analysis, to test whether the FLAG-tagged MS2cp becomes crosslinked to the NGFR promoter via circZNF287-2XMS2. The NGFR promoter signal was significantly higher in HEK293 cells harboring the circZNF827-2XMS2 expression cassette, when compared to both control cell lines and background signal on the GAPDH promoter remained constant in all three cell types , signals from the four mammalian neurotrophins (Nerve Growth Factor (NGF)), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NTF3) and neurotrophin 4 (NTF4) -end-labeled oligonucleotides  according to a standard PEI transfection protocol. 5 hr after transfection, MS2 coat protein and circRNA expression were induced by addition of 100 ng/ml tetracyclin to the cell culture medium. The ChIP assay including crosslinking and harvest of cells were carried out using the Pierce Magnetic ChIP Kit (Thermo Scientific) according to the manufacturer’s protocol except that sonication was carried out on a Covaris S2 ultrasonicator  and 10 μl Anti-FLAG M2 beads (Sigma-Aldrich) blocked with 10 ng/ml FLAG-peptid (Sigma-Aldrich) were used for IP of the FLAG-tagged MS2 coat protein. The antibodies used for the ChIP assay are listed in Lentiviral transduction and RA-mediated differentiation of L-AN-5 cells were carried out as described in the section ‘circRNA knockdown and differentiation of L-AN-5 cells’. HEK293 Flp-In T-Rex, HEK293 Flp-In T-Rex In biochemical assays , the significance of difference between samples were calculated by a two-tailed Student’s t test to test the null hypothesis of no difference between the two compared groups. The assumption of equal variances was tested by an F test. p<0.05 was considered statistically significant. Data are presented as mean ± SD. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Circular RNAs have been previously shown to be noncoding RNAs capable of titrating away microRNAs or RNA binding proteins. Here the authors show a role for a circular RNA at chromatin for control of gene expression. circZNF877 is shown to interact with its host zinc finger protein and two RNA binding proteins to nucleate a gene repressor complex at target gene loci.Decision letter after peer review:circZNF827 nucleates a transcription inhibitory complex to balance neuronal differentiation" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and James Manley as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife". Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at Summary:circZNF827 increases the expression of several neuronal genes and reduces the S-phase fraction. Later, the authors found that circ-FP827 controls the expression of two retinoid receptors (RARa and RARg) posttranscriptionally. Using Nanostring, the authors showed that circZNF827 knockdown increases the expression of several neuronal genes  and subsequently a known downstream gene of NGFR, c-fos. To understand how circZNF827 regulates the expression of downstream genes transcriptionally or post-transcriptionally, the authors performed pull-down followed by LC-MS/MS and identified hnRNP K and L as its main interactors. Next, the authors found out that knocking down of circZFP827 increases the cytoplasmic level of hnRNP K and overexpressing of circZFP827, in contrast, generates multiple distinct nuclear foci. Finally, the authors showed that circZNF287 downregulates NGFR transcription by interacting with hnRNP k inhibitory complex and the ZNF827 protein.Hollensen and coworkers identified the circRNA at different various neuronal differentiation stages using three different circRNA prediction tools and found circZFP827 together with other circRNAs have temporal expression patterns during differentiation. The authors decided to focus on circZFP827 and showed that downregulation of circZNF827 and its mechanism of action through affecting nuclear RNA binding proteins hnRNPK and hnRNPL.While the reviewers find the paper to be of interest, there are several points that require clarification with additional data before the paper can be considered for publication. The key points relate to the copy number of nuclear Essential revisions:circZNF827 and show that the wildtype circRNA rescues the NGFR expression phenotypes but a circRNA with mutated K and L binding sites does not. The authors should also use a technique like ChIRP to show direct binding of the circRNA to the NGFR locus.1) The authors' model is that the small (10%) amount of the circRNA that is present in the nucleus has a functional role via interacting with hnRNP K and L. This is possible but more direct functional experiments should be included to justify the model. For example, the authors should express dishRNA-resistant versions of 2) The retinoic acid receptor data  is poorly integrated into the rest of the manuscript and the data quality is weak. The supplemental figure shows that RARalpha has a slight (20%) increase in transcription that is insufficient to explain the mRNA effect sizes in the main figure. There is no significant change in RARgamma transcription or decay rates, yet the mRNA expression is increased. Most critically, the authors have failed to connect the proposed hnRNP K/L based mechanism  