This conference brought together a highly dynamic community of researchers with the common aim to understand how chromatin organization controls gene expression, development, and plant responses to the environment. New evidence showing how epigenetic states are set, perpetuated, and inherited were presented, and novel data related to the three-dimensional organization of chromatin within the nucleus were discussed. At the level of the nucleosome, its composition by different histone variants and their specialized histone deposition complexes were addressed as well as the mechanisms involved in histone post-translational modifications and their role in gene expression. The keynote lecture on plant DNA methylation by Julie Law (SALK Institute) and the tribute session to Lars Hennig, honoring the memory of one of the founders of the EWPC who contributed to promote the plant chromatin and epigenetic field in Europe, added a very special note to this gathering. In this perspective article we summarize some of the most outstanding data and advances on plant chromatin research presented at this workshop.In June 2019, more than a hundred plant researchers met in Cologne, Germany, for the 6 A total of 110 researchers met to present the most recent focuses, advances, and challenges in the plant chromatin and epigenetics field during this 2-day workshop that comprised more than 25 standard talks and a similar number of short PechaKucha-style talks. Many other topics were talked over during the poster sessions in which the participants had the opportunity to discuss new discoveries and concepts in plant chromatin science in a thriving atmosphere.Last year, the Max Planck Institute for Plant Breeding Research in Cologne hosted the 6Several talks emphasized the complexity of chromatin organization within the three-dimensional space of the nucleus and presented cutting-edge techniques developed to provide a deeper and higher-resolution view of chromatin structure  was invited to present the keynote lecture, which highlighted some of the most important past and present contributions to the DNA methylation field from her laboratory. Julie gave an overview of the crucial roles played by DNA methylation in gene regulation and transposon silencing. In addition, she reported that a family of four putative chromatin remodeling factors, CLASSY (CLSY) 1–4, associate with the RNA-directed DNA methylation (RdDM) pathway components Pol-IV and SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1) . FurtherThe EWPC was also the perfect venue for honoring the memory of Professor Lars Hennig who has recently passed away . TogetheThis perspective article summarizes the main topics discussed during the EWPC 2019 and provides insight into the future paths that the plant epigenetic community will follow in the next years. We thank all the laboratories, which have contributed to the EWPC with recently published or unpublished data, and we apologize to the researchers whose work could not be cited due to space limitations.The key bearer of genetic information in eukaryotic cells is chromatin, which is non-randomly distributed inside the nucleus and shows an extraordinary degree of compaction and spatial organization. Nuclear organization is achieved by many factors, including histone proteins, modifiers and readers, as well as structural components of the nuclear periphery and nuclear bodies, which together dynamically control the nuclear architecture and may form nuclear domains . The firThe core histones have been structurally conserved through evolution and have evolved to accomplish two conflicting and yet vital tasks: on one hand, the long DNA molecules have to be packaged within the limits of the eukaryotic nucleus, preventing knots and tangles and protecting the genome from physical damage; on the other hand, the information that is encoded in the DNA needs to be accessed at appropriate times . The linFrédéric Pontvianne  focused on the nucleolus, the largest nuclear body, which is well known as the site of ribosomal RNA (rRNA) gene transcription, rRNA processing, and ribosome biogenesis . In a prcrwn1 crwn2 double mutants in Arabidopsis. The interaction between PWO1 and CRWN1 suggests a role of the nuclear periphery in PRC2-mediated gene regulation in Arabidopsis  presented data showing a putative link between Polycomb Group (PcG)-mediated repression and the nuclear periphery in Arabidopsis. PWWP INTERACTOR OF POLYCOMB (PWO1) is a PWWP-domain containing protein able to interact with any of the three possible POLYCOMB REPRESSIVE COMPLEX 2 (PRC2) histone methyltransferases in Y2H, and PWO1-CURLY LEAF (CLF) interaction was confirmed in planta . Among tbidopsis . The SchThe post-translational modifications of telomere histones in plants have been investigated by Katerina Adamusova . Among the canonical and non-canonical telomeres in plants, the authors found two kinds of epigenetic patterns regardless of the differences in telomere length and telomeric sequences used. One of them corresponds to the Arabidopsis-like pattern, where telomere histones are marked predominantly with H3K9me2. The other one is the tobacco-like pattern marked predominantly with H3K27me3 .ADM and AHL10 in Arabidopsis triploid seeds results in H3K9me2 hypermethylation in MARs. Furthermore, AHL10-mediated H3K9me2 hypermethylation at MARs is independent of DNA methylation  discussed the role of AT-hook proteins in the regulation of gene expression by mediating the H3K9me2 heterochromatic mark at the nuclear matrix-associated regions (MARs). They identified AT-Hook Like 10 (AHL10), a member of the AT-hook family in Arabidopsis, and the SET domain containing SU(VAR)3-9 homolog (SUVH9) as interacting partners of ADMETOS (ADM), which functions in establishing the postzygotic hybridization barrier in Arabidopsis. Significantly increased expression of hylation . Apart fHDACs display similar abnormal phenotypes as mea mutants, suggesting an interplay between histone methylation and acetylation during early seed development.Recent advances in our understanding of inter-generational inheritance of epigenetic and chromatin marks have revealed a variety of plant peculiarities, rendering this topic an exciting field of study with impact on our fundamental understanding of inheritance, phenotypic plasticity, population dynamics, and evolution . NeverthThe double fertilization process of plants generates an additional complication in the understanding of trans-generational inheritance and maternal and paternal contributions to the next generation. Thus, being able to distinguish events taking place in the endosperm from other plant tissues will be crucial to understand the peculiarities of this triploid tissue. An exciting technical advance in this direction was presented by Vikash Kumar Yadav . He performed modified high-throughput chromatin conformation (mHi-C) on purified endosperm nuclei isolated by the INTACT method , thus enHeinrich Bente  focused his presentation on yet another aspect of epigenetic inheritance, the phenomenon of paramutation, characterized by interallelic communication between epialleles at a single locus that results in stable and heritable silencing. Employing an epigenetically regulated resistance marker for hygromycin in Arabidopsis, Heinrich and his co-workers found that paramutation becomes apparent in F2 progeny of tetraploid hybrids but not in diploid ones. Small RNA profiles differ between the two epialleles, as do DNA methylation and chromatin marks. The fact that the paramutation is not observed at low temperatures, where also small RNA production is reduced , supportfreak show (fks), a novel missense mutant of the RNA Polymerase V-specific subunit NRPE5A , are the plant-specific histone variants H2A.W.6, H2A.W.7, and H2A.W.12, highly enriched in heterochromatin and involved in chromatin fiber–fiber interactions . These h complex . Indeed,is locus .hira mutants show reduced nucleosomal occupancy both at genes and in heterochromatin translating into reactivation of transposable elements. While some H3 histone chaperones are highly conserved, species-specific chaperones deposit the centromeric histone CenH3   and the ne CenH3 . So far,ne CenH3 , the nuc protein .Professor Lars Hennig passed away last year, leaving a gap in the fields of chromatin biology and plant development . Sessionclf and swinger (clf swn)  as one subunit of the CHROMATIN ASSEMBLY FACTOR 1 (CAF-1) chaperone complex . Lars fuclf swn) , which ilhp1 mutant, which is similar to what occurs in the top1α lhp1 mutant (ANTAGONIST OF LHP1 (ALP1), which was identified in a suppressor screening of the clf mutant  . Studyin1 mutant . She prof mutant . The intf mutant . As Harberfamily , this ispDORNRÖSCHEN-LIKE::GFP in the apetala1 cauliflower mutant background were used  presented a very elegant approach in which Fluorescence Activated Cell Sorting (FACS) was combined with the Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq), which allowed for the creation of maps with higher resolution than with DNase-seq from low cell numbers. Lines expressing the ere used , which aere used . LOFCs-aere used .Genome-wide approaches were also used to identify light-induced chromatin dynamics that occur at a very specific developmental switch, such as photomorphogenesis, which corresponds to the first perception of light after germination . DE-ETIOGH1-HMGA1 gene were impaired in development, with an overall size reduction due to smaller roots and leaves and a decrease in stem length, while gh1-hmga2 mutants were phenotypically normal. gh1-hmga1 mutants also showed shorter telomeres as a result of telomere instability (gh1-hmga1 mutants suggested a contribution of GH1-HMGA1 proteins to gene expression control.High Mobility Group A (HMGA) proteins have also been proposed to create a more permissive chromatin structure competing with linker histone H1 . Simon Atability , and a tS2Lb affect the steady state levels of only a few of its target genes. Therefore, as part of AtCOMPASS, S2Lb may be required