Reversible phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (Pol II) is essential for gene expression control. How altering the phosphorylation of the CTD contributes to gene expression in mammalian systems remains poorly understood.Methods: Primary mouse embryonic fibroblasts, hepatocytes, and embryonic stem cells were isolated from conditional Ssu72f/f mice. To knockout the mouse Ssu72 gene, we infected the cells with adenoviruses of incorporated luciferase and Cre recombinase, respectively. RNA sequencing, ChIP sequencing, ChIP assay, immunoblot analyses, qRT-PCR assay, and immunostaining were performed to gain insights into the functional mechanisms of Ssu72 loss in Pol II dynamics.Results: Using primary cells isolated from Ssu72 conditional knockout and transgenic mice, we found that mammalian Ssu72-mediated transcriptional elongation rather than polyadenylation or RNA processing contributed to the transcriptional regulation of various genes. Depletion of Ssu72 resulted in aberrant Pol II pausing and elongation defects. Reduced transcriptional elongation efficiency tended to preferentially affect expression levels of actively transcribed genes in a tissue-specific manner. Furthermore, Ssu72 CTD phosphatase seemed to regulate the phosphorylation levels of CTD Ser2 and Thr4 through accurate modulation of P-TEFb activity and recruitment.Conclusions: Our findings demonstrate that mammalian Ssu72 contributes to the transcription of tissue-specific actively transcribed gene expression by regulating reciprocal phosphorylation of Pol II CTD. Transcription begins with the assembly of a pre-initiation complex at individual gene promoters. This is followed by the initiation of messenger RNA (mRNA) synthesis and the beginning of the elongation process. For all coding genes and several non-coding RNAs , recruitment of RNA polymerase II (Pol II) to a promoter is an essential step in transcriptional activation. An interesting and distinct feature of Pol II is the presence of C-terminal domain (CTD) repeats (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7) on its largest subunit (Rpb1), known to play an important role in coordinating accurate transcription, mRNA processing, and histone modification.The CTD itself is also subjected to extensive post-translational modification during the transcriptional process. The phosphorylation state of Pol II CTD is particularly critical for regulating the recruitment of appropriate transcription-associated factors. When transcription is initiated, Ser5 in the CTD is rapidly phosphorylated by TFIIH-associated kinase Cdk7 (Kin28 in yeast). Levels of phosphorylated Ser5 (pSer5) are decreased during transition from initiation to elongation, concomitant with an increase in the level of pSer2 Ssu72 has been identified as a Pol II CTD phosphatase with a preference for pSer5 and pSer7. Depletion of Ssu72 in yeast has led to increased levels of both pSer5 and pSer7 before transcriptional termination More recently, the genome-wide occupancy pattern of mammalian Ssu72 in human embryonic stem (ES) cells was found to be opposite to that seen in yeast. The highest peaks are detected in the promoter regions of genes, while small peaks are detected in the termination regions In yeast, Ssu72 has been implicated in cellular viability and efficient transcription of snoRNAs and specific mRNAs but has not been implicated in global gene expression Therefore, the objective of this study was to use primary cell lines isolated from Ssu72 conditional knockout (KO) and transgenic mice to examine the specific role of mammalian Ssu72 in regulating RNA Pol II processing during transcription. Loss of mammalian Ssu72 was found to be related to defective transcriptional elongation rather than to failure of transcriptional termination or polyadenylation. In addition, reduced transcriptional elongation seemed to preferentially affect distinct patterns of gene expression of actively transcribed genes in a tissue-specific manner. Furthermore, depletion of Ssu72 resulted in defects of Pol II elongation by reducing CTD Ser2 and Thr4 phosphorylation, which induced Pol II accumulation at the proximal promoter region by increasing CTD Ser5 and Ser7 phosphorylation of actively transcribed genes in a tissue-specific manner. Our results revealed a fundamental role of mammalian Ssu72 in regulating RNA Pol II-mediated transcriptional plasticity for cell-type-specific homeostatic maintenance.in vivo function of Ssu72 in the transcription and development of mammals, we targeted murine Ssu72 gene by homologous recombination in mouse embryonic stem cells  mice by flanking the Ssu72 exon 1 with loxP sites, allowing Cre-mediated exon 1 deletion  analyses of Ssu72 in three different cell types depending on their proliferation capabilities: ES cells (highly proliferating), MEFs (moderate proliferating), and hepatocytes . The Ssu72 deletion was induced by infection with a Cre recombinase adenovirus (Ad-Cre) or by intercrossing with albumin-Cre  mice A-C. Infeof Ssu72 A-C.p < 0.05; false discovery rate (FDR) <0.1)] of 902 and 1,135 genes in MEFs, 646 and 654 genes in hepatocytes, and 302 and 125 genes in ES cells, respectively  analysis confirmed that Ssu72 had a tendency to transcriptionally regulate active genes in a cell-type-specific manner, including genes such as s Figure F. Interenhibited G. These Phyhd1 and Pold2 in MEFs, and Adh1 and Akr1c6 in hepatocytes  coupled with high-throughput sequencing (ChIP-Seq) Figure A. IntereTo further validate the recruitment of Ssu72 to the promoter regions, ChIP analysis coupled with qPCR (ChIP-qPCR) was performed for the selected Ssu72-bound genes in the MEFs and hepatocytes Figure D. Howevep < 0.1). Interestingly, a large percentage of Ssu72-bound genes (36.7%) overlapped the genes down-regulated by Ssu72 depletion in MEFs, whereas a small percentage of Ssu72-bound genes (2.1%) overlapped the up-regulated genes  tail length assay to measure the poly(A) tail length of multiple mRNAs in MEFs and hepatocytes. Unexpectedly, we did not observe remarkable changes to actively transcribed genes in either the control or the Ssu72-depleted cells Figure E. We thuSsu72f/f MEFs and hepatocytes. As Ssu72 is capable of dephosphorylating both pSer5 and pSer7, we speculated that Ssu72 might regulate gene expression throughout the transcriptional initiation stage. An increase in Pol II density at the proximal promoter region is known to be correlated with a pause in transcriptional initiation Pold2) gene body. Pold2 was one of selective Ssu72-bound genes in MEFs  in s Figure C, left. s Figure C, right.re Pold2 A. In connt cells B.Next, to quantitatively assess the effect of Ssu72 on Pol II occupancy in gene bodies, we calculated the traveling ratio (TR) of Pol II occupancy. This TR represents the ratio of Pol II density in the promoter-proximal region to that in the expanded gene body and also denotes the transcriptional elongation activity c-fos gene is one such actively transcribed gene (a Ssu72 target gene) in MEFs regulated by mammalian Ssu72  at multiple time points  with dramatic attenuation in the middle and termination regions of this gene (p3 to p6 primer sites) at 10 min after EGF administration in Ssu72 depleted MEFs. At 30 min after EGF administration, the mRNA expression levels in the middle and termination regions of the c-fos gene were moderately restored in Ssu72 depleted MEFs, implying that Ssu72 depletion caused defects or delays in transcriptional elongation. Similar results were observed in other EGF-target genes  -responsive genes in MEFs. The 2 Figure E. Hence,s Figure A-B. EGF s Figure B. The act Figure C. Althoud Ccne1) B.c-fos gene was dramatically attenuated by Ssu72 depletion after a series of Pol II pausing events on the c-fos gene  under an EGF-treated condition for 10 min Figure D. After e Figure D. This rc-fos proximal promoter. Clear elongation of Pol II was observed at gene body after EGF treatment in Ssu72 WT MEFs, whereas Ssu72 depletion resulted in stalling of Pol II in the promoter region after EGF treatment  was stimulated with H2O2 treatment , and NELF to the reatment C, indicaGiven the relevance of Ssu72 in Pol II accumulation at the promoter and its involvement in transcription elongation, we next examined the effect of Ssu72 depletion on genome-wide phosphorylation of Pol II and Pol II CTD phosphorylation  Figure A, indicaPold2 and Phyhd1 (Ssu72 target genes in MEFs) as well as Akr1c6 (a Ssu72 target gene in hepatocytes) revealed that the levels of pSer5 and pSer7 were increased at all regions with a concomitant decrease in the levels of pSer2 and pThr4 within the gene body in Ssu72-deficient MEFs  and Ssu72-deficient (∆/∆Ssu72) embryos. The results of the immunostaining analyses using blastocysts revealed that ∆/∆Ssu72 embryonic cells showed a clear increase in phosphorylated forms of CTD at Ser5 and Ser7  compared to the +/+Ssu72 embryonic cells    E. By conctively) E. Taken To further compare the phosphorylation profiles of each CTD residue, we immunoprecipitated the Pol II complex with an anti-pSer5 antibody. High levels of pSer5 and pSer7 were detected in the immuno-complex in Ssu72-depleted MEFs, whereas pSer2 and pThr4 levels in the pSer5-mediated immuno-complex were significantly decreased in the Ssu72-depleted MEFs compared to the control MEFs A. Consisf/f MEFs. Immunoblotting with anti-phospho-CTD antibodies revealed that depletion of Ssu72 led to an acidic shift of pSer5- and pSer7-containing polypeptides, confirming the hyperphosphorylation of Ser5 and Ser7 residues by Ssu72 depletion  in MEFs caused reductions in pSer5 and pSer7 levels, whereas the levels of pSer2 and pThr4 were almost unaffected by Ssu72 overexpression. As a negative control, the overexpression of an Ssu72 phosphatase‐dead mutant (Ssu72 C12S) showed very similar patterns of phosphorylation profiles of CTD codes to the control B, indicaepletion D. We alsepletion D. As a rMany effects of Ssu72 depletion on Pol II transcriptional activity at the elongation factor recruitment step could be explained by decreased recruitment of P-TEFb. Previous studies have demonstrated that productive elongation begins with the recruitment of P-TEFb, which phosphorylates NELF and DSIF, resulting in a release of the transcriptional pause This raises the possibility that Ssu72 may regulate kinase activity of P-TEFb, thereby contributing to phosphorylation of DSIF and Ser2 residues in CTD for effective transcriptional elongation. To test this possibility, we first examined whether direct interactions between Ssu72 and P-TEFb were involved in the Cdk9-Cyclin T1 complex. Cell extracts were immunoprecipitated with Cdk9 and Ssu72 antibodies or normal IgG. Immunoblotting was then performed with antibodies against Ssu72, Cdk9, Cyclin T1, and Pol II Figure B-C. Notain vitro kinase assay of P-TEFb using recombinant Ssu72 WT and Ssu72 C12S. Interestingly, both recombinant Ssu72 WT and C12S were significantly increased the kinase activity of P-TEFb, implying that the P-TEFb kinase activity might be induced by the protein interaction between Ssu72 and P-TEFb rather than the Ssu72 phosphatase activity , and Hexim1 occupancy ChIP analyses. Interestingly, recruitment of the P-TEFb complex onto chromatin was markedly reduced in Ssu72-depleted cells whereas chromatin binding levels of the 7SK snRNP complex (Larp7 and Hexim1) were almost unaffected by Ssu72 depletion Figure A. In addSsu72 is highly conserved from yeast to mammals. It is essential for yeast cell viability and an efficient transcriptional process vice versa. This effect is likely to be linked to inhibition of P-TEFb and elongation factor (DSIF) recruitment onto the gene body regions, thereby causing severe defects in transcriptional elongation during Pol II-mediated gene expression of Ssu72-bound genes (actively transcribed genes).Unexpectedly, this study also provided evidence that mammalian Ssu72 was associated with both direct and indirect mechanisms of phosphorylation dynamics of the CTD Ser2 and Thr4 residues through