to RAR transcriptional regulation.3) The biochemical data supporting a complex comprising hnRNP K and L is weak and confusing. hnRNP K can pull down a little bit of L, but hnRNP L pull down does not appear to pull down K . This is relevant to the author's main model as the circRNA can efficiently bind L, but hnRNP K is the one that seems to be having the transcriptional control role.circZNF827 represses neuronal genes and knocking down circZNF827 upregulates pan neuronal genes , it is counterintuitive to see the circ expression level upregulated during differentiation by RT-qPCR ?4) Some circRNAs in group 4  do not have the same expression pattern upon validation in qPCR  . If circZNF827 . Would the deletions or mutations of hnRNP-L or K binding sites in circZNF827 disrupt its interactions with hnRNP-L or K and later the circZNF827-hnRNPK/L-ZNF827 binding to the promoter of NGFR?5) The authors showed that there are putative hnRNP-L or K binding sites in  Essential revisions:1) The authors' model is that the small (10%) amount of the circRNA that is present in the nucleus has a functional role via interacting with hnRNP K and L. This is possible but more direct functional experiments should be included to justify the model. For example, the authors should express dishRNA-resistant versions of circZNF827 and show that the wildtype circRNA rescues the NGFR expression phenotypes but a circRNA with mutated K and L binding sites does not.circZNF827 is regulating NGFR at the transcriptional level, which could potentially be an indirect effect, although hnRNPK  binding to the NGFR promoter is decreased upon circZNF827 knockdown, and although we observe nucleation/phase separation of endogenous hnRNPK and -L upon overexpression of circZNF827.We agree that a rescue experiment, if successful, would be an important addition. Our model is based on the finding that circZNF827 (on top of the endogenous level) from a lentiviral Laccase2-driven circRNA expression system in differentiated L-AN-5 cells. Curiously, this did not alter expression from NGFR, NEFL, TrkB, MAP2 or TUBB3, suggesting either of three scenarios : 1) that the level of circZNF827 is already saturated, 2) that high expression will lead to non-physiological titration effects, or 3) that the backsplicing junction found only in the endogenous circRNA is important for its structure, regulation and function.Previously, we had already conducted experiments with overexpression of case-by-case trade-off between efficiently producing a significantly different circRNA with potentially altered structure and function and a more subtle mutant with low circularization efficiency.Rescue experiments are inherently difficult to conduct for circRNAs compared to protein-coding mRNAs, for 2 main reasons: 1) It is difficult to control exogenous circRNA expression levels due to its ultra-high stability, and 2) Since efficient circRNA expression vectors that generate high-levels of circRNA (relative to undesired linear precursor RNA or concatemer RNAs) often have predesigned splice sites (e.g. the Laccase2 system), which will produce a circRNA with a different backsplicing junction, this structure may be important for the function of the endogenous circRNA. Thus, with current circRNA expression vector technologies, there is a NGFR by expressing two newly cloned variants of dishRNA-resistent (changed backsplicing junction) circRNAs from a laccase2-driven lentiviral vector: Full-length circZNF827 or a hnRNPK/L binding site deletion mutant (very poorly bound by hnRNPK/L in RNA-immunoprecipitation experiments – see below in comments to point 5). High expression (using 3 different MOI’s) of the full-length laccase2-driven circZNF827, left NGFR expression unaltered, at best only marginally lowered as judged by western blotting analysis . Similarly, at the level of RT-qPCR, we did not observe a normalization of the upregulation of NGFR expression. However, the hnRNP K/-L binding site mutant circRNA was, as would be expected, unable to change NGFR expression levels back to the normal.With these limitations in mind, and to directly address the reviewer’s point, we conducted a series of knockdowns using dishRNAs and attempted to rescue upregulation of NGFR upregulation, leaving additional time-consuming optimizations of MOI’s and timings of superinfections very unfeasible.As stated above, we conclude that there are likely sequences, or structures, surrounding the backsplicing junction, that are important for the function of the produced dishRNA-resistant circRNA. Also, expression levels might still be too high and difficult to decrease without leaving a large proportion of cells untransduced, which is not a feasible option, when re-introducing expression and assaying biochemically from bulk culture. Additionally, we predict that the timing of re-expression relative to knockdown could also be important not to exceed a “point of commitment” of circZNF827 and ZNF827 itself, since the circRNA host-encoded protein is an integral part of the circRNA-hnRNPK/L complex. In support of our model, the double knockdown further increases the output from NGFR significantly, as was also