for appropriate transcriptional dynamics but is not essential for gene expression. Interestingly, S2Lb recruitment and H3K4me3 deposition at target genes are independent of H2Bub, indicating that AtCOMPASS-S2Lb activity does not require H2Bub in contrast to yeast. Whether there is a crosstalk between this histone mark and other methyltransferases is still an open question  adds this mark as part of COMPlex of proteins ASsociated with SET1 (COMPASS). Another subunit of this complex, Swd2, is needed for the recruitment of COMPASS to specific chromatin domains enriched in H2Bub . In Arabquestion .Whether it is by changing large-scale chromatin conformation, nucleosome composition, and occupancy or histone post-translational modifications, chromatin regulation can impact plant development in a variety of ways, as highlighted in the previous sessions. This complexity becomes more evident when, despite being highly conserved among the plant species, the function of chromatin regulators, as well as their target genes, also depends on the context in which they act . The lasChromatin-based regulation allows us to quickly and reversibly switch genes on and off through the concerted action of antagonistic regulators. One example was presented by Cristel Carles  with her latest work on ULTRAPETALA1 (ULT1). It was known that ULT1 antagonizes the activity of PRC2 and regulates levels of H3K27me3 at genes involved in flowering and meristem determination . The newFLC locus and is required for PRC2 nucleation in cold-induced vernalization -derived epigenetic recombinant inbred lines (epiRILs), Ioanna Kakoulidou  showed that chromatin states can also impact subsequent generations. Previous work from the laboratory had shown that epiHybrids exhibit strong heterosis in several developmental traits, which correlates to DMRs in the parental lines  and TF accessibility to target DNA. An interesting example was presented by Sebastian Marquardt , who showed that the histone chaperone complex FACT is required for the repression of cryptic intragenic Transcriptional Start Sites (TSSs) during RNAPII-mediated transcription. In their repressed state, these TSSs are enriched in H3K4me1, a hallmark for RNAPII elongation, while, in the iversity . Moreoveal lines . RecentlIn summary, the EWPC2019 encouraged discussion about the most recent advances in epigenetics, chromatin-related mechanisms, and nuclear architecture in relation to the regulation of transcription and its impact on plants traits. Particularly, how the nuclear space is organized and how specific histones and structures within the nucleus, such as the nucleolus and the nuclear periphery, relate to specific chromatin domains was thoroughly discussed in various talks. However, we are still far from understanding the complexity that is shrouded by the nuclear envelope. The extent of interplay between DNA methylation, various histone modifications, histone variants, and regulatory RNAs taking place during epigenetic inheritance processes remains to be elucidated. Likewise, much remains to be understood on the importance of core histone variants and their chaperones in chromatin structure through the control of nucleosome assembly and occupancy or the role of linker histones and other dynamic DNA-binding proteins. While histone modifications have so far rarely been considered in a variant-specific manner, combinations of histone variants with their particular marks constitute an additional layer of complexity to fine-tune chromatin regulation that is now just emerging and that will most certainly require further studies. While different presentations exposed the complexity of chromatin-based regulatory mechanisms in plants, it became clear that we need to investigate how chromatin-associated proteins are regulated in different tissues, developmental stages, and under specific environmental conditions, in order to fully understand their role in transcriptional regulation. Novel technical advances making use of CRISPR/Cas9 based strategies or new developments in 3C techniques together with a deeper characterization of multi-subunit complexes and their functions will help to better our understanding of the organization of plant genomes and nuclear protein networks in the near future. Simultaneously, studying the interplay between different regulators with the help of emerging technologies, such as the development of imaging and image-processing solutions that take into account the challenges of plant systems  and otheThe memory of Lars Hennig imbued specifically one of the meeting sessions but was also present in many other talks, demonstrating that the contributions of this excellent scientist and mentor will last over time. His work had a tremendous impact on the understanding of chromatin regulation and plant development, particularly concerning our knowledge of the PcG pathway, and will perpetuate through the ongoing contributions of many of his alumni who are still actively investigating these questions.SF and AP created JM-R is supported by funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 797473, financial support from the Spanish Ministry of Economy and Competitiveness through the “Severo Ochoa Program for Centres of Excellence in R&D” 2016-2019 (SEV‐2015‐0533), and by the CERCA Programme/Generalitat de Catalunya. AP, K, and SF acknowledge networking support from the COST Action CA16212 “Impact of nuclear domains on gene expression and plant traits (INDEPTH).” The EWPC was supported by grants from The German Research Foundation (TU126/14-1) and The Company of Biologists (EA1772). The organizers also acknowledge the generous contributions from industrial sponsors Zeiss Microscopy GmbH (Germany), Diagenode SA (Belgium), and Dispendix GmbH (Germany).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.


In this review, we will illustrate the role played by pancRNAs in cancer biology and will discuss the latest findings that connect pancRNAs with cancer risk and progression. The molecular mechanisms involved in the function of pancRNAs may open the path to novel therapeutic opportunities, thus expanding the repertoire of targets to be tested as anticancer agents in the near future.“Cancer” includes a heterogeneous group of diseases characterized by abnormal growth beyond natural boundaries. Neoplastic transformation of cells is orchestrated by multiple molecular players, including oncogenic transcription factors, epigenetic modifiers, RNA binding proteins, and coding and noncoding transcripts. The use of computational methods for global and quantitative analysis of RNA processing regulation provides new insights into the genomic and epigenomic features of the cancer transcriptome. In particular, noncoding RNAs are emerging as key molecular players in oncogenesis. Among them, the promoter-associated noncoding RNAs (pancRNAs) are noncoding transcripts acting in  Tumor formation is a multistep process characterized by transformation of the cell from a physiological to a neoplastic stage. During this process, cells lose checkpoints that control their growth in response to internal and external cues and acquire oncogenic features that allow them to evade growth-inhibitory signals, to invade neighboring tissues, and to colonize distant new sites by promoting neo-angiogenesis and by escaping cell death . Tumor cThe extensive implementation of high-throughput sequencing technologies has revealed that less than 2% of the genome encodes proteins whereas 75% is actively transcribed into noncoding RNAs . ExcludiLncRNAs are evolutionarily less conserved than protein-coding transcripts  and dispcis-acting elements and regulate transcription of their host genes. PancRNAs display tissue specificity and contribute to many biological processes . hactivity .Thus, pancRNAs orchestrate gene expression by intervening in different biological aspects, ranging from promoter regulation and transcriptional control to chromatin structure arrangement. The comprehension of these molecular mechanisms might provide novel insights into fundamental aspects of gene regulation.Mounting evidence indicates that lncRNAs are promising biomarkers in the diagnosis and prognosis of cancer, especially their presence in body fluids. These findings highlight their diagnostic and prognostic potentials and open the path for novel therapeutic strategies .EF1α) promoter. Interestingly, degradation of this pancRNA by antisense phosphorothioate oligodeoxynucleotides (ODN) exhibited high specificity, since it did not affect EF1α mRNA transcript [MYC gene, leading to profound inhibition of the development of prostate tumor xenografts [Khps1 led to impaired cell migration, invasion, and clonogenicity of cancer cells. As mentioned above, Khps1 is synthesized in antisense orientation to the proto-oncogene SPHK1 and is required for the activation of SPHK1 transcription by establishing a transcription-permissive chromatin structure and by increasing CBP/p300 occupancy and H3K27 acetylation to ensure E2F1 binding [Khps1 as a biomarker but also represents a promising step toward therapeutic intervention.As widely discussed in the previous sections, pancRNAs modulate gene expression through several mechanistic strategies. Nevertheless, their functional role is strictly confined to the promoter regions where they are embedded, thus providing specificity for the modulation of their host gene. The ability to selectively activate or inhibit gene expression is fundamental to understanding cellular systems and developing therapeutics. In line with this, pancRNAs transcribed from the promoter of genes involved in cancer could represent alternative molecular candidates for RNA-based anticancer strategies. The use of small interference RNA (siRNA) is becoming an emerging tool for its therapeutic potential . In vitranscript . This wonografts ,79. Anotnografts ,80. Tran binding ,80. The CDH1 promoter [RASSF1A promoter increase de novo DNA methylation and gene silencing [Similar to the pancRNA molecular proceeding, multiple strategies have