regulation of P-TEFb activity and recruitment on chromatin during transcriptional elongation. A principal mechanism by which P-TEFb influences transcriptional events is phosphorylation of the Pol II CTD Ser2 and Thr4 residues to recruit factors that regulate elongation, RNA processing, and chromatin modification bona fide protein phosphatase that can regulate finely-tuned Pol II pause release and active elongation.During transcription elongation, the CTD phosphorylation state is highly dynamic. It takes place within a short time, almost simultaneously. During this process, the levels of pSer5 and pSer7 are sharply decreased whereas pSer2 levels are increased across gene bodies Recent reports on Thr4 phosphorylation required for transcriptional elongation of protein-coding genes During the development and maintenance of multicellular organs, each type of cell acquires its specific fate through precise control of gene expression. Some researchers have reported that this process is mediated by tissue-specific functions of broadly expressed transcription factors as well as spatial contacts between a distal enhancer and a proximal promoter cis configuration of the Ser5-Pro6 motif within CTD through the cis/trans conversion activity of Pin1 isomerase, different from other CTD phosphatases If so, how does mammalian Ssu72 phosphatase control the expression of distinct sets of genes in a cell-type-specific manner? One possible explanation is that Ssu72 has distinct localization patterns in various cell types, including quiescent and proliferating cells. During the transcription cycle, many CTD-specific kinases and phosphatases are recruited to Pol II CTD. The dynamics of hyper-phosphorylation and hypo-phosphorylation is a major regulatory mechanism involved in CTD modification. Various phosphatases and kinases also have different specificities toward isomeric states of proline residues adjacent to various phosphorylation sites. According to recent reports, Ssu72 can selectively recognize the cis conformation of Pol II CTD. This complex is likely to affect the transcriptional machinery and mRNA expression of actively transcribed genes depending on cell type. Our ChIP-Seq results showed that Ssu72 occupied core promoters of a substantial proportion of actively transcribed and bivalent genes in all cell types tested. However, the density of the Ssu72 signal in each cell type was slightly different. The occupancy of Ssu72 was significantly increased at the 3'-end regions as much as in the promoter regions of ES cells. However, it was mostly observed in the promoter regions of MEFs and hepatocytes. Therefore, Ssu72 might be specifically localized in a distinct cis configuration of Pol II for actively transcribed genes in each cell type, thereby regulating different sets of transcribed genes in different cell types.In line with this evidence, Ssu72 may localize with Pin1 at the Another possibility is that Ssu72 may regulate the expression of cell type-specific gene families containing bidirectional genes by controlling gene loops. Recent reports have suggested that gene loops may bring distal regulatory elements such as enhancer and 3'-end processing components into proximity to promoter regions in vitro and in vivo. It also shows that Ssu72 deficiency can cause aberrant Pol II pausing and elongation defects during the transcriptional cycle, with diversity dependent upon the cell type based on gene transcription.Altogether, this study demonstrates that mammalian Ssu72 can mediate tissue-specific transcriptional activity by regulating reciprocal phosphorylation of CTD both loxP site was inserted into exon 1, and a puromycin selection cassette flanked by FRT sites with a single loxP site was inserted between exons 1 and 2  library. To construct the targeting vector for Ssu72, a single  1 and 2 A. The tain vitro culture of pre-implantation embryo, blastocysts were collected on E3.5 by flushing the uterus of each mouse with M2 medium, followed by culturing in conditioned ES media . For immunostaining of mouse embryos, blastocysts were rinsed with PBS and fixed with fresh 4% formaldehyde in PBS for 30 min at room temperature. DNA was denatured after permeabilization with 2 M HCl and 0.5% Triton X-100 for 20 min at RT. After washing extensively with PBS containing 1.0% BSA, blastocysts were collected and incubated with the indicated primary and secondary antibodies. Slides were stained with DAPI, washed with PBS, air-dried, mounted, and processed for confocal microscopy. Fluorescence intensity levels of each embryo were quantified using ZEN software.For Z-stack module. Deconvolution was performed using Huygens software.For immunostaining, cells were cultured directly on glass coverslips, washed with PBS, fixed in 4% paraformaldehyde, incubated with indicated primary and fluorescent-labeled secondary antibodies , and then stained with DAPI. Images were acquired on a LSM510 META confocal microscope fitted with a ×20 NA 0.75 objective lens and an AxioCam camera. Z series of 0.3 μm stacks were acquired using AxioVision Release 3.x software with a For histological analysis, liver tissues were fixed with formalin overnight at 4 °C and embedded in paraffin, and cut into 4 μm sections. After deparaffinization and dehydration, antigen retrieval was performed by boiling the sections in 10 mM citric acid buffer (pH 6.0) for 15 min, and then cooled to room temperature for 30 min. Immunohistochemistry was performed with anti-BrdU and anti-Ki67 antibodies. For BrdU immunofluorescence staining, 5-bromo-2'-deoxyuridine (BrdU) (Sigma-Aldrich) was administered to the mice by intraperitoneal injection 2 h before harvesting the livers. Paraffin-embedded liver sections were stained with anti-BrdU antibody, DAPI solution and anti-Alexa Fluor 488 (Invitrogen Life Technologies). Images were acquired on an AxioImager M2 microscope (Carl Zeiss) with AxioVision 4.82 software (Carl Zeiss) and analyzed for hepatocyte nuclei numbers and sizes using NIH ImageJ software.One day prior to recombinase adenovirus infection, MEFs, hepatocytes, and ES cells were plated onto dishes. On the next day, the cells were infected with Ad-Luc or Ad-Cre in low-FBS serum containing 100 nM protein transduction domain (PTD) peptide (Sequence: GLNGPDIYKGVYQFKSVEFD) to enhance the efficiency of adenoviral infection.Ssu72f/f and Alb-Cre;Ssu72f/f (KO) mice by liver perfusion with collagenase IV. After isolation, the hepatocytes were seeded in M199 medium supplemented with 10% FBS, 23 mM HEPES, and 10 nM dexamethasone.MEFs were isolated from embryonic day 13.5 embryos according to a standard protocol. Each embryo was dissected in 10 ml sterile PBS and sheared through an 18G syringe in the presence of 0.25% trypsin and 1 mM EDTA. After 10 min of incubation at 37 ºC with gentle shaking, the cells were plated onto dishes and maintained at 37 ºC. Adherent cells were used as MEFs. Hepatocytes were isolated from 5-week-old . We added 1 μL of a 1:50,000 dilution of ERCC RNA spike‐in mix (Thermo Fisher) to all samples prior to library preparation. The transcriptome sequencing libraries were prepared using TruSeq® Stranded Total RNA Library Prep Kit (Illumina) by following the manufacturer's procedure. RNA-seq libraries were sequenced on an Illumina HiSeq 2000 or 3000/4000 using paired-end with read lengths of 150 bp. An initial sequence-level quality assessment was performed using FastQC. The Fastq files with 150 bp paired-end reads were processed using Partek Flow. Briefly, the raw reads were aligned and mapped to the mouse mm10 reference genome using Tophat. The aligned reads are quantified to an annotation model through Partek E/M. The normalization method used here is counts per million (CPM) through Partek Flow. The normalized counts were then subjected to statistical analysis using ANOVA. Differential gene expression analysis was performed using the package DESeq2. Read counts were used and modeled based on a negative binomial distribution. We considered genes as differentially expressed if: (1) the FDR was less than 0.05, (2) the expression ratio among three independent samples was > 2-folds change, (3) there was agreement with DESeq2. Data were obtained from three independent experiments and processed using DAVID Bioinformatics Resources 6.7 software for Gene ontology, BIOCARTA, Cytoscape, KEGG pathway and GSEA.Total RNA from Ad-Luc or Ad-Cre infected MEFs, hepatocytes, and ES cells were extracted with Trizol reagent and subjected to RNA-Seq analysesFor immunoblot assays, primary cells were harvested by scraping and then resuspended in TNN buffer  containing a protease inhibitor cocktail followed by brief sonication. Equal amounts of proteins from each sample were separated by SDS-PAGE, transferred to a nitrocellulose membrane, blocked, and analyzed by immunoblotting with appropriate antibodies .in vitro kinase assay, purified GST-fused CTD proteins were incubated with kinase buffer , and 0.5 μg of recombinant TFIIH, P-TEFb, and Plk3 kinase at 30 ºC for 1 h. The reaction was halted by addition of SDS sample buffer, resolved by SDS-PAGE, and visualized by Ponceau S staining and immunoblotting analyses. For P-TEFb IP-kinase assay, total cell lysates prepared from MEFs were incubated with Cdk9 antibody and Protein A/G agarose beads to obtain immuno-P-TEFb complexes. Purified GST-CTD (1 μg) was used as a substrate of the P-TEFb precipitate in subsequent kinase reaction as described above. For the phosphatase analyses, purified GST-Ssu72 proteins were cleaved by thrombin digestion and then incubated in a phosphatase buffer  containing hyper-phosphorylated GST-CTD substrates.For immunoprecipitation using total cell extracts, Ad-Luc- and Ad-Cre-infected MEF cells were resuspended in buffer A (100 mM Tris-HCl (pH 7.5), 20 mM EDTA, 1% NP40, 1 mM PMSF, 1 mM DTT, and a protease inhibitor cocktail). The supernatants (soluble cytoplasmic fractions) were obtained. The cell pellets were resuspended in buffer B , briefly sonicated, and centrifuged to obtain the supernatants (soluble pellet fractions). Mixed extracts (soluble cytoplasmic and pellet supernatants) were diluted with a salt‐free buffer to reduce salt concentration to 150 mM. These samples were centrifuged and analyzed by immunoprecipitation. For the Total RNAs from Ad-Luc or Ad-Cre infected MEFs were extracted with Trizol reagent and subjected to G/I tailing using Poly(A) Tail Length Assay Kit following the manufacturer's protocol. The Poly(G/I)-tailed RNA product was subjected to RT-PCR and the reaction product was loaded onto a 2.5% agarose TBE gel.2, 3 mM CaCl2, 250 units of SUPERaseIn, and 10% glycerol] and incubated on ice for 10 min. Lysates were then mechanically lysed by passing the suspension 10 times through a 26G needle. Nuclei were pelleted by centrifugation and resuspended in 1 ml of nuclear extraction buffer followed by gentle pipetting up and down 10 times. The nuclei were centrifuged and resuspended in 100 μL of storage solution . For nuclear run-on analyses, nuclei were incubated with 300 μL of nuclear run-on reaction buffer [ at 4 °C for 20 min. Isolated nuclei were re-suspended in nuclei lysis buffer  and sonicated. The samples were immunoprecipitated with the appropriate antibodies in combination with Protein-A-Sepharose beads. These precipitates were extracted with elution buffer . The eluted samples were pooled and heated at 65 °C overnight to reverse the formaldehyde cross-linking. The DNA fragments were purified with a QIAquick spin kit. The qPCR reactions were performed with SYBR Green PCR Master Mix in a MicroAmp optical reaction plate using QuantStudio 3. Primers used for qPCR are summarized in For ChIP analysis, MEFs and hepatocytes were crosslinked with 1% formaldehyde at room temperature for 20 min. The reaction was halted with 125 mM glycine. These cells were washed twice in PBS and collected by centrifugation. The nuclei were isolated by resuspending the cells in cell lysis buffer  were removed using BEDTools. To generate average occupancy plots against ChIP-Seq results of Ssu72, Pol