the case for hnRNPK/L and circZNF827 co-depletions. In addition, we have now performed biological triplicate ChIP assays using both hnRNP K and ZNF827 antibodies to show that these factors are indeed recruited to the NGFR promoter, and that this recruitment is significantly lowered upon circZNF827 knockdown. We believe that these new results, and further results below, support our model and strengthen our conclusions considerably. The results have been integrated into Figure 6C-D, F-H and Figure 6—figure supplement 1D.Still, to further solidify our model, we have now performed knockdown of both The authors should also use a technique like ChIRP to show direct binding of the circRNA to the NGFR locus.circZNF827 with the NGFR promoter  is circumstantial and could be strengthened. In order to address this issue, we find the suggested ChIRP-DNA strategy unsuitable, in this particular case, for two main reasons: First, the circRNA of interest is only 402 nt long and is predicted to be bound along almost the entire one side of its suggested structure by hnRNPK/L at presumably single-stranded and accessible regions – this is now confirmed by our new RIP analyses using the hnRNPK/L binding site mutant of circZNF827 (see below in comments to point 5). This leaves very little room for design of a suitable number of appropriate tiling probes and control probes (probes that have been shifted to control for direct probe binding and off-target effects). We have previously performed ChIRP to capture another short circRNA for subsequent mass spectrometry analyses without positive results. In comparison, the Chang lab, who carefully developed the procedure, used 48 probes to map genomic interactions sites for HOTAIR lncRNA, which is 2200 nt in length . In addition, 43 probes were used to the interacting proteome for the 17000 nt long XIST lncRNA and a prominent ChIRP-MS study conducted on circRNA targets , used 8 and 15 probes, respectively, to pull-down two exogenously expressed circRNAs of approximately 1500 nt each. We therefore predict a poor pull-down efficiency given the limited ability of designing a suitable number of probes. Second, another caveat using this strategy, is that any probes designed to target the short circZNF827, will also target its linear host pre-mRNA and its fully processed mRNA, potentially giving rise to false positive signals or out-titration of the probes. Thus, we anticipate that ChIRP will be inefficient and take much optimization and therefore remains unfeasible.We agree that the evidence for a direct (or hnRNPK/L/ZNF827 complex-mediated) interaction of circZNF827, which retains its putative hnRNPK/L binding sites . We established inducible stable HEK293S Flp-In T-Rex cell lines expressing these constructs. Using these cells for transfections with FLAG-MS2 coat protein expression constructs, we performed anti-FLAG ChIP RT-qPCR with amplicons for the NGFR promoter region. By this setup, we observed an enriched NGFR promoter signal for the MS2tagged circZNF827 cell line as compared to non-MS2 tagged or non-circRNA controls, while the low control GAPDH promoter-signal remained constant in all cases . We conclude that the MS2-tagged circZNF287 is also localized to the NGFR promoter region in accordance with our model. Importantly, hnRNPK and -L both interact with MS2-tagged circRNA in RNA immunoprecipitation assays (similar to circZNF827 WT affinity) . Second, since ZNF827 interacts with hnRNPK/L and circZNF827, we performed ChIP of endogenous ZNF827 in L-AN-5 cells to address whether the host protein also interacts with the NGFR promoter. Indeed, our results showed an enrichment of ZNF827 at the promoter when circZNF827 is present in cells, while its knockdown significantly diminishes the occupancy of ZNF827 at the promoter . This places hnRNPK, ZNF827 and importantly, circZNF827 at the NGFR promoter, consistent with a likely nucleation-mechanism and repression of transcription. Most importantly, the recruitment of both ZNF827 and hnRNP K/L to the NGFR promoter is dependent on circZNF827. These results have considerably strengthened our model and have been integrated into Figure 6 and 7, along with a discussion in the main text as indicated.As alternative approaches, we conducted two independent experiments: First, we cloned a 2 x MS2 hairpintagged version of 2) The retinoic acid receptor data  is poorly integrated into the rest of the manuscript and the data quality is weak. The supplemental figure shows that RARalpha has a slight (20%) increase in transcription that is insufficient to explain the mRNA effect sizes in the main figure. There is no significant change in RARgamma transcription or decay rates, yet the mRNA expression is increased. Most critically, the authors have failed to connect the proposed hnRNP K/L based mechanism  to RAR transcriptional regulation.NGFR promoter, we detect less docking of hnRNPK and ZNF827 during circZNF827 knockdown, indicating that the hnRNPK/L-regulatory model is likely not limited to the NGFR locus. The reason for the small, yet significant, increase in BrU incorporation (RARa) upon circZNF827 knockdown (compared to steady-state level) might