been designed. Several groups reported that targeting promoter regions with dsRNAs was effective in inducing transcriptional silencing by triggering histone modification and/or DNA methylation. This method was successfully used to downregulate CDH1 expression in breast cancer cells . Notablypromoter . A simililencing . These rPancRNAs can either repress or activate gene expression of their host genes. Likewise, small double-stranded RNA (dsRNA) molecules were developed to either repress or activate endogenous gene transcription. These last molecules were termed small activating RNA (saRNA) and consist of 21-nt dsRNAs targeting selected promoter regions of human genes such as p21WAF1/CIP1 (p21), E-cadherin, and VEGF . SaRNAs were able to induce an increase in mRNA and protein levels when transfected in vitro . SaRNA tSimilarly, saRNA mediating upregulation of the endogenous E-cadherin reduced cell proliferation, promoted apoptosis, decreased mobility, and inhibited tumor growth in breast cancer xenograft mice . TherapeCollectively, these reports highlight the potential of therapeutic use of dsRNA in targeting gene activation or repression at promoters, analogously to pancRNA proceeding. Moreover, saRNA could be further applied in combination with other drugs to improve treatment efficacy. Indeed, saRNAs targeting p21 enhance the sensitivity of A549 non-small-cell lung carcinoma to cisplatin . OverallDuring the last decade, considerable developments have been achieved in understanding the regulatory role of pancRNAs in physiological and pathological processes. Gene activation or repression by pancRNAs finely shape tissue-specific patterns but can also contribute to the transformation process. Recent advances showed that pancRNAs are targetable molecules and that modulation of their expression can be used for therapeutic purpose. Notably, pancRNA-mediated epigenetic dynamics can be mimicked by RNA oligonucleotides, exploitable for therapeutic purpose.To this regard, several therapeutic oligonucleotides have been recently developed for clinical use ,91,92,93


These results provide a roadmap for future engineering efforts to further expand and generalize the scope of bacterial CRISPRa.In bacterial systems, CRISPR-Cas transcriptional activation (CRISPRa) has the potential to dramatically expand our ability to regulate gene expression, but we lack predictive rules for designing effective gRNA target sites. Here, we identify multiple features of bacterial promoters that impose stringent requirements on CRISPRa target sites. Notably, we observe narrow, 2–4 base windows of effective sites with a periodicity corresponding to one helical turn of DNA, spanning ~40 bases and centered ~80 bases upstream of the TSS. However, we also identify two features suggesting the potential for broad scope: CRISPRa is effective at a broad range of σ The use of CRISPRa in bacteria lacks predictive rules for identifying effective gRNA target sites. Here the authors define features of bacterial promoters that impose stringent requirements on effective sites. In metabolic engineering, regulating the timing and levels of the expression of complex multi-gene pathways is critical for reducing cellular burden and improving production of valuable metabolites2. To enable these goals, we recently developed a CRISPR-Cas transcriptional activation (CRISPRa) system that is effective in Escherichiacoli. Our system can be combined with CRISPRi gene repression to programmably target multiple genes for simultaneous activation and repression3. Although our CRISPRa system can be used with heterologous genes, an outstanding challenge is to understand the rules that define effective target sites at arbitrary promoters in the genome.Developing tools to activate the expression of arbitrary genes has been transformative for biotechnology and biological research4. To enable simultaneous activation, we use modified guide RNAs, termed scaffold RNAs (scRNAs), that include a 3’ MS2 hairpin to recruit a transcriptional activator fused to the MS2 coat protein (MCP)3. We can express multiple gRNAs and scRNAs to inhibit and activate genes simultaneously; gRNAs targeted to a promoter or ORF result in CRISPRi and scRNAs targeted to an appropriate site upstream of a minimal promoter result in CRISPRa.To programmably downregulate target genes, we use nuclease defective Cas9 (dCas9) with a guide RNA (gRNA) that specifies a target site on the DNA. Targeting this complex to a promoter or an open reading frame (ORF) results in gene repression (CRISPRi)E. coli are surprisingly stringent. In prior work, we found that CRISPRa in E. coli was effective at target sites located in a narrow 40 base window between 60 and 100 bases upstream of the transcriptional start site (TSS)3. Here, we show that multiple factors combine to make the requirements for effective sites even stricter. We demonstrate that the basal promoter strength of the target gene and the sequence composition between the target site and the minimal promoter can have marked effects on gene activation. Further, by scanning the 40 base window at single base resolution, we find sharp peaks of activity and broad regions of inactivity that occur in a periodic 10–11 base pattern, corresponding to one helical turn along the DNA target. The observation that only a few precisely positioned target sites upstream of the TSS are effective for CRISPRa poses a significant challenge, as many genes will likely lack an NGG PAM sequence at exactly the right position necessary for Streptococcuspyogenes dCas9. These stringent requirements may explain why CRISPRa and other tools for gene activation in bacteria have lagged far behind comparable tools in eukaryotic systems, where such strict target site requirements are absent5.We demonstrate here that the rules for targeting CRISPRa to effective sites in 70-dependent genes, CRISPRa can activate expression from genes that use the σ70 family members σ38, σ32, and σ24. We further demonstrate that the strict requirement for a precisely positioned PAM site can be partially overcome using a re-engineered dCas9 protein that targets an expanded set of PAM sequences6. Recently, some of the rules that we describe here were independently reported for an alternative bacterial CRISPRa system that can target genes regulated by σ54 promoters7. Our results demonstrate that this behavior applies to a much broader range of σ70 family promoters, which cover the majority of the E. coli genome8. The availability of these complementary systems should further extend the scope of bacterial CRISPRa. More broadly, by systematically defining the rules for effective CRISPRa sites, we identify strategies for improving and generalizing synthetic gene regulation in bacteria.Although the requirements for bacterial CRISPRa target sites pose challenges, our data also demonstrate CRISPRa has the potential to be effective at a broad range of target genes. In addition to σE. coli transcription factor that directly binds DNA and activates endogenous gene targets as part of a stress response program3. We previously demonstrated that point mutations in the SoxS DNA-binding site can reduce activation of endogenous SoxS targets while maintaining CRISPRa activity at a heterologous reporter gene. However, the most effective single point mutants, R93A and S101A, did not completely abolish activity at endogenous targets. To further minimize off-target SoxS activity, we tested a double mutant SoxS(R93A/S101A). This double mutant SoxS retained full CRISPRa activity and showed a reduction in endogenous SoxS-dependent gene expression to levels indistinguishable from background   Fig. . We obse16.The SoxS activator binds directly to the α subunit of RNA polymerase17, which suggests that our CRISPRa system could be compatible with genes that are controlled by non-housekeeping sigma factors. To investigate this possibility, we built synthetic promoters regulated by σ38 (RpoS), σ32 (RpoH), σ24 (RpoE), and σ54 (RpoN) to compare with our original housekeeping σ70 (RpoD) promoter , which releases TetR from the DNA, restored CRISPRa activity to the levels observed when TetR was not present  to 10 or 20 aa, but even with these longer linkers we observed the same sharp dependence on the target site position as with the original 5 aa linker Fig. . We obtaAnother potential approach to expand the range of effective CRISPRa sites would be to change the spatial position of the MCP-SoxS protein by altering the position of the MS2 hairpin that binds MCP. We therefore tested multiple alternative scRNA designs that present the MS2 hairpin at different locations. Extending the MS2 stem by 2, 5, 10, and 20 bp resulted in progressively lower CRISPRa activity, but no change in the position of the target sites that were most effective  to target these reporters. dxCas9(3.7) maintained the ability to target the AGG PAM and showed significantly increased levels of activation at alternative PAM sites compared to dCas9  showed similar distance and phase dependent target site preferences as dCas9 -compatible PAM sites than NGG PAM sites  for CRISPRa at sites inaccessible to dCas9, we constructed a reporter plasmid that contains an AGG PAM site at the original position with maximum CRISPRa activity and an AGT PAM five bases downstream. Using this reporter, we observe that both dCas9 and dxCas9(3.7) are effective for CRISPRa at the optimally positioned NGG PAM site, but neither is capable of activating the AGT PAM site, which is five bases out of phase from the optimal site Fig. . We theners Fig. , and a rE. coli demonstrates that candidate genes must have a targetable PAM site located at one of the sharp peaks of activity upstream of the TSS. In hindsight, the scRNA sites at endogenous genes that we initially targeted in Fig. E. coli genes, we surveyed the genome for candidate genes with appropriately positioned, dxCas9(3.7)-compatible PAM sites (Supplementary Methods) . We also included an additional site predicted to be out of phase and ineffective for CRISPRa. We