II and phosho-Pol II in the MEFs and hepatocytes, we selected all genes with a distance of at least 4kb from any other known annotation  by using mouse mm10 Refseq gene annotations. Based on selected genes, we retrieved the occupancy values from - 4 kb upstream of TSS to + 4 kb downstream of TES for total genes selected with a distance of at least 4 kb. Mapped reads were extended to 150 bp toward the interior of the sequenced fragment and input normalized to total reads aligned. The average coverage was binned in 20-bp intervals. Peak calling was performed using MACS 1.4.2 to determine statistical enrichment at a stringent p-value (p < 1E-5 ~p < 1E-7). Peaks within 1000 bp to the nearest TSS were set as promoter. The distribution of peaks  was annotated using findMotifsGenome.pl. Metagene plots of ChIP-seq were performed using NGSplot. Density plots were generated by counting tags using indicated window size with 20 bp bin. Each plot was involved in 4 kb upstream of TSS, the gene body, and 4 kb downstream of the TES. Different occupancy-profiling categories  were calculated and classified based on the average ChIP signal from 4kb upstream of the TSS to 4kb downstream of the 3' end of all selected genes for each experiment. For example, 515 of Ssu72-bound genes were selected in MEFs, 1994 in hepatocytes and 680 in ES cells through ChIP-Seq results. The 515 genes selected in MEFs were subdivided into groups of 155, 103 and 257 based on the average ChIP signal; 1994 genes selected in hepatocytes were subdivided into groups of 599, 398 and 997; 680 genes selected in ES cells were subdivided into 204, 136 and 340, as represented by differentially colored lines. Heatmaps of signal intensity of the ChIP samples were generated using deepTools. In brief, computeMatrix was used to calculate the signal intensity scores per ChIP sample in a given genome region that was specified by bed files. The output of computeMatrix was a matrix file of scores. The heatmap generated using plotHeatmap.To validate that the anti-Ssu72 antibody  is feasible for ChIP-seq, we performed Western blotting after immunoprecipitating the formaldehyde fixed cells using the anti-Ssu72 antibody. We observed the pull-down of endogenous Ssu72 in Ad-Luc- and Ad-Cre-infected MEFs, indicating that this antibody is able to pull down crosslinked endogenous Ssu72 protein. Then, 30 to 50 million MEFs, hepatocytes and ES cells were crosslinked with 1% paraformaldehyde in PBS for 10 min at RT and were quenched with 0.2 M glycine for 5 min. For sonication of cells fixed for 10 min, the optimal sonication conditions were generated a DNA smear with more than about 90% of total DNA fragments less than 1 kb. To normalize the ChIP-seq signal, we employed a ChIP-seq spike in, using Drosophila chromatin (Active Motif) according to the manufacturer's instructions, for the Ssu72, Pol II and phospho-Pol II ChIP-Seq analyses. The ChIP-seq DNA libraries were multiplexed and sequenced using a TruSeq ChIP Samples Prep Kit on an Illumina NextSeq machine. A total of 50,000,000-103,000,000 unique reads were generated per sample. To generate the ChIP-seq data, ChIP enriched DNA reads were mapped to UCSC mouse genome (mm10) using BWA. All duplicate tags with identical sequences and possible sequencing artifacts were filtered out with the PICRAD 1.76 pipeline. The ChIP-Seq read density values were normalized per million mapped reads. The peaks of ChIP-Seq that overlapped with the possible anomalous artifact regions (such as high-mapping regions or satellite repeats) blacklisted by the ENCODE consortium (All raw and processed sequencing data that support the findings of this study have been deposited in Gene Expression Omnibus (GEO). The accession number is GSE122101.∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; n.s., p > 0.05.Data are presented as mean ± SD. Statistical significance was tested with Student's t-test for two groups or one-way ANOVA for multiple groups with similar size. When exact p values are not indicated, they are represented as follows: Supplementary figures and methods.Click here for additional data file.Supplementary table 1. List of down- and up-regulated genes by Ssu72 depletion in MEFs, hepatocytes, and ES cells.Click here for additional data file.Supplementary table 2. Ssu72 mainly controls the expression of actively transcribed gene groups with cell-type-specificity.Click here for additional data file.Supplementary table 3. Gene set enrichment analysis (GSEA) by Ssu72 depletion in MEFs, hepatocytes, and ES cells.Click here for additional data file.Supplementary table 4. Gene ontology results of RNA-Seq and Chip-Seq analyses.Click here for additional data file.Supplementary table 5. The peaks and target genes of Ssu72 ChIP-Seq data in MEFs, hepatocytes, and ES cells.Click here for additional data file.Supplementary table 6. Ssu72 binding proteomes.Click here for additional data file.Supplementary table 7. The primers for mouse genotyping.Click here for additional data file.Supplementary table 8. List of used antibody.Click here for additional data file.Supplementary table 9. The primers for qRT-PCR and ChIP analyses.Click here for additional data file.


Transcription by RNA polymerase II (Pol II) is regulated by different processes, including alterations in chromatin structure, interactions between distal regulatory elements and promoters, formation of transcription domains enriched for Pol II and co-regulators, and mechanisms involved in the initiation, elongation, and termination steps of transcription. Transcription factor TFII-I, originally identified as an initiator (INR)-binding protein, contains multiple protein–protein interaction domains and plays diverse roles in the regulation of transcription. Genome-wide analysis revealed that TFII-I associates with expressed as well as repressed genes. Consistently, TFII-I interacts with co-regulators that either positively or negatively regulate the transcription. Furthermore, TFII-I has been shown to regulate transcription pausing by interacting with proteins that promote or inhibit the elongation step of transcription. Changes in TFII-I expression in humans are associated with neurological and immunological diseases as well as cancer. Furthermore, TFII-I is essential for the development of mice and represents a barrier for the induction of pluripotency. Here, we review the known functions of TFII-I related to the regulation of Pol II transcription at the stages of initiation and elongation. The initiator is a pyrimidine-rich DNA sequence that overlaps with the sequence of the TSS and was shown to be able to direct accurate transcription in the absence of a TATA box. Transcription factor TFII-I was one of the early proteins identified to interact with the initiator and to recruit transcription complexes to TATA-less promoters  DNA-binding domain, a nuclear localization sequence (NLS), and multiple protein–protein interaction domains, including a leucine zipper (LZ) and six helix-loop-helix (HLH)-like domains, also referred to as I-repeats R1–R6, . TFII-I in vitro . Some ofin vitro , 1993a. Since these initial studies, subsequent work has demonstrated that TFII-I is a multifunctional protein that exerts activities in both cytoplasm and nucleus . In the GTF2IRD1, is located in close proximity to the GTF2I gene and encodes BEN (binding factor of early enhancer) , which are expressed in a ubiquitous or cell type-specific manner . The Δ-inhancer) . WilliamD1 genes . IndividD1 genes .TFII-I has been implicated in a variety of diseases, including neurological abnormalities. This has recently been reviewed by Mutations in TFII-I are associated with a number of different tumors, including T-cell lymphoma and thymus epithelial tumors TETs; . Point mTranscription factors impact gene expression in many different ways. They can bind to and act in close proximity to the genes they regulate, or they act at a distance by binding to enhancer or insulator sequences. Recent advances in our understanding of the organization of genes within the nucleus demonstrate that the genome is organized in defined topologically associating domains (TADs) that are often multiple Mb-long and are characterized by frequent chromosomal interactions within TADs and limited contacts between TADs . TADs arvia interactions with specific DNA elements, only 8% of genomic binding sites for TFII-I in IPSCs correspond to nearby genes that change expression in response to TFII-I depletion , lysine-specific demethylase 1 (LSD1), topoisomerases, and transcription elongation factors as TFII-I-interacting proteins, suggesting diverse functions of TFII-I during the regulation of transcription . LSD1 reAs mentioned before, TFII-I interacts with negative and positive co-regulators. At some gene loci, perhaps at those involved in stress response or in cell cycle control, TFII-I may play negative and positive roles at different stages of induction . It is cAt a subset of genes, Pol II pauses near the TSS and several activities have been identified to mediate the transition from pausing to productive elongation . At mRNAin vitro studies demonstrated that Elongin A stimulates the elongation step of transcription (The Elongin complex has been shown to regulate Pol II transcription elongation activity . Studiescription . Recent cription . Interescription .TFII-I has been shown to interact with NELF as well as Elongin A . It appeDrosophila, other IBPs have also been shown to modulate Pol II pausing at distantly located genes. This is mediated by their common cofactor CP190 (As mentioned before, TFII-I was shown to interact with the insulator protein CTCF and to regulate genes in response to metabolic stress . This isor CP190 .The above-discussed studies implicate TFII-I in the positive regulation of the elongation step of transcription. A recent study implicates TFII-I in the negative regulation of genes induced by TGF-β . At a suRecently, it was shown that NELF forms nuclear condensates in response to stress . FormatiBecause of its unique structure, its multiple functions in thenucleus and cytoplasm, and its implication in cancer as well as neurological and immunological disorders, there is interest and a significant requirement to elucidate the mechanisms by which TFII-I affects gene expression patterns and other cellular functions during development and differentiation. It is clear that TFII-I is a DNA-binding protein that interacts with co-regulators to positively or negatively affect the transcription of specific target genes, but many aspects of TFII-I function remain enigmatic. It seems that a fraction of TFII-I chromatin associations is mediated by interactions with other DNA-binding proteins, including associations with E-box sequences together with HLH proteins, associations with E2F sites together with E2F transcription factors, and associations with insulator sequences together with CTCF and perhaps components of cohesin. In addition, TFII-I interacts with a variety of proteins that regulate different steps in the process of transcription, including histone-modifying enzymes, topoisomerases, and transcription elongation factors. Due to its relatively large size and the presence of multiple protein–protein interaction domains, it is possible that TFII-I functions as a hub to regulate the coordinated recruitment of activities involved in gene regulation. Its putative involvement in the creation of chromatin domains and insulated chromatin neighborhoods will be an exciting focus of future research. Furthermore, the recent findings implicating TFII-I in regulating the elongation step of transcription suggest that it will be important to determine its function in response to stress or other signals. As mentioned before, TFII-I has been shown to interact with TAF15, which is capable of forming phase-separated domains . Recent JB, JS, RN, NL, and AT discussed the contents and outline of the review. All authors contributed to the writing and generation of figures.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.