be a temporal issue, when comparing steadystate levels (5-6 days in the making – knockdown/differentiation) to short-pulse-measured transcription rates at that given point in time. This discrepancy is now discussed in the manuscript as indicated by track-changes. We believe that we have now integrated the observed, albeit moderate, effects on the RARa expression with the hnRNPK/L-ZNF827 model, and that this justifies its inclusion.We agree that the effects on Retinoic Acid Receptor α (RARa) and γ (RARg) expression are only moderate and in the latter case not significant in terms of BrU pulse-measured transcription. To strengthen these data and to integrate them better into the hnRNP K/L-ZNF827 regulatory model as suggested, we have performed ChIP-qPCR of hnRNPK and ZNF827 on the promoter of the RARa gene . Importantly, and as observed with the 3) The biochemical data supporting a complex comprising hnRNP K and L is weak and confusing. hnRNP K can pull down a little bit of L, but hnRNP L pull down does not appear to pull down K . This is relevant to the author's main model as the circRNA can efficiently bind L, but hnRNP K is the one that seems to be having the transcriptional control role.bona fide interaction between hnRNP K and -L, as documented by others before us.We agree that the results of the co-immunoprecipitations were not entirely clear. Of note, several previous studies have detected a direct interaction between hnRNPK and -L in different cellular settings . Regarding the co-IPs using FLAG-tagged versions shown in Figure 5D, we do observe that the longer isoform of hnRNP L pulls down hnRNP K. Longer exposures of the other mentioned co-IPs  indeed show that hnRNP L can co-immunoprecipitate hnRNP K well above IgG control. New and extra panels with longer exposures have been included in Figures 5E and 6E. The reason for less hnRNP K pulldown by hnRNP L, than the other way around, is probably due to the fact that the anti-hnRNP L antibody is less efficient as an IP-grade antibody than the anti-hnRNP K antibody. This will inevitably produce lower signal in the hnRNP K westerns. In addition, both proteins are very abundant with multiple functions and many other interaction partners, which explains that not all of either protein population will interact with the other hnRNP-protein . In addition, this type of regulation at the transcriptional level, likely does not require many of such hnRNP-circRNA complexes per cell, so the observed “seemingly weak” interactions in these biochemical assays are likely physiologically relevant. Our additional biochemical data using RIP and ChIP also supports this interaction. We believe that the new figures now demonstrate a 4) Some circRNAs in group 4  do not have the same expression pattern upon validation in qPCR  . If circZNF827 represses neuronal genes and knocking down circZNF827 upregulates pan neuronal genes , it is counterintuitive to see the circ expression level upregulated during differentiation by RT-qPCR ?circZNF827 might function to “tap on the brakes” in the beginning of differentiation until certain processes have been correctly initiated and then at a later stage, after commitment to differentiation, become downregulated again. This issue is now included in the discussion. To align the RTqPCR results better with the RNA-seq data, we have chosen another normalization gene, Vinculin, which is more constant. These data are now included and the color scale has been modified for a clearer distinction between circRNAs.We agree that the color scaling is poor and this has now been changed. We also agree that the expression patterns are not identical based on RNA-seq and RT-qPCR analyses, respectively (e.g. circEZH2 and circZFP827), which is likely due differences in data normalization. While in the RT-qPCR transformed Ct-values are normalized to GAPDH the heatmap displays RPM-normalized reads . These two profiles will become different and as GAPDH also changes slightly upon differentiation (likely slightly changed metabolism in cells starting to exit the cell cycle), resulting in a somewhat skewed picture. Assessing both methods, it is clear that both circZFP827 and circEZH2 become upregulated from mESCs to neuronal progenitor cells (NPCs) and then expression either flattens or goes slightly down at the neuron stage. We do not believe that it is necessarily counterintuitive that an apparent inhibitor of differentiation initially becomes upregulated upon differentiation only to be reduced at the latest stage of this process. 5) The authors showed that there are putative hnRNP-L or K binding sites in circZNF827 . Would the deletions or mutations of hnRNP-L or K binding sites in circZNF827 disrupt its interactions with hnRNP-L or K and later the circZNF827-hnRNPK/L-ZNF827 binding to the promoter of NGFR?NGFR upregulation by knockdown of the endogenous circZNF827 as expected.Corroborating our models, we have now conducted RNA-immunoprecipitations with hnRNP K/L deletion mutant and found that they no longer efficiently interact with neither hnRNP K nor hnRNP L. Likewise, the rescue experiments described in 1) show that this mutant cannot inhibit the