observed significant, ~4–6-fold gene activation for the two sites located at the predicted peak of activity at −80/−81, and no activation at the out of phase site at −87  and uxuR (approximately twofold) . For the remaining two candidate genes, ansB was modestly repressed at one of the target sites and we did not observe a statistically significant difference in expression at ppiD. Similarly, one of the ldhA sites that we targeted in initial experiments  Fig. . We valiAlthough any success at endogenous gene activation is encouraging, significant challenges remain for predictable CRISPRa in bacteria. Our results suggest that even with a precise distance metric for effective target sites, some genes will not be predictably activated. There are several possible explanations: (1) tightly bound negative regulators could interfere with CRISPRa Fig. , and (2)38. Other factors are plausible given our understanding of bacterial transcription. Sigma factor levels are regulated to control gene expression in response to cell state and external signals16, so it is reasonable that we observed variable levels of activation from promoters with alternative sigma factors. Many bacterial genes are controlled by negative regulators39, and different sequences upstream of the minimal promoter could be recruiting repressors.Bacterial CRISPRa is sensitive to a number of factors, including (i) the strength of the target promoter, (ii) the sigma factor regulating the promoter, (iii) the sequence composition immediately upstream of the minimal promoter, (iv) the composition of the scRNA target sequence, (v) the position of the scRNA target site with respect to the TSS at single base resolution. Some of these factors, such as promoter strength and scRNA target sequence composition, are also relevant in eukaryotic systems40, possibly because eukaryotic activators typically recruit transcription factors and chromatin modifying machinery rather than directly recruiting RNA polymerase. There is precedent for bacterial transcriptional activators that are sensitive to target site periodicity34, but the marked changes in activity with only single base shifts is surprising. Moreover, it is puzzling that we were unable to predictably alter or broaden the range of sites that are effective. Our models for how activators interact with bacterial transcription machinery may be incomplete. It will likely be productive to continue screening for activity at out-of-phase target sites using additional systematic modifications to the CRISPRa complex structure, alternative CRISPR–Cas systems, and additional candidate transcriptional activation domains.The most unexpected property that we observed with bacterial CRISPRa was its sharp, periodic dependence on-target site position. This behavior is quite distinct from CRISPRa in eukaryotes, where a broad range of sites upstream of the TSS are effectiveE. coli, our systematic characterization provides a framework for immediate practical applications and a path for future improvements. We now have a clear understanding of the criteria needed to design synthetic promoters that can be regulated by CRISPRa, which will enable the construction of complex, tunable synthetic multi-gene circuits. To extend the scope of CRISPRa to endogenous target genes, expanded PAM variants like dxCas9(3.7)6, or orthologous dCas9 proteins with alternate PAM specificities42 will open more DNA sites for targeting, increasing the likelihood of finding a targetable site at an optimal position relative to the TSS. These strategies lay the groundwork for more widespread use of bacterial CRISPRa in basic research and practical applications including functional genomics screens, metabolic engineering, and synthetic microbial communities.Despite the challenges described above for identifying effective CRISPRa sites in S. pyogenes dCas9 (Sp-dCas9) or dxCas9(3.7) were expressed from the endogenous Sp.pCas9 promoter in a p15A vector. MCP-SoxS containing wild-type and mutant SoxS were expressed using the BBa_J23107 promoter (http://parts.igem.org) in the same plasmid with dCas9. The scRNAs were expressed using the BBa_J23119 promoter, either in the same plasmid with the dCas9 protein and the activation domain or in a separate ColE1 plasmid. The scRNA.b1 or scRNA.b2 designs, where the endogenous tracr terminator hairpin upstream of MS2 was removed3, were used in all experiments except otherwise noted. The zwfp-lacZ and fumCp-lacZ reporter plasmids were generated in a previous study3. mRFP1 and sfGFP reporters were expressed from the weak BBa_J23117 minimal promoter (http://parts.igem.org) in a low-copy pSC101** vector. Variant versions of reporter genes are described in the Supplementary Methods. Plasmid libraries containing N26 sequences between the scRNA target site and BBa_J23117 minimal promoter were constructed by PCR amplification using mixed bases oligos (IDT). The dxCas9(3.7)-VPR plasmid was a gift from David Liu (Addgene #108383)6.Plasmids were cloned using standard molecular biology protocols. Bacterial strains with sfGFP or mRFP1 reporter strains are described in Supplementary Table Single colonies from LB plates were inoculated in 500 μL EZ-RDM (Teknova) supplemented with appropriate antibiotics and grown in 96-deep-well plates at 37 °C and shaking. Cultures were grown overnight at 37 °C and shaking and then diluted in 1:50 in Dulbecco’s phosphate-buffered saline and analyzed on a MACSQuant VYB flow cytometer with the MACSQuantify 2.8 software (Miltenyi Biotec). A side scatter threshold trigger (SSC-H) was applied to enrich for single cells until 10000 events were collected. The FlowJo 10.0.7 software was used to apply a narrow gate along the diagonal line on the SSC-H vs SSC-A plot was selected to exclude the events where multiple cells were grouped together. Within the selected population, events that appeared on the edges of the FSC-A vs. SSC-A plot and the fluorescence histogram were excluded.E. coli promoter collection36 the activation domain was placed under the control of a tet-inducible promoter. Attempts to use constitutive CRISPRa were unsuccessful due to plasmid instability, possibly because of toxicity arising from increased expression of the target genes. Single colonies from LB plates were inoculated in 500 μL EZ-RDM supplemented with appropriate antibiotics and 400 nM anhydrotetracycline (aTc) and grown in 96-deep-well plates at 37 °C and shaking overnight. 150 μL of the overnight culture were transferred into a flat, clear-bottomed black 96-well plate and the OD600 and fluorescence were measured in a Biotek Synergy HTX plate reader and analyzed using the BioTek Gen5 2.07.17 software. For mRFP1 detection, the excitation wavelength was 540 nm and emission wavelength was 600 nm. For sfGFP detection, the excitation wavelength was 485 nm and emission wavelength was 528 nm.Single colonies from LB plates were inoculated in 500 μL EZ-RDM (Teknova) supplemented with appropriate antibiotics and grown in 96-deep-well plates at 37 °C and shaking overnight. For experiments with the 600 of 0.5 (using 150 μL of culture in a 96-well plate) was reached. For the experiments targeting yajG and poxB, the activation domain was placed under the control of a tet-inducible promoter and cultures in EZ-RDM were supplemented with 400 nM aTc. Cultures were pelleted and total RNA was extracted using the Aurum Total RNA Mini Kit (Bio-rad). Reverse transcription reactions were performed from 1 μg RNA in 20 μL reactions using iScript reverse transcriptase (Bio-Rad). qPCR reactions were prepared in triplicate in a final volume of 10 μL using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad), 0.5–5 ng of cDNA and 400 nm primers. The reaction was performed in a CFX Connect (Bio-Rad) with a 58 °C annealing temperature and 30 s extension time. A list of the qPCR primer sequences is provided in Supplementary Table 43.Single colonies from LB plates were inoculated in 5 mL LB containing appropriate antibiotics and grown overnight at 37 °C and shaking. Overnight cultures were diluted 1:100 into 5 mL EZ-RDM supplemented with appropriate antibiotics and grown at 37 °C and shaking until an ODt tests. To ensure reproducibility, experiments were performed using n = 3 biologically independent samples, unless otherwise noted.Statistical significance was calculated using two-tailed unpaired Welch’s Further information on research design is available in the Supplementary InformationReporting SummaryPeer Review File


Xenopus gastrulae. We show that Sox17 functionally interacts with the canonical Wnt pathway to specify and pattern the endoderm while repressing alternative mesectoderm fates. Sox17 and β-catenin co-occupy hundreds of key enhancers. In some cases, Sox17 and β-catenin synergistically activate transcription apparently independent of Tcfs, whereas on other enhancers, Sox17 represses β-catenin/Tcf-mediated transcription to spatially restrict gene expression domains. Our findings establish Sox17 as a tissue-specific modifier of Wnt responses and point to a novel paradigm where genomic specificity of Wnt/β-catenin transcription is determined through functional interactions between lineage-specific Sox TFs and β-catenin/Tcf transcriptional complexes. Given the ubiquitous nature of Sox TFs and Wnt signaling, this mechanism has important implications across a diverse range of developmental and disease contexts.Lineage specification is governed by gene regulatory networks (GRNs) that integrate the activity of signaling effectors and transcription factors (TFs) on enhancers. Sox17 is a key transcriptional regulator of definitive endoderm development, and yet, its genomic targets remain largely uncharacterized. Here, using genomic approaches and epistasis experiments, we define the Sox17-governed endoderm GRN in  During embryogenesis, the pluripotent zygote progressively gives rise to specialized cell types expressing distinct sets of genes that, in turn, define the cell’s identity and encode proteins necessary for its function. Lineage-specific gene