Drosophila as a major component of constitutive heterochromatin, required for stable epigenetic gene silencing in many species including humans. Over the years, several studies have highlighted additional roles of HP1 in different cellular processes including telomere maintenance, DNA replication and repair, chromosome segregation and, surprisingly, positive regulation of gene expression. In this review, we briefly summarize past research and recent results supporting the unexpected and emerging role of HP1 in activating gene expression. In particular, we discuss the role of HP1 in post-transcriptional regulation of mRNA processing because it has proved decisive in the control of germline stem cells homeostasis in Drosophila and has certainly added a new dimension to our understanding on HP1 targeting and functions in epigenetic regulation of stem cell behaviour.Heterochromatin protein 1 (HP1) is a non-histone chromosomal protein first identified in  Su(var)2–5 gene, is an evolutionarily conserved chromosomal protein first identified in Drosophila melanogaster by its association with constitutive heterochromatin domains and through mutations acting as dosage-dependent modifiers of position-effect variegation (James and Elgin Schizosaccharomyces pombe (Swi6)   , encoded by the Schizosaccharomyces pombe, Drosophila and mammals have proposed a new mechanism for heterochromatin compartmentalization and spreading based on the ability of higher-order HP1 oligomers to aggregate into liquid-phase droplets that contribute to heterochromatin phase separation and promote epigenetic gene silencing , important for the binding to the N-terminal tail of histone H3 when it is di- or trimethylated  that are very important in modulating both HP1 interactions and chromatin binding ability  to heterochromatin  control the stable recruitment of the histone chaperone complex FACT (facilitates chromatin transcription) on active chromatin, thus promoting gene expression , multiple layers of post-transcriptional regulation, including alternative splicing, RNA modifications and translational repression, orchestrate the balance between self-renewal and differentiation and ensure proper germline stem cell homeostasis , piwi and bag of marbles (bam)  are important for maintaining self-renewal of embryonic stem cells in mice and adult germline stem cells in Drosophila, respectively  Fig.  confirmeSince its identification, during 1980s, the multifunctionality of HP1 is still a subject of new discoveries. The ability of HP1 to maintain a silenced state or promote rapid transcription upon cell insult or cell fate program is a very fascinating field. What molecular mechanisms are responsible for the functional versatility of this protein? The details of how HP1 regulates active transcription remain largely unknown. Post-translational modifications certainly play a key role in modulating HP1 functions because they can differentially regulate HP1 activity, localization and chromatin interactions. Similarly, the identification of HP1 binding partners would help to provide some explanation on how it works in different chromatin contexts and cellular processes.epigenetics reader, might specifically recognize and bind, through its chromodomain, methylated residues on target RNAs, thus directly regulating their metabolism and processing. The methylated residues in RNA sequences could in fact allow HP1 to discriminate between different transcripts and to specifically regulate their metabolism and processing , could play a dual role: on one hand, it protects the newly synthesized RNA from degradation; on the other hand, it provides the machinery that enables accurate RNA processing in a temporally and spatially regulated fashion, thus reinforcing gene expression Fig. . It remaing Fig. . In conc


APA was initially characterized for a few genes, but in the past decade, has been found to be the rule for metazoan genes. While numerous differences in APA profiles have been catalogued across genetic conditions, perturbations, and diseases, our knowledge of APA mechanisms and biology is far from complete. In this review, we highlight recent findings regarding the role of the conserved ELAV/Hu family of RNA binding proteins (RBPs) in generating the broad landscape of lengthened 3' UTRs that is characteristic of neurons. We relate this to their established roles in alternative splicing, and summarize ongoing directions that will further elucidate the molecular strategies for neural APA, the  The 3' untranslated region (3' UTR) is a hub of post-transcriptional regulation by RNA binding proteins and miRNAs, which collectively mediate diverse functional impacts including alteration of mRNA stability, directing transcript subcellular localization, modulating translational efficiency and/or changing protein function . Since aAnother dominant source of transcriptome diversity arises from alternative splicing. Compared to APA, we know more about the molecular control of alternative splice isoforms and their biological impacts . This isRecently, members of the conserved ELAV/Hu RNA binding protein (RBP) family have received growing attention as global mediators of both neural-specific splicing and APA programs . In thisMALAT1 and MEN β) (With the exception of histone mRNAs and some unique noncoding RNAs (such as d MEN β) , maturatd MEN β) . More thd MEN β) . In mammd MEN β) . 1) The d MEN β) . In partd MEN β) , which gd MEN β) , generatd MEN β) . Multipld MEN β) . CFIIm id MEN β) , and intd MEN β) . Howeverd MEN β) . 3) The d MEN β) . 4) Addid MEN β) , interacMore generally, as 3'-end formation is a co-transcriptional process, one can imagine that other chromatin or transcriptional mechanisms could influence 3' end formation, either through indirect impacts or via specific functional couplings. For example, epigenetic modifications and nucleosomal organization , Pol II Drosophila Dscam1, such that distinct protein-encoding isoforms intrinsically bear different post-transcriptional content  . Since b content . It is l content  will revCPA is initiated by recognizing poly(A) signals (PAS) on nascent transcripts. PAS are located 10–30 nt upstream of cleavage sites, and defined by the canonical hexamer AAUAAA , along wCFIm25 (Nudt21) alters 3' isoforms in the hippocampus and yields behavioral defects , generates mRNA isoforms with the same coding region but with different 3' UTR lengths . The alt defects . The dosDrosophila gene lola, which plays diverse roles in neural development and differentiation  machinery. For example, core CPA genes activity . Thus, gAPA is widespread across diverse multicellular eukaryotes, including both animals and plants . The expA particularly notable setting is the expression of hundreds of extended 3' UTR isoforms in neural settings , both inDrosophila and mammals  was initially studied genetically as a locus that is required for viability as well as the differentiation and survival of certain neural tissues, including the eye , with a hinge region connecting RRM2 and 3 , and Elavl2/3/4 (HuB/C/D) . HuR is c lethal , and conc lethal , germinac lethal , adipogec lethal , and colc lethal . In contc lethal  and HuD c lethal  are viaber birth . Overaller birth , maintaier birth , and shaer birth  includiner birth . Beyond er birth .Drosophila and mammals might suggest there could be orthology of specific fly and mammalian family members (Panorpida), other arthropods, as well as C. elegans, encode only a single member ; it is conceivable these are orthologs (Drosophila ELAV/Hu genes (fne and rbp9) harbor introns, and some of these reside in similar positions as the vertebrate ELAV/Hu genes , reveals highly complex alternative splicing and variable levels of nuclear and cytoplasmic proteins in different settings , neuroglian (nrg), and armadillo (arm) were recognized to exhibit unique neural-specific alternative pre-mRNA splicing that was regulated by Elav  variety  were found to be regulated by inhibition of 3' end processing at PAS of proximal ALE isoforms  brains and hila CNS ; an analogenesis . For neantiation . Subsequiation . Moreover, all three family members were capable of conferring globally lengthened 3' UTRs in S2 cells. Although these cells are not converted into neurons per se, they do exhibit largescale shifts to neural-preferred cassette exons, distal ALE usage, and extended 3' UTRs, and often utilize the particular isoform expressed in the nervous system  has only subtle defects on its own but turns out to be a critical regulator when its paralog Elav is not available.Mechanistically, CLIP studies and motif analysis provide evidence that Elav and its characteristic U-rich binding sites are enriched downstream of proximal PAS that are prone to bypass in neurons or in settings of ectopic ELAV/Hu RBPs  Figure . Prior iage site , and curage site . Finallysmic Fne . These sDrosophila genetics have revealed that ELAV/Hu family members are necessary and sufficient to direct neural splicing and APA patterns   , and it r (DSCP) .In the future, additional experiments will be helpful to determine the contribution of paused promoter-mediated recruitment of Elav to its functional targets. In particular, it may be informative to assess Elav-mediated 3' UTR extension under conditions of genetically reduced pausing (e.g. by manipulating GAGA or the pause-inducing NELF complex). Moreover, now that Fne is also recognized as a critical factor in neural TUTR-APA, the recruitment mechanism may be expected to accommodate Fne as well. Finally, the model should accommodate if promoter recruitment of ELAV/Hu members can distinguish amongst multiple functional outcomes, including alternative cassette exon splicing, ALE splicing and tandem 3' UTR isoforms.Amongst numerous setting-specific APA programs that have been documented in mammals, we know of relatively few trans-acting factors  that directly alter 3' UTR landscapes . The higDrosophila ELAV/Hu family RBPs inform the ongoing study of their mammalian counterparts. On the loss-of-function side, the fly genetics clearly indicate that ELAV/Hu RBPs can have overlapping activities in mRNA processing. Metazoan ELAV/Hu RBPs bind relatively similar sequences, and mammalian neural ELAV/Hu paralogs are sufficiently similar that available antibodies do not distinguish them well. Thus, while nearly all genetic studies of mammalian ELAV/Hu RBPs manipulate only a single member, it seems that it is critical to consider the possibility of overlapping activities. Consistent with this, HuC/D dKO mice exhibit phenotypic enhancement over the single mutants  could greatly facilitate such an endeavor.It is also possible that other classes of trans-acting factors are involved . ExistinFinally, it is worth considering the potential involvement of core CPA factors on tissue-specific APA. Since mRNA 3' ends involve differential recognition and utilization of multiple PASs in an individual transcription unit, it is reasonable to infer that the modulation of CPA factors might influence APA. For example, knockdown studies of a couple dozen individual CPA and splicing factors  collectiBeyond the mechanics and genomics of generating tissue-specific alternative transcriptomes, a fundamental question remains about whether this matters for normal biology? It is clear that alternative splicing creates new isoforms with critical functions, but it is somewhat less clear how broad the phenotypic impacts of 3' UTR isoforms are, especially within endogenous contexts. Since 1) hundreds of genes are processed into longer 3' UTR isoforms specifically in neurons, 2) these are often kilobases in length, 3) these frequently utilize conserved, canonical PAS, 4) the overall feature of extended neural 3' UTRs is broadly conserved between flies and mammals, it seems certain that neural APA is not a fortuitous process, but rather is implemented for specific regulatory and/or functional outcomes that are of utility in neurons. However, this does not directly implicate that alternative neural 3' UTRs are necessarily of phenotypically relevant developmental or behavioral consequences.3' UTRs certainly do harbor substantial sites for miRNAs and RBPs, which can be demonstrated functional in a variety of experimental assays. However, there are increasing efforts that show that specific mutation of endogenous 3' UTR binding sites or of entire 3' UTRs did not recapitulate the expected effects . Thus, sDrosophila genetics has been informative in this regard. For example, using transgene rescues, the extended neural 3' UTR of the calcium/calmodulin-dependent protein kinase II gene (CaMKII), which was shown to be required for normal synaptic plasticity  enables regulation by the neural miRNA locus mir-iab-4/8, since many high affinity sites are located within its neural-specific 3' UTR extension  was generated. This mouse mutant maintains expression of the proximal 3' UTR isoform, but exhibits disorganized dorsal root ganglion and reduces Calm1 expression (Several exciting precedents for endogenous functions of neural 3' UTRs and/or explicitly neural APA also exist in mice. For example, the long neural 3' UTR of Importin β1 mediates its axonal localization, and specific deletion of this region impairs recovery from nerve injury . In anotpression .in vitro differentiated neurons (There is no shortage of loci to nominate for hypothesis-driven assessments, e.g., to remove endogenous 3' UTRs or specifically of alternative neural 3' UTR extensions. With the obvious suitability of CRISPR/Cas9 for this task , we may  neurons . These mDrosophila genetics and extending to human autoantigens. Since then, we have learned a great deal on their biological requirements in diverse aspects of neural specification, differentiation and behavior. In addition, we also have learned that ELAV/Hu factors broadly determine multiple aspects of neural expression and the alternative neural transcriptome, including usage of alternative cassette exons, distal alternative exons, and extended 3' UTR isoforms. Further knowledge on the biochemical and genetic functions of ELAV/Hu RBPs will illuminate basic mechanisms of mRNA processing, development and function of the nervous system, and likely human neurological disorders.Neural ELAV/Hu genes and their RBP products have been studied since the mid 1980s, starting with


As part of ribonucleoprotein complexes, the CPEB proteins participate in mRNA transport and localization to different sub-cellular compartments. The CPEB proteins are evolutionarily conserved and have similar functions in vertebrates and invertebrates. In the nervous system, the CPEB proteins are involved in cell division, neural development, learning, and memory. Here we consider the functional features of these proteins in the nervous system of phylogenetically distant organisms: Drosophila, a well-studied model, and mammals. Disruption of the CPEB proteins functioning is associated with various pathologies, such as autism spectrum disorder and brain cancer. At the same time, CPEB gene regulation can provide for a recovery of the brain function in patients with fragile X syndrome and Huntington's disease, making the CPEB genes promising targets for gene therapy.Posttranscriptional gene regulation includes mRNA transport, localization, translation, and regulation of mRNA stability. CPEB ( The functioning of the nervous system is based on the ability of neurons to perceive, transmit, and store information encoded in electrical and chemical signals. The molecular basis for this function in response to stimulation includes alterations in the intracellular distribution of proteins and RNAs and changes in the number and quality of membrane receptors in synapses. One of the most important mechanisms underlying changes in the intracellular architecture of both an individual neuron and entire brain networks is the activation of translation of localized mRNAs mediated by cytoplasmic polyadenylation. Critical regulatory elements that encode signals for transport, anchoring and translational regulation including signals for regulating cytoplasmic polyadenylation (cytoplasmic polyadenylation elements: CPE) are present in the 3′-untranslated region (3′UTR) of mRNA. CPE sequences are recognized by CPEB family proteins, which are widespread in the animal kingdom and show extensive homology among different organisms.Xenopus laevis , a U/GU-rich downstream sequence element, and a cleavage site positioned at a distance of 15–30 nt from the hexamer [Drosophila [The position of CPEs in the 3′UTR of mRNA influences the efficiency of CPEB function. In vertebrates, a polyadenylation site (PAS) is located at the end of the 3′UTR. PAS consists of several  hexamer  , CREB1, CREB2, MAPK (Mek1/2 in mice), and CPEB—perform similar functions in other animals, such as Drosophila and mice [Short-term and long-term memory are distinguished based on the duration of information storage. At the molecular level, the distinction is that activation or inhibition of gene expression is required for long-term memory, but not for short-term memory . Synaptiand mice .Aplysia, serotonin is released in response to stimulation of the presynaptic terminal of a sensory neuron  and induces the synthesis of cAMP from ATP [In from ATP . cAMP acfrom ATP . Thus, ofrom ATP . Repetitfrom ATP , triggerfrom ATP ). The nefrom ATP . Using afrom ATP . The synfrom ATP , 39. Profrom ATP .One potential problem with the mechanism that involves protein synthesis in synapses as a basis for maintaining long-term memory is how this memory could be stored for days or years if protein decay would occur within a shorter period of time. In 1998, Peter Tompa and Peter Friedrich proposed a prion theory of memory . The theDrosophila brain is 100 times higher than the concentration of Orb2A required for a local regulation of translation [Both neurogenesis and the normal functioning of the brain require the transport and localization of mRNAs from the cell body of neurons to dendrites and axons Fig. a. The innslation , 46.Fig.Drosophila, Orb2A is phosphorylated and stabilized during synaptic activation by protein kinase LimK, which thus causes a local increase in Orb2A concentration near synapses [The mRNAs that are transported and localized by CPEB-containing RNP complexes are in a translationally dormant state. For example, mouse mRNAs are localized in P-bodies, which contain monomeric CPEB3 . CPEB3 isynapses . An incrsynapses .Drosophila ortholog of Neurl1  is also required for the formation of long-term memory [Like Orb2A, CPEB3 forms aggregates in response to neuronal stimulation. CPEB3 interaction with the actin cytoskeleton is required for prion formation, while CPEB3 oligomers activate translation of actin mRNA . The resm memory . The actm memory , 54. Them memory . Conditim memory . Moreovem memory . Since aDrosophila. Li et al. [Drosophila resulted in a significant increase in the rate of acquisition of long-term memory. Evidence that the maintenance of memory also requires Orb2 comes from studies on a fly strain carrying an orb2 gene that contains recognition sequences for tobacco mosaic virus protease [de novo Orb2 expression when the memory is recalled. However, activation of the protease before or during training, with its subsequent inactivation, was found to disrupt memory formation. This suggests that Orb2 is also necessary for the formation of memory [The most detailed experiments to confirm the role of the CPEB proteins in long-term memory were carried out on i et al.  found thprotease . When thf memory .The CPEB proteins were also found to regulate expression and the functioning of AMPA receptors. The CPEB2 protein activates translation of mRNAs encoding the endosomal protein GRASP1, implicated in recycling and maintaining the surface level of the AMPA receptors . The numAs indicated in Table Autism spectrum disorder (ASD) is a polygenic disease in which variants of hundreds of genes are involved, each making a minimal individual contribution to the development of the disease. ASD is a developmental disability that manifests itself in childhood and is characterized by a persistent deficit in the ability to initiate and maintain social interaction, as well as limited interests and often repetitive behaviors. A recent study on humans and mice showed that the disorder correlates with impaired CPEB4 mRNA splicing . The amoKip1, activating its translation. CPEB1 competes for binding sites in the p27Kip1 3′UTR with the oncogenic miR-221 and miR-222 microRNAs which repress translation of the p27Kip mRNAs [The CPEB family proteins play an important role in cell proliferation and differentiation; therefore, they are involved in the development of tumors, which makes them potential targets for gene therapy. Expression of the CPEB1 and CPEB4 genes influences the formation of gliomas in the brain. Glioblastoma multiforme, the most common and aggressive brain tumor, has a significantly reduced level of CPEB1 gene expression as compared with normal brain cells , 65. Indip mRNAs . The difXenopus cyclin B1 3′UTR (AdCPE virus), which contains two consensus CPEs and one nonconsensus CPE. This combination of CPEs provides translational repression of the transcript by unphosphorylated CPEB1 and activation upon binding with CPEB4. Intravenous injection of the AdCPE virus in mice resulted in a significant reduction in tumor growth and was less toxic than injection of the AdWT virus. The levels of the pre-mRNA of the AdWT and AdCPE viruses were the same in healthy animal cells, while the mature mRNA level was reduced in cells infected with the AdCPE virus, suggesting a destabilizing effect of CPEB1 on the E1A mRNA [Unlike with CPEB1, expression of CPEB4 is increased in gliomas –68. A deE1A mRNA .FMR1  gene underlies fragile X syndrome and results from an increase in the number of CGG repeats in the 5'UTR of the gene [FMR1 gene is a translational repressor. The inactivation of FMR1 is accompanied by a 20% increase in protein synthesis in the hippocampus [FMR1 knockout), mutations of the CPEB1 gene was found to reduce the pathological processes associated with the syndrome [FMR1 and CPEB1) occurs at the level of RNA stabilization. In the cerebral cortex of FMR1 knockout mice, the synthesis and maturation of mRNA remain at the same level, while an increase in the rate of mRNA degradation is observed for about 700 genes [Aplysia while the synapses are inactive, thereby participating in the maintenance of synaptic plasticity [A current trend is to study the CPEB proteins for potential use as a target for gene therapy. Fragile X syndrome (Martin–Bell syndrome) is an inherited genetic disorder that manifests itself in intellectual disability and is associated with a mutation in one gene located on the X chromosome . Inactivthe gene . The RNApocampus . When mosyndrome . Recent 00 genes . A CPEB100 genes . Studies00 genes . miR-22 asticity .huntingtin (htt). In a pathological process, the number of glutamine coding triplets in htt varies from 36 to 180 [Drosophila cell culture revealed absorption of the Orb2A protein on the surface of HttQ138 aggregates [Drosophila mortality rate at the pupal stage in the presence of HttQ138, increasing the number of hatched flies from 4.8% to 60 and 51% . The amount of HttQ138 aggregates did not decrease, but the balance of protein synthesis in cells was restored [Huntington's disease is a chronic neurodegenerative disease associated with progressive death of brain cells . The cau6 to 180 . The polThe functioning of the CPEB family proteins is essential at all stages of ontogeny. CPEBs play an important role in the formation and maintenance of cell polarity, participating in mRNA transport and localization, translational repression or activation of target mRNAs , 81–83. The CPEB proteins participate in the translational control of a wide range of mRNAs and, therefore, are involved in pathologies of the nervous system. Moreover, disturbances in the functioning of the CPEB proteins cause other pathological processes, including carcinogenesis, tumor invasion, and angiogenesis. In the case of rectal cancer, breast cancer, and gliomas, the expression levels of several CPEB proteins change simultaneously, which is indicative of interactions between them in the oncological process , 84–86. Thus, investigation of the role of the CPEB proteins is an extremely important fundamental task that opens up prospects for understanding the molecular mechanisms of the formation and functioning of the nervous system and other body systems, as well as for finding ways to treat a wide range of diseases.