expression is controlled by the genomic integration of signaling pathways and transcription factors (TFs) on DNA cis-regulatory modules (CRMs), such as enhancers, that control transcription . An impoXenopus where endoderm specification is well studied, the Nodal and Wnt pathways interact at several levels. First, maternal (m) Wnt/Bcat, active on the dorsal side of the blastula, is required for high levels of nodal expression at the onset of zygotic transcription  is expressed on the ventral side of the gastrula where, together with Nodal signaling, it promotes ventral and posterior mesendoderm identities  segregate the transient mesendoderm into endoderm and mesoderm, (2) pattern the nascent endoderm into spatially distinct subtypes and (3) execute the downstream differentiation program to give rise to endoderm-derived lineages. But mechanistically how the Wnt signaling machinery interacts with core endoderm TFs to execute this differentiation cascade is unresolved. The transcriptional targets of the endoderm TFs are largely unknown, and it is unclear exactly how Wnt/Bcat regulates distinct spatiotemporal transcription programs in the embryo. Indeed, how the canonical Wnt pathway elicits context-specific transcriptional responses in its multitude of different biological roles in development, homeostasis and cancer is still poorly understood. According to the dogma of canonical Wnt signaling, Wnt-activated Fzd-Lrp5/6 receptor complexes sequester the Bcat degradation complex, resulting in the stabilization and translocation of Bcat to the nucleus. There, it interacts with one of four HMG-box Tcf TFs  at Wnt-responsive CRMs , ultimatXenopus endodermal GRN. In all vertebrate embryos, Sox17 is specifically expressed in the gastrula endoderm where it is required for early gut development   . In Xenoap cells , and co-ap cells . Sox17 cap cells . HoweverXenopus gastrula. In addition to promoting expression of endoderm genes, Sox17 also represses ectoderm and mesoderm gene transcription, and acts in a negative feedback loop to restrain Nodal signaling. We demonstrate that functional interactions with canonical Wnt signaling is a key feature of the Sox17-regulated GRN. Over a third of all Bcat and Sox17 genomic binding in the gastrula occur at the same CRMs. In some instances, Sox17 suppresses Bcat-Tcf mediated transcription, while in other cases, Sox17 and Bcat synergistically activates enhancers apparently independently of Tcfs, indicating a novel mode of regulation. These results provide new insight into the GRN controlling endoderm development and have implications for how Sox TFs and Bcat might interact in diverse biological contexts from development to cancer.Here, we define the genomic targets of Sox17 in the Xenopus tropicalis embryos at multiple time points during blastula and gastrula stages (NF9-12) when the endoderm germ layer is being specified. In Xenopus tropicalis, there are three redundant genes: sox17a, sox17b.1 and sox17b.2 (collectively sox17) with indistinguishable activities and identical expression in presumptive vegetal endoderm cells of gastrula embryos  on control and Sox17-depleted  embryos . Microin embryos . The Sox embryos  and phen embryos . Injecti embryos .>2 fold change, FDR< 5%) ; a key epithelial signal in gut organogenesis. This confirms that Sox17 sits atop of the regulatory hierarchy regulating endoderm differentiation.Differential expression analysis of control-MO and Sox17-MO embryos identified 1023 Sox17-regulated genes (FDR< 5%) . Gene OnFDR< 5%) . In totaFDR< 5%) . Differend hnf1b , and parnd hnf1b  such as osr1, hnf1b, dhh and slc5a8 , endoderm-enriched Nodal pathway genes  and Wnt-regulated organizer genes (dkk and fst), suggesting direct transcriptional repression , 1321 of which were downregulated and 1247 upregulated in the Bcat-MO embryos  were enriched for ectoderm-specific transcripts, consistent with the expansion of ectoderm and loss of neural tissue in ventralized embryos   . Most ofn group)  but weresox17, foxa, eomes, gata4, mix1 and mixer  compared to 31% in background genomic sequences (six1), suggesting that Bcat might be recruited to these loci independent of Tcfs. Only 3/191 peaks had Tcf but no Sox motifs. Moreover, co-occupied enhancers often contained multiple Sox17 motifs (>5), which was also significantly enriched over random  were bound and regulated by both Sox17 and Bcat . This isequences . In contr random . In addisix1, dkk1, lhx5 and gli1) showed enrichment compared to IgG demonstrating that these genomic loci (~150 bp in length) were simultaneously bound by Sox17 and Bcat in endodermal cells. In contrast, a negative control locus gtse1 which is bound by Bcat but not Sox17 was not enriched in the ChIP-reChIP experiment  and tended to have endoderm enriched expression, 27/84 were activated by Bcat but repressed by Sox17 (e.g. dkk1) and these tended to be enriched in the organizer mesendoderm, 19/84 were positively regulated by Sox17 and negatively regulated by Bcat (up in the Bcat MO) and finally 6/84 were negatively regulated by both Sox17 and Bcat. The observation that Bcat positively regulated most of these genes  was consistent with its known role in transcriptional activation. Sox17 positively regulated 60% of targets and repressed 40% of targets, suggesting context-dependent activity. For further analysis, we focused on the two major regulatory groups; genes/enhancers activated by both Sox17/Bcat and those activated by Bcat but repressed by Sox17 Bcat mRNA rescued the normal expression of foxa1 but not six1, osr1, lhx5 or dkk1 in Sox17-morphants indicating that the defects in their expression were not simply due to reduced Wnt signaling  a proximal dkk1 enhancer located −1.6 kb from its TSS, (c) a distal −7.5 kb dkk1 enhancer and (d) a −1 kb lhx5 enhancer  a proximal enhancer . We focusix1 enhancer is evolutionarily conserved from Xenopus to human containing three Sox-binding sites but no predicted Tcf-binding sites  reporter constructs and tested whether they require Sox17 and Bcat for enhancer activity in  embryos . Targeteectoderm . Depleti embryos . Howeverox17-MOs . These eenhancer . In contnhancers .mSox17 but not caBcat mRNA was sufficient to stimulate the −1 kb six1:luc and human SIX1:luc reporters in the ectoderm, but co-injection of caBcat along with mSox17 synergistically activated the reporters . Together these results provide novel insights into the establishment of the endoderm GRN and suggest a new paradigm where Sox17 acts as a tissue-specific modifier of Wnt-signaling responses.Sox17 is a key regulator of vertebrate endoderm development and morphogenesis, yet surprisingly, the transcriptional program that it controls has been poorly characterized until now. Here, we defined the Sox17-regulated GRN in nodal ligands (A/T)CAA(A/T)3’, which is similar but distinct from Tcf DNA-binding sites 5’-T(A/T)(A/T)CAAG 3’ . SOX TFsMany Sox proteins, including Sox17, can bind to recombinant Bcat and Tcfs in vitro and modulate Wnt/Bcat-stimulated transcription of synthetic reporters assays with multimerized Tcf-sites (pTOPFlash) . While asix1, igf3 and +7 kb osr1, where Sox17 and Bcat synergistically stimulate transcription, we postulate that Sox17 and Bcat recruit Ep300/Cbp to chromatin, analogous to Tcf-Bcat in canonical Wnt-signaling. Consistent with this, structure-function studies have found that a conserved 10 amino-acid domain (VDXXEFEQYL) in the Sox17 C-terminus is required for both Bcat-binding and transcriptional transactivation (At enhancers like tivation . On the tivation  might astivation .osr1). To negatively regulate Bcat-Tcf activity (e.g. dkk1) Sox17 might destabilize Bcat-Tcf-Ep300-DNA or stabilize Tcf-Tle complexes. This idea is supported by work in mouse cortical neuron progenitors where Sox2 binds within 4–10 bp of Tcf/Lef sites and recruits Tle/Gro corepressors to repress Tcf/Bcatmediated Ccnd1 expression  and experiments were performed according to CCHMC IACUC approved protocols. Embryos were staged according to CCTCTTACCTCAGTTACAATTTATA-3’;Standard control MO: 5’Xenopus tropicalis ß-catenin MO: 5’ TTTCAACAGTTTCCAAAGAACCAGG 3’;Xenopus tropicalis sox17a MO: 5’-AGCCACCATCAGGGCTGCTCATGGT-3’Xenopus tropicalis sox17b.1/2 MO: 5’-AGCCACCATCTGGGCTGCTCATGGT-3’Xenopus laevis ß-catenin MO: 5’TTTCAACCGTTTCCAAAGAACCAGG-3’ , 8 ng standard control MO.Total amounts of MO injected into Synthetic mRNA for injections was generated using the Message Machine SP6 transcription kit (Thermo Fisher AM1340) using the following plasmids: pCS2+ human B-catenin S37A caBCAT ; pcDNA6-Xenopus tropicalis embryos was performed as described , 3.7% methanol-free paraformaldehyde for 1.5 hr at 4°C and then dehydrated directly into ice-cold Dent’s post-fixative (80%Methanol/20% DMSO) and stored at −20°C. Embryos were serially rehydrated on ice into PBS+0.1% TritonX-100 (PBSTr) and bisected through the dorsal-ventral axis with a fine razor blade. Embryo halves were subjected to antigen retrieval in 1x R-Universal epitope recovery buffer (Electron Microscopy Services #62719–10) for 30 min at 55–60°C, washed 2 × 10 min in PBSTr, blocked for 2 hr in PBSTr + 10% normal donkey serum (Jackson Immunoresearch 017-000-001) + 0.2% DMSO at room temperature, and then incubated overnight at 4°C in this blocking solution + the following primary antibodies: chicken anti-GFP , rabbit anti-Sox17a/b N-terminal (1:350), rabbit anti-B-catenin . After extensive washing in PBSTr, samples were incubated overnight at 4°C in PBSTr + secondary antibodies: donkey antiChicken 488, donkey anti-rabbit Cy3 .TO-PRO3 nuclear stain (ThermoFisher #T3605) was included in one 30 min PBSTr wash after secondary antibody incubation. Samples were extensively washed in PBSTr, dehydrated into 100% methanol, cleared and imaged in Murray’s Clear .For luc2/miniP vector. (Promega E8411). Xenopus laevis embryos were used for luciferase assays because of the larger size embryos and slower developmental rate. Enhancer activity was assayed by co-injecting embryos with 5 pg of pRL-TKrenilla luciferase vector (Promega E2241), 50 pg of the pGL4.23 luc2/miniPenhancer:luciferase and combinations of the following MOS or mRNAs: ca-Bcat; 5 pg, mSox17; 25 pg, mSox17 M76A; 25 pg, Tcf3-VP16; 10 pg, Xl sox17a+b1/2 MO; 10 ng each, Xl bcat MO; 10 ng. For each condition three tubes of 5 NF10.5 embryos were collected. Embryos were lysed in 100 µL of 100 mM TRIS-Cl pH7.5, centrifuged for 10 min at ~13,000 x g and then 25 µL of the clear supernatant lysate was used separately in Firefly (Biotium #30085–1) and Renilla (Biotium 300821) luciferase assays according to the manufacturer’s instructions. The average relative Luciferase activity was normalized Renilla levels for each sample and statistical significance was determined by pairwise Student T-test. Each luciferase experiment was repeated two to four times with similar results and a representative is shown.Putative enhancers (wildtype as well as Sox and Tcf DNA-binding site mutants) were synthesized (supplementary sequence file S1) and cloned into the pGL4.23 X. tropicalis embryos were injected with 8 ng of either control-MO or Sox17-MO, or 2 ng of control-MO and Bcat-MO and 10 sibling embryos were collected at every time point from blastula to late gastrula in biological duplicate. Total RNA was extracted with the Nucleo-spin RNA kit (Machery-Nagel). RNA-seq libraries were generated using Smart-seq2 cDNA synthesis followed by tagmentation, quality-tested using an Agilent Bioanalyzer 2100, quantified using KAPA qPCR and sequenced using Illumina sequencers at the UC Irvine Genomics High Throughput Facility.Approximately 200 Xenopus tropicalis genome version 9.0  were identified by a pairwise comparison between control-MO versus Sox17-MO and control-MO versus Bcat-MO sibling embryos at each stage using RUVSeq (R package) (http://geneontology.org) and default parameters.Raw reads were quality checked using FastQC and adapters/low-quality reads were removed with Trimmomatic . Reads wsion 9.0  with bowsion 9.0  and repoR_003280 . To visuR_003280 . Differepackage) . Gene onTo assess the spatial expression of transcripts, we examined the RNA-seq TPM expression values of dissected stage 10.5 embryo published in GEO GSE81458 . EnricheX. tropicalis embryos and 30 µg rabbit anti-Sox17b-Cterminal antibody. ChIP-seq libraries were generated using Nextflex ChIP-seq kit (Bio Scientific), analyzed using an Agilent Bioanalyzer 2100, quantified using KAPA qPCR and sequenced using Illumina instruments at the UC Irvine Genomics High Throughput Facility.Large-scale Sox17 ChIP was carried out as previously described  using 20Xenopus tropicalis embryos were fixed in 1% formaldehyde in 0.1x MBS at room temperature for 30 min. Sonication was carried out in a Diagenode Bioruptor Pico for 30 cycles of 30 s ON, 30 s OFF. Chromatin extracts were incubated with 10 µg rabbit anti-Sox17-Cterminal antibody  supplemented with 1x Protease Inhibitor Cocktail and 1 mM DTT. The 2Xenopus tropicalis genome (http://broadinstitute.github.io/picard/) and samtools As the editors have judged that your manuscript is of interest, but as described below, that additional experiments may be required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at Summary:eLife.This paper by Mukherjee et al. addresses functional interactions of Sox17 and Wnt signaling in endodermal gene regulation. The results reveal that Sox17 factors act as endoderm-specific modifiers of Wnt-signaling responses, providing strong evidence for a TCF independent role for β-catenin/Sox17 in promoting endodermal and suppressing mesodermal/ectodermal gene expression. The reviewers felt that this interesting study addresses important questions and is well-executed, but that there are instances where the conclusions seem overgeneralized, and that some additional analysis is needed before the article would be appropriate for publication in Essential revisions:1) Interpreting the Sox17 and β-catenin knock down results requires assessment of the efficiency knock downs – is occupancy at high affinity sites being decreased or only low affinity? Ideally this would be shown by ChIP following knockdown.six1, and only by gain-of function experiments. Data showing that other enhancers behave like six1, and possibly also using a Tcf7l1-EnR fusion, are needed to support this conclusion. Absent such data, conclusions should relate only to Six1 regulation.2) The generalized finding that "Sox17 and β-catenin co-occupy a subset of enhancers to synergistically stimulate transcription in the absence of Tcfs" is not fully supported by the data presented. Although a subset of enhancers lack predicted Tcf binding motifs, functional data is shown only for 3) Please add discussion of the dynamics of Sox17 binding/function. Could this explain differences with reported enrichment in other studies such as Nakamura et al. and Kjolby et al.? Those differences should be discussed.4) It might be useful to extend the analysis to look at collaborating transcription factors  by cross-referencing with published data sets .5) In the first section of the Results, the text should reflect the possibility that some of the observed transcriptional effects after Sox17 knockdown are caused by indirect mechanisms. Also, please avoid using the non-specific term "regulates", and instead use "inhibits/increases".6) Motif frequencies in peaks cannot be compared directly between motifs or different versions of the motifs, as seems to have been done in Figure 4—figure supplement 2A because the baseline for motif occurrence (in random genomic sequences) is highly motif-specific.7) Please indicate the application of multiple testing correction  where applicable . Also indicate p-values for all enrichments and intersections .8) Including a summary of the temporal role of Sox17 in context of other signals and transcription factors, including Nodal/Smad2, BMP, VegT would be useful.eLife. Your revised article has been evaluated by Edward Morrisey (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Sox17 and β-catenin co-occupy Wnt-responsive enhancers to govern the endoderm gene regulatory network" for further consideration by The manuscript has been greatly improved but there is one remaining issue that should be addressed before acceptance. This concerns the motif analysis in Figure 4—figure supplement 2. Examining background frequencies via scrambling of peak sequences while maintaining di-nucleotide frequencies has strengthened this analysis. However, ranking the motifs by hypergeometric p-value (provided by HOMER) is perhaps not most informative way to represent these data. We suggest instead plotting the 2log fold enrichment over background, in combination with a statistical test . Also, it is felt that a discussion of background frequencies is needed where the co-occurring motifs are discussed and Figure 4—figure supplement 2D should be revised by calculating for background sequences. Essential revisions:1) Interpreting the Sox17 and β-catenin knock down results requires assessment of the efficiency knock downs – is occupancy at high affinity sites being decreased or only low affinity? Ideally this would be shown by ChIP following knockdown.We agree that this is an important point. We have now performed ChIP-qPCR experiments on control, Sox17-MO and β-catenin-MO embryos as requested. The new data added to Figure 2—figure supplement 1D-E shows a dramatic loss of ChIP-qPCR signal at all loci tested, down to negative control background levels in most cases. This data supports our immunostaining  showing a complete loss of nuclear Sox17 and β-catenin in knockdown embryos. Together from all the data we estimate that the knockdown efficiency is greater than 90%.foxi2 and snai2) or low-affinity Sox17 sites (nrp1 and lmbx1). ChIP-qPCR shows that Sox17 occupancy dramatically decreased by several orders of magnitude at all four enhancers, after Sox17-MO knockdown. However, there was still some signal above background at foxi2. We prefer to not make any conclusions about this result, because these experiments lack the rigorous quantitative measurement of DNA-binding affinity needed for such claims, and this is not really the point of the current study. In the future we plan to systematically examine the impact of DNA-affinity which is complicated because Sox monomers bind to DNA with low sequence specificity and low affinity and are thought to gain specificity by other interaction with co-occupying transcription factors . We have added this point to the Discussion.The issue of high-affinity versus low-affinity site is very interesting and we understand the reviewers point that, with a partial knockdown, high-affinity enhancers might still be active. Since our knockdown is very efficient, we don't expect this will play much of a role, but we cannot totally rule it out. This is difficult to test since in vivo high-affinity and low-affinity sites for Sox17 (or Tcfs) have not yet been characterized in gastrula embryos. In an attempt to identify high-affinity vs. low-affinity Sox17 binding sites, we analyzed published protein binding microarray data from the CIS-BP database where recombinant DNA-binding domains of hundreds of TFs were challenged to all possible combinations of DNA 8-mers . Per the original criteria set out by Badis et al., we classified 8-mer sequences that had an enrichment (E)-score > 0.45 for Sox17 binding as “high affinity” sequences and those with an E-score < 0.2 as “low affinity” sequences. To assess if any of these sequences were present in our in vivo ChIP-seq data, we scanned Sox17 bound peaks for the presence of potential Sox binding sites. We then selected candidate enhancers for ChIP-qPCR in Figure 2—figure supplement 1D to include some that were predicted to contain only high-affinity Sox-sites (2) The generalized finding that "Sox17 and β-catenin co-occupy a subset of enhancers to synergistically stimulate transcription in the absence of Tcfs" is not fully supported by the data presented. Although a subset of enhancers lack predicted Tcf binding motifs, functional data is shown only