Setd1b conditional knockout (cKO) oocytes using ultra-low input ChIP-seq, with comparisons to DNA methylation and gene expression analyses. H3K4me3 was redistributed in Setd1b cKO oocytes showing losses at active gene promoters associated with downregulated gene expression. Remarkably, many regions also gained H3K4me3, in particular those that were DNA hypomethylated, transcriptionally inactive and CpG-rich, which are hallmarks of MLL2 targets. Consequently, loss of SETD1B disrupts the balance between MLL2 and de novo DNA methyltransferases in determining the epigenetic landscape during oogenesis. Our work reveals two distinct, complementary mechanisms of genomic targeting of H3K4me3 in oogenesis, with SETD1B linked to gene expression and MLL2 to CpG content.Histone 3 lysine 4 trimethylation (H3K4me3) is an epigenetic mark found at gene promoters and CpG islands. H3K4me3 is essential for mammalian development, yet mechanisms underlying its genomic targeting are poorly understood. H3K4me3 methyltransferases SETD1B and MLL2 (KMT2B) are essential for oogenesis. We investigated changes in H3K4me3 in   Despite islands ,14. Inde islands .Due to the importance of H3K4me3 in development, fertility and disease, there is a need to understand the mechanisms that govern the placement and maintenance of H3K4me3 in chromatin. Several mechanisms that do not rely on positioning by sequence-specific DNA binding proteins (i.e. transcription factors) have been identified. In yeast, the sole H3K4 methyltransferase, Set1 ,16, assoIn mammals, the mechanisms that target H3K4me3 in chromatin have been additionally challenging to disentangle because mammals have six partially redundant H3K4 methyltransferase enzymes  ,27–29. ECxxc1 and Setd1b knockouts  oocytes to elucidate the changes in the H3K4me3 landscape  and FDSetd1b/FD or FDCSetd1b/+;Gdf9-Cre   .Ovaries were collected from 21-day-old females and digested with 2 mg/ml collagenase (Sigma) and 0.02% trypsin solution, agitated at 37°C for 30 min. GV oocytes were collected in KSOM medium (Millipore) and washed with PBS. Four GV oocytes for each genotype were collected for single cell RNA-seq (scRNA-seq). Approximately 100 GV oocytes were collected for each of three biological replicates of each genotype for post-bisulfite adaptor tagging (PBAT). Approximately 200 GV oocytes were collected for each of four biological replicates of each genotype for H3K4me3 ULI-nChIP-seq. All molecular experiments were performed as previously described  but are Setd1b WT. Libraries were multiplexed for 75 bp single-read sequencing on an Illumina HiSeq 2000.Cells were lysed and RNA was reverse transcribed and amplified according to the SMARTer Ultra Low RNA Kit for Illumina Sequencing . Libraries were prepared from cDNA using the NEBNext Ultra DNA library preparation for Illumina sequencing with indexed adaptors (New England Biolabs). Library preparation failed for one replicate of Cells were lysed with 0.5% SDS in EB buffer and bisulfite treated with the Imprint DNA Modification Kit (Sigma). The resulting DNA was purified using columns and reagents from the EZ DNA Methylation Direct Kit (Zymo Research). First-strand synthesis was performed with Klenow Exo- enzyme (New England Biolabs) using a customized biotin-conjugated adaptor containing standard Illumina adaptor sequences and 9 bp of random sequences (9N), as previously described . FollowiThe ULI-nChIP-seq protocol was performed using our previous protocol , which iFastq sequence files were quality and adaptor trimmed with trim galore v0.4.2 using default parameters. For PBAT libraries, the -clip option was used. Mapping of ChIP-seq data was performed with Bowtie v2.2.9 against the mouse GRCm38 genome assembly. The resulting hits were filtered to remove mappings with a MAPQ scores < 20. Mapping of RNA-seq data was performed with Hisat v2.0.5 against the mouse GRCm38 genome assembly, as guided by known splice sites taken from Ensembl v68. Hits were again filtered to remove mappings with MAPQ scores < 20. Sequencing depths for all libraries are provided in Mapping and methylation calling of bisulphite-seq data were performed using Bismark v0.16.3 in PBAT mode against the mouse GRCm38 genome assembly. Trimmed reads were first aligned to the genome in paired-end mode to be able to detect and discard overlapping parts of the reads while writing out unmapped singleton reads; in a second step remaining singleton reads were aligned in single-end mode. Alignments were carried out with Bismark  with theDnmt3 cDKO and Dnmt3 WT GV oocytes, and RNA-seq from Mll2 cKO and WT GV oocytes (GSE93941) (Setd1b cKO and WT MII oocytes (GSE85360) (Cxxc1 cKO and WT GV oocytes (GSE85019) (Cxxc1 cKO and WT GV oocytes (GSE159581) (Publicly available datasets were downloaded from Gene Expression Omnibus (GEO), including: H3K27me3 and H3K27ac ChIP-seq in GV oocytes, H3K4me3 ChIP-seq from d5 non-growing oocytes, d10 growing oocytes, d15 GV oocytes, d25 GV oocytes, SE93941) , RNA-seqSE93941) , RNA-seqSE93941) , RNA-seqSE85360) , RNA-seqE159581) , and bisSetd1b WT and one replicate of Setd1b cKO. Poor enrichment was defined as a cumulative distribution plot reflecting a signal-to-noise similar to input and presenting as an outlier in hierarchical clustering. For all subsequent analyses, including the use of publically available H3K4me3 ChIP-seq data, valid 5 kb windows were quantitated using enrichment normalization of RPKM in SeqMonk, as previously described (P < 0.05 corrected for multiple comparisons) was used to identify differentially enriched H3K4me3 windows between Setd1b cKO and WT GV oocytes, using an average normalized RPKM for replicates within each group. Overlapping TSSs and promoters (TSS ±500 bp) were classified as previously described (Running windows (5 kb) were used for quantitative analysis of H3K4me3 ChIP-seq data using RPKM, with mapping artifacts excluded from analysis (RPKM > 4 in any input sample). Poor quality H3K4me3 ChIP-seq libraries were excluded from analysis, which included one replicate of escribed . LIMMA sescribed . In brieescribed  or markeP < 0.05 corrected for multiple comparisons) and a >2-fold change in expression: (i) Setd1b cKO and WT GV oocytes, (ii) 10–30 μm oocytes (non-growing oocytes) and GV oocytes, (iii) Cxxc1 cKO and WT GV oocytes, and (iv) Mll2 cKO and WT GV oocytes. Fold enrichment for genes upregulated in oogenesis and DEGs among the Setd1b, Cxxc1 and Mll2 cKOs was calculated relative to all oocyte transcripts and increase in observed over expected overlap between DEGs from different conditions was statistically compared using Chi-square. Gene ontology analysis was done using DAVID (https://david.ncifcrf.gov/) for up- and downregulated DEGs, using the default settings with the addition of the UP_TISSUE category.RNA-seq datasets were quantitated using the RNA-seq quantitation pipeline in Seqmonk, over previously defined oocyte transcripts . TranscrSetd1b cKO and a random set) was assessed using compter (https://www.bioinformatics.babraham.ac.uk/projects/compter/). The mouse genome was used as the background and values were expressed as log2 observed/expected. Enrichment values for each dimer in each condition were summarized by taking the mean, and the difference in mean log ratio (Setd1b cKO - Random) was calculated and plotted.Dimer composition of candidate 5 kb windows (gains and losses of H3K4me3 in Setd1b cKO and WT GV oocytes using logistic regression (P < 0.05 corrected for multiple comparisons) and a > 20% difference in methylation.DNA methylation datasets were analysed using 100-CpG running windows, with a minimum coverage of at least 10-CpGs, using the bisulfite-seq quantitation pipeline in SeqMonk. Differentially methylated regions (DMRs) were identified between Setd1b driven by Gdf9-Cre (Setd1b cKO (N = 3) and Setd1b WT (N = 3) GV oocytes  oocytes from the ovaries of 21-day-old mice after oocyte-specific ablation of Gdf9-Cre ,43. PoolGdf9-Cre . The gens Figure , in concs Figure . However cluster , and we  cluster ,B. The m cluster . However cluster . Promoten Figure . Conversn Figure . This trn Figure .Setd1b cKO and found a highly significant enrichment for CpG content compared to a random set of regions  between Setd1b cKO (N = 4) and Setd1b WT (N = 3) GV oocytes, with 594 genes downregulated and 925 gene upregulated in the Setd1b cKO , while there was no difference at upregulated DEGs   Figure . Neverth) Figure , reinforSetd1b cKO MII oocytes  but not upregulated DEGs  in Setd1b cKO oocytes  but not upregulated DEGs   (Setd1b cKO and Cxxc1 cKO for Setd1b DEGs (As CXXC1 and SETD1B function together in the SETD1B complex  and have oocytes . Similar) Figure . We see  0.0001) . There id1b DEGs . These dSetd1b cKO oocytes, we evaluated the dynamics of promoter H3K4me3 for genes upregulated in oogenesis that were downregulated DEGs compared to genes not called as differentially expressed (not DEGs) in Setd1b cKO oocytes . Promoters of Setd1b cKO downregulated DEGs gained the majority of H3K4me3 early in oogenesis (between d5 and d10), while the promoters of genes that were not DEGs continued to gain H3K4me3 throughout oogenesis until d25 , we assessed genome-wide patterns of DNA methylation using post-bisulphite adaptor tagging (PBAT). DNA methylation in Setd1b cKO and WT GV oocytes was distinct  GV oocytes , a , a de noO Figure ,E. TogetSetd1b cKO oocytes. Previously, we discovered that MLL2 deposits broad, transcription-independent H3K4me3 domains that are uniquely and abundantly found on the mouse oocyte genome  ectopic accumulation of H3K4me3 acts to block de novo DNMTs, or (ii) delayed de novo DNA methylation allows encroachment of H3K4me3. It is also possible that the two mechanisms exist side by side to mutually shape the H3K4me3/DNA methylation landscape. The generalized loss of DNA methylation suggests a delay in the normal de novo methylation process in Setd1b cKO oocytes, although we did not detect reduced transcript abundance of the key Dnmts. In support of this model, we found that gains in H3K4me3 seen in Setd1b cKO oocytes partially recapitulate trends seen in oocytes lacking all DNA methylation. Furthermore, the high CpG content of regions that gain H3K4me3 in the Setd1b cKO may support a model where MLL2 is titrated away from CpG-poor inter-genic sites to more favorable CpG-rich sites when DNA methylation is delayed. Alternatively, because de novo methylation and non-promoter H3K4me3 deposition are mutually exclusive and occur in parallel in growing oocytes .gkac051_Supplemental_FileClick here for additional data file.


The pioneer factor hypothesis (PFH) states that pioneer factors (PFs) are a subclass of transcription factors (TFs) that bind to and open inaccessible sites and then recruit non-pioneer factors (non-PFs) that activate batteries of silent genes. The PFH predicts that ectopic gene activation requires the sequential activity of qualitatively different TFs. We tested the PFH by expressing the endodermal PF FOXA1 and non-PF HNF4A in K562 lymphoblast cells. While co-expression of FOXA1 and HNF4A activated a burst of endoderm-specific gene expression, we found no evidence for a functional distinction between these two TFs. When expressed independently, both TFs bound and opened inaccessible sites, activated endodermal genes, and ‘pioneered’ for each other, although FOXA1 required fewer copies of its motif for binding. A subset of targets required both TFs, but the predominant mode of action at these targets did not conform to the sequential activity predicted by the PFH. From these results, we hypothesize an alternative to the PFH where ‘pioneer activity’ depends not on categorically different TFs but rather on the affinity of interaction between TF and DNA. Cells only use a fraction of their genetic information to make the proteins they need. The rest is carefully packaged away and tightly bundled in structures called nucleosomes. This physically shields the DNA from being accessed by transcription factors – the molecular actors that can read genes and kickstart the protein production process. Effectively, the genetic sequences inside nucleosomes are being silenced.However, during development, transcription factors must overcome this nucleosome barrier and activate silent genes to program cells. The pioneer factor hypothesis describes how this may be possible: first, ‘pioneer’ transcription factors can bind to and ‘open up’ nucleosomes to make target genes accessible. Then, non-pioneer factors can access the genetic sequence and recruit cofactors that begin copying the now-exposed genetic information.The widely accepted theory is based on studies of two proteins – FOXA1, an archetypal pioneer factor, and HNF4A, a non-pioneer factor – but the predictions of the pioneer factor hypothesis have yet to be explicitly tested. To do so, Hansen et al. expressed FOXA1 and HNF4A, separately and together, in cells which do not usually make these proteins. They then assessed how the proteins could bind to DNA and impact gene accessibility and transcription.The experiments demonstrate that FOXA1 and HNF4A do not necessarily follow the two-step activation predicted by the pioneer factor hypothesis. When expressed independently, both transcription factors bound and opened inaccessible sites, activated target genes, and ‘pioneered’ for each other. Similar patterns were observed across the genome. The only notable distinction between the two factors was that FOXA1, the archetypal pioneering factor, required fewer copies of its target sequence to bind DNA than HNF4A.These findings led Hansen et al. to propose an alternative theory to the pioneer factor hypothesis which eliminates the categorical distinction between pioneer and non-pioneer factors. Overall, this work has implications for how biologists understand the way that transcription factors activate silent genes during development. Transcription factors (TFs) face steric hindrance when instances of their motifs are occluded by nucleosomes . This baPFs also play a primary role in cellular reprogramming by first engaging silent regulatory sites of ectopic lineages . ContinuThe PFH makes strong predictions about the activities of ectopically expressed PFs and non-PFs. Because PFs are defined by their ability to bind nucleosome-occluded instances of their motifs, the PFH predicts that PFs should bind to a large fraction of their motifs. However, similar to other TFs, PFs only bind a limited subset of their inaccessible motifs . There aTo test these predictions, we expressed FOXA1 and HNF4A separately and together in K562 lymphoblast cells and then measured their effects on DNA-binding, chromatin accessibility, and gene activation. In contrast to the predictions of the PFH, we found that both FOXA1 and HNF4A could independently bind to inaccessible instances of their motifs, induce chromatin accessibility, and activate endoderm-specific gene expression. The only notable distinction between the two factors was that HNF4A required more copies of its motif to bind. When expressed together, co-binding could only be explained in a minority of cases by sequential FOXA1 and HNF4A activity. Instead, most co-bound sites required concurrent co-expression of both factors, which suggests cooperativity between these TFs at certain repressive genomic locations. We suggest that our findings present an alternative to the PFH that eliminates the categorical distinction between PFs and non-PFs and instead posits that the energy required to pioneer occluded sites (‘pioneer activity’) comes from the affinity of interaction between TFs and DNA.We tested predictions of the PFH using FOXA1 as a model endoderm PF and HNF4A as a model non-PF. Because PFs are defined by their behavior in ectopic settings, we expressed FOXA1 and HNF4A in mesoderm-derived K562 lymphoblast cells. These cells express neither FOXA1 nor HNF4A and present a different complement of chromatin and cofactors. Thus, any ectopic signature that we observe is due primarily to the TFs themselves. We focused only on the initial response to TF expression to capture primary mechanisms of TF behavior and not the secondary effects that can lead to cellular conversion and that may confound our analyses.FOXA1 and HNF4A, and their respective target genes ALB and APOB, to reach a plateau of expression  and 38 intestinal genes   . The duaometric) . We concALB, FOXA1 and HNF4A co-bound at inaccessible sites and increased accessibility  , and bot–4, cumulative hypergeometric) and weak intestinal-specific enrichment (not significant)  and strong intestinal-specific enrichment   , while Hometric) . ImportaWhen expressed individually, FOXA1 and HNF4A activated largely independent sets of liver genes  and inteARG1, a liver-specific gene that is silent in K562 cells  in Iscove’s Modified Dulbecco Serum supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% nonessential amino acids. We used these cells to generate our clonal lines , and we thank Washington University in St. Louis Genome Engineering and the iPSC Center for their help confirming K562 identity with STR profiling and testing for mycoplasma contamination. When it was time to conduct one of our functional assays, we split FOXA1-, HNF4A-, or FOXA1-HNF4A-expressing cells into replicate flasks and then treated with either ±0.5 µg/ml or 0.05 µg/ml doxycycline (Sigma #D9891-1G) for 24 hr.We used PCR to add V5 epitope tags to the 3′ end of FOXA1  and HNF4A (Addgene #120450) constructs and then used HiFi DNA Assembly  to clone each construct into a pINDUCER21 doxycycline-inducible lentiviral vector (Addgene #46948). All primers are listed in We extracted RNA from 1e6 cells/sample with the PureLink RNA Mini  column extraction kit and completed on-column DNA digestion with PureLink DNase (Invitrogen #12185010). We quantified and assessed the quality of the RNA with an Agilent 2200 Tapestation instrument and then either froze down pure RNA for later RNA-sequencing library preparation or used ReadyScript cDNA Synthesis Mix (Sigma #RDRT-100RXN) to produce cDNA for qPCR. We performed qPCR with SYBR Green PCR Master Mix  and gene-specific and housekeeping primers .We generated three replicates of ±doxycycline-treated RNA-sequencing libraries with the NEBNext Ultra II Directional RNA Library Prep Kit (NEB #E7765S). We quantified and assessed the quality of the libraries with an Agilent 2200 Tapestation instrument, size selected with AMPure XP beads , and then sequenced the libraries with 75 bp paired-end reads on an Illumina NextSeq 500 instrument.We quantified transcripts with Salmon , filtereWe followed the Omni-ATAC protocol  to gener--local -X2000, generated RPKM normalized BigWig files for visualization with deepTools bamCoverage  to generate two replicates of low-background CUT&Tag libraries. We isolated 1e5 cells/sample, extracted nuclei with Concanavalin A paramagnetic beads (EpiCypher #21-1401), and then either used rabbit anti-human FOXA1 monoclonal antibody , mouse anti-human HNF4A monoclonal antibody (Invitrogen #MA1-199), or rabbit anti-human histone H3K4me3 polyclonal antibody (EpiCypher #13-0041) as a positive control. We amplified this signal with either goat anti-rabbit (EpiCypher #13-0047) or goat anti-mouse (EpiCypher #13-0048) polyclonal secondary antibodies. For a negative control, we omitted the primary antibody and checked for any nonspecific pull-down. Finally, we used CUTANA pAG-Tn5 (EpiCypher #15-1017) to tagment the genomic regions surrounding each bound antibody complex. We quantified and assessed the quality of the libraries with an Agilent 2200 Tapestation instrument, size selected with AMPure XP beads, and then sequenced the libraries with 150 bp paired-end reads on an Illumina NextSeq 500 instrument. --very-sensitive --end-to-end --no-mixed --no-discordant -I 10X700, created RPKM normalized BigWig files with deepTools bamCoverage  are ectopically expressed. These results provide a new standard for testing the degree to which a TF acts as a pioneer factor. Moreover, they suggest that cis elements  are critical to open a locus upon transcription factor expression. How motif density and TF binding contribute to nucleosome eviction will be interesting to unravel in future studies. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Jessica Tyler as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "A Test of the Pioneer Factor Hypothesis" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) Tone down and clarify conclusions. See reviewer comments below for details. In particular, the title is misleading and should be edited to reflect the more narrow scope of the manuscript. Also, the limitations of the experimental system should be made clear. Finally, there should be more of an attempt to reconcile their findings with prior studies.2) Various data analyses to address reviewer comments, specifically address the following ones:A) Are the sites that are independently activated by FOXA1 or HNF4A bound by other TFs as well? Specifically, is it the case that FOXA1 and HNF4A are acting like solo pioneering factors at these sites, or are FOXA1 and HNF4A similarly "cooperatively co-bound" at these sites with TFs that are endogenously expressed in K562 cells but on their own do not open up these elements?B) A more thorough analysis could substantially strengthen the manuscript. Most features in this manuscript are classified in a Boolean OFF/ON manner, which creates clean analysis, but limits the scope of interpretations. By oversimplifying the assumptions, this limits the ability to explain the results in the context of sequence affinity. Do Foxa1 and Hnf4a bind with equal amounts to co-bound accessible sites? Does their binding enrichment (characterized by CUT&Run) correlate with differential pre- and post- induction ATAC-seq enrichment? It is mentioned in the discussion that "not all sites containing multiple motifs were pioneered in K652 cells" and suggests many possible explanations such as (1) TF affinity to its motif, (2) sequence context, (3) motif orientation relative to the nucleosome. However, aside from running FIMO to scan for motif instances, there is limited follow-up analysis. At the very least, FIMO could be used to quantitatively score motif matches in the genome in order to explore the role of sequence affinity.3) Determine how TF concentration impacts the results. The TF concentration very likely determines the degree by which chromatin is made accessible. Since the authors have the perfect experimental system to address this question, it would be straightforward to determine whether changing TF concentration influences the ability of Hnf4a to open chromatin comparably to Foxa1.Reviewer #2 (Recommendations for the authors):Please provide data on the level of expression of FOXA1 and HNF4A relative to their physiological expression levels in human tissue/cell lines.The