for six1, and only by gain-of function experiments. Data showing that other enhancers behave like six1, and possibly also using a Tcf7l1-EnR fusion, are needed to support this conclusion. Absent such data, conclusions should relate only to Six1 regulation.osr1 and 2) +3kb igf3 enhancer that shared similar characteristics to six1; . The new Figure 6—figure supplement 1 shows that Sox17 and β-catenin cooperatively activate both enhancers, but neither was activated by Tcf7l1-VP16 (similar to six1 but unlike the dkk1 and lhx5 enhancers which contain Tcf-sites and are activated by Tcf7l1-VP16). These new data further support our conclusion that “Sox17 and b-catenin co-occupy a subset of enhancers to synergistically stimulate transcription independently of Tcfs”. We have not tried the Tcf7l1-EnR fusion. This reagent is problematic as it is well known to dominantly block all maternal Wnt/β-catenin-targets including nodal. Thus the embryos arrest without ever initiating endoderm fate or sox17 expression, precluding the analysis. Acknowledging that we don’t have a Tcf loss of function, we have now tempered our conclusions to say “apparently independent of Tcfs” in the Abstract and throughout the paper. We also point out in the paper that the majority of Sox17-β-catenin cobound enhancers do have Tcf sites (even those that are activated). In the future we plan to fully characterize the role of Tcfs at these enhancers. We emphasize the enhancers without detectable Tcf-regulation since this is the most novel aspect of the work.We have now extended our analysis to include two additional enhancers: 1) a +7kb 3) Please add discussion of the dynamics of Sox17 binding/function. Could this explain differences with reported enrichment in other studies such as Nakamura et al. and Kjolby et al.? Those differences should be discussed.Xenopustropicalis ChIP-seq of endogenous β-catenin). Although our processing pipeline is a little bit different than theirs, like them we find >11,000 IDR peaks and our motif analysis shows Sox (2nd ranked) being more enriched than Tcfs (5th ranked). This is almost exactly what Nakamura et. al reported. Using the same pipeline and motif analysis of β-catenin bound peaks of the Gentsch et al., 2019 dataset (β-catenin ChIP-seq at stage 10+), we find a similar ranking of Sox (3rd ranked) and Tcf (6th ranked) motifs. In the case of Kjolby et al., they performed ChIP-seq on slightly later stage NF11.5 Xenopus laevis using an overexpressed 3xFlag-tagged β-catenin reporting only 855 reproducible peaks. HOMER de novo motif analysis of the Kjolby data shows Lef1 as the 1st top motif and Sox as the 3rd ranked . So essentially there is not much difference in the motif analysis reported in these different studies. In both cases, Sox and Tcf motifs are among the top 5 enriched in β-catenin bound peaks, these other studies just did not follow up on the high rank of Sox motifs. The minor differences in motif calling between papers could be due to many things: the number of peaks in the analysis; >10,000 peaks vs. 855 peaks, developmental timing; 10 vs. 11.5, species; X. tropicalis vs. X. laevis, overexpression vs. endogenous, antibody; β-catenin vs. Flag or different lab ChIP protocols. Since we have no way of knowing the source of variation, we prefer not to speculate in the discussion.As requested, we have added a section in the Discussion on the temporal dynamics of Sox17 binding/function. Moreover, we have added a new panel to Figure 3—figure supplement 2 comparing our motif enrichment analysis to that of Kjolby et al., 2017 and Gentsch et al., 2019. We should point out that we use the published β-catenin ChIP-seq data reported in Nakamura et al., 2016 (stage 10.5 4) It might be useful to extend the analysis to look at collaborating transcription factors  by cross-referencing with published data sets .Per the reviewers’ suggestion, we extended our analysis to all other publicly available datasets of endodermal TFs . This new analysis presented in Figure 4—figure supplement 1, shows that Sox17 and β-catenin co-occupied peaks were indeed enriched for binding of other endodermal TFs including Foxa4, Smad1, Smad2, Sox3 and VegT. We have described this in the Results and Discussion sections explaining how it is likely that combinatorial activity of multiple lineage-specific TFs may cooperate with Sox17/β-catenin on super enhancers to regulate the endoderm GRN.5) In the first section of the Results, the text should reflect the possibility that some of the observed transcriptional effects after Sox17 knockdown are caused by indirect mechanisms. Also, please avoid using the non-specific term "regulates", and instead use "inhibits/increases"We have now updated the text as requested and explicitly indicated that changes in transcript level after Sox17-depletion could be indirect.6) Motif frequencies in peaks cannot be compared directly between motifs or different versions of the motifs, as seems to have been done in Figure 4—figure supplement 2A because the baseline for motif occurrence (in random genomic sequences) is highly motif-specific.We thank the reviewers for bringing this to our attention. We agree that since the occurrence of different motifs or version of motifs in random genomic sequences is variable, making comparisons not really possible. In the revisions, we have modified our analysis to not directly compare between Sox and Tcf motif frequencies, but to ask whether the frequency of Sox or Tcf motifs in a given group or peaks is significant over random. In addition we used a library of all predicted Sox or Tcf motifs rather than a specific motif.To assesses motif frequency in different categories of peaks we used a library of all known Sox and Tcf motifs combining HOMER and an exhaustive search of the CIS-BP database containing all experimentally defined sites from protein-DNA binding microarrays . Combining these  we generated a library of position weight matrices (PWMs) of all potential Sox17, Tcf7l1, Tcf7l2, Tcf7, Lef1 motifs and scanned peak scoring the number of PWMs present above a 70% threshold. A list of all the motifs from the CIS-BP database that we’ve included in our analysis is show in Author response table 1. We had not explained this properly in the original version of the paper and have now updated the Materials and methods.Author response table 1Then for each wild-type test peak we generated a random “shuffled” version where the wild-type peak sequences were randomly scrambled ten times maintaining dinucleotide frequencies. These represented random genomic sequences with the same GC content as the test case. For each iteration, we determine the frequency of the Sox and Tcf motifs in the “wild-type” and “shuffled” peaks. We then perform enrichment analysis using Fisher’s exact test to determine whether the frequency of Sox or Tcf motifs in a given group of “wild-type” peaks was enriched over the random “shuffled” sequences. Our results reveal Sox17 motifs are more frequently enriched in Sox17 bound and Sox17/β-catenin bound peaks over random, while Tcf motifs are not enriched over random in any of the categories of sequences. We now incorporate this new analysis in the revised results and new Figure 4—figure supplement 2. In the future we will explore the biological impact of number and frequency of motifs in enhancers, but for the current paper this is a very minor observation, from which we do not make any major conclusions.7) Please indicate the application of multiple testing correction  where applicable . Also indicate p-values for all enrichments and intersections .We have now updated the figures, legends and Materials and methods to indicate the statistical tests used and p-values for all enrichments and intersections.8) Including a summary of the temporal role of Sox17 in context of other signals and transcription factors, including Nodal/Smad2, BMP, VegT would be useful.We have now incorporated the requested summary in the Discussion.The manuscript has been greatly improved but there is one remaining issue that should be addressed before acceptance. This concerns the motif analysis in Figure 4—figure supplement 2. Examining background frequencies via scrambling of peak sequences while maintaining di-nucleotide frequencies has strengthened this analysis. However, ranking the motifs by hypergeometric p-value (provided by HOMER) is perhaps not most informative way to represent these data. We suggest instead plotting the 2log fold enrichment over background, in combination with a statistical test .As requested, we added a new panel Figure 4—figure supplement 2D plotting the log2 fold enrichment of Sox and Tcf site frequency for each class of peak compared to random shuffled sequence with a Fisher Exact test to show the enrichment and statistical significance. We agree that some readers may find this way of viewing the data easier. Other naïve test readers preferred the original display and so we kept that too.Also, it is felt that a discussion of background frequencies is needed where the co-occurring motifs are discussed and Figure 4—figure supplement 2D should be revised by calculating for background sequences.As requested, we modified this panel  to also show random genomic background sequence. A Kolmogorov-Smirnov test comparing the motif distribution of each group to background, showed that the “Sox17 and β-catenin” and “Sox17 Only” motif distribution was significantly different to random background. We made minor modifications to the text to reflect this.We want to stress that the motif frequency and co-occurrence is very minor part of the paper. The only points we want to make are (1) that most peaks contain both Sox and Tcf sites while some have just Sox sites and (2) that when Sox motifs are present there are usually multiple sites. We did not make any assertions based on the statistics. In many respects we could remove Figure 4—figure supplement 2 completely and it would not change any of our conclusions. We just included these observations as the reader might be interested.