presentation of Supplementary Figure 2 and 3 is confusing. Is Supplementary Figure 3a stating that there are ~35,000 FoxA1 CUT&Tag peaks in uninduced cells? Or is this showing the ATAC-seq chromatin accessibility in uninduced cells for FoxA1 CUT&Tag peaks that were identified in induced cells?Page 6, line 137. The line "The vast majority of both FoxA1 and Hnf4a binding sites fell within inaccessible regions…" is confusing, as it implies that the FoxA1 and Hnf4a CUT&Tag peaks were outside of ATAC-seq peaks in the same sample. I am assuming that you meant to say that the "The vast majority of both FoxA1 and Hnf4a binding sites in the induced sample matched inaccessible regions in the uninduced sample…"Please provide quality summary statistics for ATAC-seq and CUT&Tag data. Were these samples sequenced to a similar degree? Do the ATAC-seq samples have a similar proportion of reads surrounding TSSs, or within peaks? Were a similar number of peaks called in each ATAC-seq sample?Pg 12, line 235. "cooperative" is a better word than "collaborative" to describe this phenomenon. Similarly, the term "collaboratively co-bound" is better represented using the term "cooperatively co-bound". Note that the term cooperative does not necessitate direct protein-protein interactions between TFs, but can rather merely reflect their cooperative ability to stabilize a nucleosome free stretch of DNA (PMID 21149679).Please provide further description on how you define the list of organ-specific genes?Per convention, please use uppercase protein names as K562 is a human cell line.Are the sites that are independently activated by FOXA1 or HNF4A bound by other TFs as well? Specifically, is it the case that FOXA1 and HNF4A are acting like solo pioneering factors at these sites, or are FOXA1 and HNF4A similarly "cooperatively co-bound" at these sites with TFs that are endogenously expressed in K562 cells but on their own do not open up these elements?Reviewer #3 (Recommendations for the authors):The key weakness in this manuscript is that the authors question the pioneer factor hypothesis based on a single finding. They find that Hnf4a, which was classified previously as non-pioneer, is able to open chromatin in their assay. Furthermore, they do not sufficiently explore the similarities and differences between the binding behavior of Foxa1 and Hnf4a to provide more insights. As it is, they do not refute the pioneer factor hypothesis and thus the title is misleading. There are several main points that would strengthen the manuscript:1) If Hnf4a is also able to open chromatin, why has it previously been categorized as a non-pioneer? Is it because of its role as "differentiation TF", thus a TF that is expressed relatively late in development? Or are there physical properties of the TF that make it a non-pioneer? Some of the cited papers actually describe Hnf1a whose behavior is quite similar to pioneer TFs. Without exploring this, the simplest explanation for the observed results is that Hnf4a has previously been misclassified.2) A more thorough analysis could substantially strengthen the manuscript. Most features in this manuscript are classified in a Boolean OFF/ON manner, which creates clean analysis, but limits the scope of interpretations. By oversimplifying the assumptions, this limits the ability to explain the results in the context of sequence affinity. Do Foxa1 and Hnf4a bind with equal amounts to co-bound accessible sites? Does their binding enrichment (characterized by CUT&Run) correlate with differential pre- and post- induction ATAC-seq enrichment? It is mentioned in the discussion that "not all sites containing multiple motifs were pioneered in K652 cells" and suggests many possible explanations such as (1) TF affinity to its motif, (2) sequence context, (3) motif orientation relative to the nucleosome. However, aside from running FIMO to scan for motif instances, there is limited follow-up analysis. At the very least, FIMO could be used to quantitatively score motif matches in the genome in order to explore the role of sequence affinity.3) What is the effect of TF concentration? I understand the rationale of the experimental design to express Foxa1 and Hnf4a at similar levels in order to test their ability to open chromatin at the same concentration. However, the TF concentration very likely determines the degree by which chromatin is made accessible. Since the authors have the perfect experimental system to address this question, there is a missed opportunity to explore whether changing TF concentration influences the ability of Hnf4a to open chromatin comparably to Foxa1. Essential revisions:1) Tone down and clarify conclusions. See reviewer comments below for details. In particular, the title is misleading and should be edited to reflect the more narrow scope of the manuscript.We have changed the title and adjusted our language to reflect the focus of our study on FOXA1, HNF4A, and their role in inducing ectopic expression of liver-specific genes. We chose FOXA1 and HNF4A because they offer a clear and testable example of the PFH where a PF and nonPF have been hypothesized to act sequentially. We have also clarified that our results do not necessarily apply to all other combinations of PFs and nonPFs .Also, the limitations of the experimental system should be made clear.We have emphasized that our conclusions are based on the behavior of two factors in one cell line (line 317), but also have directly addressed reviewer comments regarding TF concentration and endogenous K562 co-factors (see below).Finally, there should be more of an attempt to reconcile their findings with prior studies.We have added a section that discusses why HNF4A may have been mis-classified as a nonPF (line 308).2) Various data analyses to address reviewer comments, specifically addressthe following ones:A) Are the sites that are independently activated by FOXA1 or HNF4A bound by other TFs as well? Specifically, is it the case that FOXA1 and HNF4A are acting like solo pioneering factors at these sites, or are FOXA1 and HNF4A similarly "cooperatively co-bound" at these sites with TFs that are endogenously expressed in K562 cells but on their own do not open up these elements?We have Figure 3—figure supplement 2 and accompanying text (line 191) that demonstrates that binding motifs for endogenous K562 factors are not more enriched at FOXA1 or HNF4a inaccessible binding sites than expected by random chance, nor are they differentially enriched between FOXA1 vs. HNF4A binding sites.B) A more thorough analysis could substantially strengthen the manuscript. Most features in this manuscript are classified in a Boolean OFF/ON manner, which creates clean analysis, but limits the scope of interpretations. By oversimplifying the assumptions, this limits the ability to explain the results in the context of sequence affinity. Do Foxa1 and Hnf4a bind with equal amounts to co-bound accessible sites? Does their binding enrichment (characterized by CUT&Run) correlate with differential pre- and post- induction ATAC-seq enrichment? It is mentioned in the discussion that "not all sites containing multiple motifs were pioneered in K652 cells" and suggests many possible explanations such as (1) TF affinity to its motif, (2) sequence context, (3) motif orientation relative to the nucleosome. However, aside from running FIMO to scan for motif instances, there is limited follow-up analysis. At the very least, FIMO could be used to quantitatively score motif matches in the genome in order to explore the role of sequence affinity.We have added Figure 5 and accompanying text to show that an affinity model more parsimoniously explains ectopic FOXA1 and HNF4A binding behavior than the Pioneer Factor Hypothesis . We show that the total motif count distinguishes between binding sites and random DNA, between inaccessible binding sites and accessible binding sites, and between sites “pioneered” or “cooperatively bound” sites. In each case, we can equally well predict FOXA1 and HNF4A binding with motif count, suggesting that the two TFs are not behaving qualitatively differently.3) Determine how TF concentration impacts the results. The TF concentration very likely determines the degree by which chromatin is made accessible. Since the authors have the perfect experimental system to address this question, it would be straightforward to determine whether changing TF concentration influences the ability of Hnf4a to open chromatin comparably to Foxa1.In response to this comment we performed new experiments in which we reduced the levels of FOXA1 and HNF4A induction by lowering the doxycycline concentration from 0.5 to 0.05 ug/ml. The lower induction conditions reduce binding and liver gene activation for both FOXA1 and HNF4A, but does not qualitatively alter the results: the effects are still the same for both FOXA1 and HNF4A .Reviewer #2 (Recommendations for the authors):Please provide data on the level of expression of FOXA1 and HNF4A relative to their physiological expression levels in human tissue/cell lines.It is unclear what expression levels of FOXA1 and HNF4A are physiological, as their levels in HepG2 or even primary fibroblasts are not physiological. It was partly for this reason that we chose to study the PFH in the context of cellular reprogramming (where it was first conceived) and why we followed protocols used during reprogramming assays. Our concentrations may be higher than during development, but they are the same as the levels used in cellular reprogramming protocols. To address the concern that our results might be an artifact of overexpression, we lowered the induction conditions 10-fold and re-measured binding and ectopic expression for both FOXA1 and HNF4A. As discussed above, the lower induction conditions reduce binding and liver gene activation for both FOXA1 and HNF4A, but does not qualitatively alter the results: the effects are still the same for both FOXA1 and HNF4A. We also have taken careful steps to limit the degree of over-expression and to express FOXA1 and HNF4A at similar levels .The presentation of Supplementary Figure 2 and 3 is confusing. Is Supplementary Figure 3a stating that there are ~35,000 FoxA1 CUT&Tag peaks in uninduced cells? Or is this showing the ATAC-seq chromatin accessibility in uninduced cells for FoxA1 CUT&Tag peaks that were identified in induced cells?We now clarify in the text that Figure 1—figure supplement 2 and Figure 3—figure supplement 1 show the chromatin accessibility in uninduced (-dox) cells for FOXA1 or HNF4A CUT&Tag peaks that were identified in induced (+dox) cells .Page 6, line 137. The line "The vast majority of both FoxA1 and Hnf4a binding sites fell within inaccessible regions…" is confusing, as it implies that the FoxA1 and Hnf4a CUT&Tag peaks were outside of ATAC-seq peaks in the same sample. I am assuming that you meant to say that the "The vast majority of both FoxA1 and Hnf4a binding sites in the induced sample matched inaccessible regions in the uninduced sample…"Yes. We have clarified this in the revised text (line 141).Please provide quality summary statistics for ATAC-seq and CUT&Tag data. Were these samples sequenced to a similar degree? Do the ATAC-seq samples have a similar proportion of reads surrounding TSSs, or within peaks? Were a similar number of peaks called in each ATAC-seq sample?We have added Supplementary Files 4-5 to address these statistics and included the analysis within our methods (lines 410 and 436).Pg 12, line 235. "cooperative" is a better word than "collaborative" to describe this phenomenon. Similarly, the term "collaboratively co-bound" is better represented using the term "cooperatively co-bound". Note that the term cooperative does not necessitate direct protein-protein interactions between TFs, but can rather merely reflect their cooperative ability to stabilize a nucleosome free stretch of DNA (PMID 21149679).Thank you for the recommendation. We have adjusted our language throughout .Please provide further description on how you define the list of organ-specific genes?We have detailed how we defined tissue-specific genes in the Methods section (line 390).Per convention, please use uppercase protein names as K562 is a human cell line.We have corrected this error throughout.