Circular RNAs (circRNAs) are stable, often highly expressed RNA transcripts with potential to modulate other regulatory RNAs. A few circRNAs have been shown to bind RNA-binding proteins (RBPs); however, little is known about the prevalence and distribution of these interactions in different biological contexts.We conduct an extensive screen of circRNA-RBP interactions in the ENCODE cell lines HepG2 and K562. We profile circRNAs in deep-sequenced total RNA samples and analyze circRNA-RBP interactions using a large set of eCLIP data with binding sites of 150 RBPs. We validate interactions for select circRNAs and RBPs by performing RNA immunoprecipitation and functionally characterize our most interesting candidates by conducting knockdown studies followed by RNA-Seq.TP53. Finally, we show that elevated levels of circCDYL are associated with overall survival of bladder cancer patients.We generate a comprehensive catalog of circRNA-RBP interactions in HepG2 and K562 cells. We show that KHSRP binding sites are enriched in flanking introns of circRNAs and that KHSRP depletion affects circRNA biogenesis. We identify circRNAs that are highly covered by RBP binding sites and experimentally validate individual circRNA-RBP interactions. We show that circCDYL, a highly expressed circRNA with clinical and functional implications in bladder cancer, is almost completely covered with GRWD1 binding sites in HepG2 cells, and that circCDYL depletion counteracts the effect of GRWD1 depletion. Furthermore, we confirm interactions between circCDYL and RBPs in bladder cancer cells and demonstrate that circCDYL depletion affects hallmarks of cancer and perturbs the expression of key cancer genes, e.g., Our study demonstrates transcriptome-wide and cell-type-specific circRNA-RBP interactions that could play important regulatory roles in tumorigenesis. Circular RNAs (circRNAs) are covalently closed RNA molecules often derived from precursor mRNA (pre-mRNA) through a backsplicing event, in which a downstream 5′ splice donor backsplices to an upstream 3′ splice acceptor . First iAlu elements, facilitate circRNA production by RNA pairing . O. O64]. OP = 0.9, T-test, Additional file P = 0.5, T-test, Additional file Since our observations could be explained by overall splicing perturbations in KHSRP KD samples, we evaluated the expression of the corresponding linear RNA of circRNAs. We found no difference in linear RNA expression between KHSRP KD and control samples for circRNAs with  increases with transcript abundance as expected  circRNA host genes, (B) genes of the same expression level, and (C) the most highly expressed genes. We divided exons into three different groups: (1) backsplice junction circularizing exons (BSJ circ-exons), which are directly involved in backsplicing; (2) circularizing exons (circ-exons) that are potentially part of circRNAs but not supported by BSJ reads; 3) and non-circ-exons, which are not part of circRNAs , we found that BSJ circ-exons were significantly more covered with RBP binding sites than internal non-circ-exons within the same genes for both K562 , we divided all genes into expression level percentiles. We randomly drew non-circ-exons, while ensuring the same expression level distribution as circ-genes, which are distributed across percentiles ~ 20–100  or similar , indicating specific circRNA-RBP interactions . Although we observed no significant results, we found that a larger fraction of RBPs that are more prone to bind to BSJ circ-exons are associated with regulation of mRNA stability (3.8 fold), translation (3.7 fold), and binding to the mRNA 5′-UTR (10.1 fold) and 3′-UTR (3.4 fold) compared to the rest of the RBPs in K562  < 0.1 for all shown RBPs, Wilcoxon rank sum test, Fig. n = 111) and K562 (n = 133) , while there are no RBPs overlapping circGSE1 in HepG2 but many RBP binding sites in GSE1 non-circularizing exons (ratio = 0.02). In both cell lines, circGSE1 is among the 1% highest expressed circRNAs and the parent gene GSE1 is found in similar gene expression percentiles in K562 (percentile 81) and HepG2 (percentile 83).To ensure our observations are not simply explained by host gene expression levels, we compared the RBP coverage of highly expressed circRNAs to non-circularizing exons within the parent gene “c. Of theP < 0.05, empirical p value). The RBM33 gene is highly expressed in both K562 (percentile 73) and HepG2 (percentile 91). The high enrichment for circRBM33 is due to ~ 100% RBP coverage of circRBM33 and no RBP binding sites in the single internal non-circ-exon in the gene. The CDYL host gene, which gives rise to the highest expressed circRNA, is itself modestly expressed in both K562 (percentile 48) and HepG2 (percentile 62). CircCDYL is 93% covered with RBP binding sites in both cell lines while the non-circ-exons in CDYL have a mean RBP coverage of only 7%  are shared between the cell lines  and circRBM33 (~ 100×) are highly covered with RBP binding sites compared to non-circ-exons in their parent gene in both cell lines Fig. c. These ZKSCAN1 or with circZKSCAN1 under certain conditions. Faint bands indicate that the circRNAs interact with other RBPs than the ones identified from the eCLIP data with lower affinity. The lack of low-affinity and nonspecific RBP binding sites in the eCLIP data could be explained by the stringent cut-off used, of 8-fold enrichment in IP compared to input  of five RBPs, IGF2BP1, IGF2BP2, GRWD1, YBX3, and UCHL5, that are predicted to bind to one or several of four highly expressed and highly RBP-covered circRNAs, circCDYL, circRBM33, circZKSCAN1, and circSMARCA5  in HepG2 with an siRNA specifically recognizing the backsplice junction of circCDYL , while IGF2BP1 binding sites are enriched in upregulated genes  and IGF2BP1 (3.8×) in the cytosol of HepG2 cells , which igenesis . Since cm ENCODE  and founPLA2G2A is an extreme outlier upon circCDYL KD with abundance increasing more than 19 times . IGF2BPl cancer , and is l cancer , 80. ConOur results suggest that circCDYL interacts with GRWD1 in HepG2 to regulate GRWD1 target genes overall consistent with an RBP sponge hypothesis. Besides, circCDYL could be involved in other regulatory mechanisms to regulate classes of genes and individual transcripts in cancer-related pathways.In a previous study, we found that circCDYL is highly expressed in patients with non-muscle invasive bladder cancer (NMIBC) and correlates positively with good prognosis independently of the parent gene . Based oCDYL mRNA  but not between the BC cells and HepG2 (P > 0.4), consistent with cell-type-specific interactions and functions of circRNAs and RBPs  and IGF2BP2 (> 5×), in both bladder cancer cell lines, supporting circCDYL’s potential to act as a sponge . Corresponding with our previous findings, high expression levels of circCDYL are positively correlated with overall survival , nucleus (n = 1), membrane (n = 1), cytosol (n = 1), and insoluble cytoplasmic fraction (n = 1).Additional file 2: File S2. Bash script containing the code to extract all K562 total RNA-Seq samples from ENCODE. The following samples were obtained for K562: total cell (n = 4), nucleus (n = 1), nucleoplasm (n = 1), nucleolus (n = 1), chromatin (n = 1), membrane (n = 1), cytosol (n = 1), and insoluble cytoplasmic fraction (n = 1).Additional file 3:Table S1. Genomic and statistical information of the highly expressed circRNAs in K562 and HepG2. First six columns provide circRNA_ID, genomic positions of circRNAs, and information of circRNAs found in circBase. BSJ_reads and linear_reads give the number of reads supporting the circRNA and its corresponding linear transcript based on the CIRI2 pipeline. BSJ_reads_CIRCexplorer holds the number of reads supporting the circRNA by the CIRCexplorer pipeline. Exonic_length and N_exons hold the exonic length of the circRNA and the number of exons comprising the circRNA. N_RBPs give the number of RBPs with binding sites in the exonic part of the circRNA. RBP_overlap_bp is the number of bp overlapped by RBP binding sites (merged) in the exonic part of the circRNA. RBP_coverage is the fraction of the circRNA (exonic part) covered by RBP binding sites (RBP_overlap_bp/exonic_length*100). N_non_circ_exon_host_gene indicates the number of non-circularizing exons in the host gene. The mean RBP-coverage of non-circularizing exons in the host gene is provided in the column mean_RBP_coverage_non_circ_exons and was added a pseudo count of 1 to calculate the ratio between circRNA RBP-coverage and RBP-coverage of non-circ-exons in the host gene. This ratio is provided in Ratio_RBP_coverage_circRNA_non_circ_exons ((RBP_coverage+ 1)/(mean_RBP_coverage_non_circ_exons+ 1)).Additional file 4:Table S2. Target sequences for siRNA-mediated knockdown of circCDYL, IGF2BP1, IGF2BP2, and GRWD1 in HepG2 and/or UMUC3 and J82.Additional file 5: Table S3. Sample information for HepG2 and K562.Additional file 6:Table S4. Count matrix of backsplice junction reads supporting circRNAs in HepG2 txt).Additional file 7:Table S5. Count matrix of backsplice junction reads supporting circRNAs in K562.Additional file 8: Fig. S1-S6. Pdf file with all supplementary figures  with corresponding figure legends.Additional file 9:File S3. Name of the RNA binding proteins with eCLIP data in HepG2.Additional file 10:File S4. Name of the RNA binding proteins with eCLIP data in K562.Additional file 11:Table S6. Genomic overlap of circRNAs and RBP binding sites in HepG2 and K562.Additional file 12:Table S7. Subcellular localization of RBPs in HepG2.Additional file 13:Table S8. GO enrichment analysis of RBPs that bind relatively more to BSJ circ-exons than non-circ-exons.Additional file 14:Table S9. Primers for validating circRNA expression.Additional file 15:Table S10. qPCR primers to detect circCDYL and CDYL host gene expression levels.