CpG islands are gene regulatory elements associated with the majority of mammalian promoters, yet how they regulate gene expression remains poorly understood. Here, we identify FBXL19 as a CpG island-binding protein in mouse embryonic stem (ES) cells and show that it associates with the CDK-Mediator complex. We discover that FBXL19 recruits CDK-Mediator to CpG island-associated promoters of non-transcribed developmental genes to prime these genes for activation during cell lineage commitment. We further show that recognition of CpG islands by FBXL19 is essential for mouse development. Together this reveals a new CpG island-centric mechanism for CDK-Mediator recruitment to developmental gene promoters in ES cells and a requirement for CDK-Mediator in priming these developmental genes for activation during cell lineage commitment. Multicellular development relies on the formation of cell-type-specific gene expression programmes that support differentiation. At the most basic level, these expression programmes are defined by cell signaling pathways that control how transcription factors bind DNA sequences in gene regulatory elements and shape RNA polymerase II (RNAPolII)-based transcription from the core gene promoter (reviewed in ). In eukIn mammalian genomes, CpG dinucleotides are pervasively methylated and this epigenetically maintained DNA modification is generally associated with transcriptional inhibition, playing a central role in silencing of repetitive and parasitic DNA elements . Most geOne of the first characterised ZF-CxxC domain-containing proteins was lysine-specific demethylase 2A (KDM2A) . KDM2A iHere, we investigate the function of FBXL19 in mouse embryonic stem (ES) cells and show that it is predominantly found in the nucleus where it localizes in a ZF-CxxC-dependent manner to CpG island promoters. Biochemical purification of FBXL19 revealed an association with the CDK-containing Mediator complex and we discover that FBXL19 can target CDK-Mediator to chromatin. Conditional removal of the CpG island-binding domain of FBXL19 in ES cells leads to a reduction in the occupancy of CDK8 at CpG islands associated with inactive developmental genes. FBXL19 and CDK-Mediator appear to play an important role in priming genes for future expression, as these genes are not appropriately activated during ES cell differentiation when the CpG island-binding capacity of FBXL19 is abolished or CDK-Mediator is disrupted. Consistent with an important role for FBXL19 in supporting normal developmental gene expression, removal of the ZF-CxxC domain of FBXL19 leads to perturbed development and embryonic lethality in mice. Together, our findings uncover an interesting new mechanism by which CDK-Mediator is recruited to gene promoters in ES cells and demonstrate a requirement for FBXL19 and CDK-Mediator in priming developmental gene expression during cell lineage commitment.FBXL19 shares extensive sequence similarity and domain architecture with the other KDM2 paralogues which are predominantly nuclear proteins . TherefoBased on the nuclear localization of FBXL19 and the fact that it encodes a highly conserved ZF-CxxC domain , we set KDM2A and KDM2B rely on defined residues in their ZF-CxxC domain to recognize non-methylated cytosine and bind CpG islands . To deteOur ChIP-seq analyses demonstrated that FBXL19 is targeted to CpG islands in a manner that is highly similar to KDM2A and KDM2B . AlthougWe next wanted to determine which region of FBXL19 is required for interaction with CDK-Mediator. To do so, we transiently expressed full length FBXL19 or versions of FBXL19 with individual domains removed and performed affinity purification followed by western blot analysis. Intact FBXL19 and a version with the ZF-CxxC domain removed interacted with CDK-Mediator, whereas removing the F-box domain resulted in a loss of this interaction . TherefoBased on a candidate approach, it was recently reported that FBXL19 could interact the RNF20/40 E3 ubiquitin ligase in ES cells and regulate histone H2B lysine 120 ubiquitylation (H2BK120ub1) . In our FBXL19 contains conserved F-box and leucine-rich repeat (LRR) domains. F-box proteins are known to function as scaffolds and substrate recognition modules for the SKP1-Cullin-F-box (SCF) protein complexes that ubiquitylate proteins for degradation by the proteasome . Based ode novo on chromatin and test their capacity to recruit additional factors Fbxl19 (ΔCXXC)   and whic (ΔCXXC)  and can, (ΔCXXC) . This mo (ΔCXXC) . Followi (ΔCXXC) , but imp (ΔCXXC) . Using t binding , indicat binding . In keep binding .ΔCXXCFbxll19 ES cells, we discovered that they tended to coincide with broad regions of CDK8 enrichment . We theES cells . Therefofl/flFbxl19 and ΔCXXCFbxl19 ES cells with RA   . We obseRNA-seq) . GO termRNA-seq) . We thenXC cells . While vXC cells . This isXC cells  and withXC cells . GO analXC cells , unlike XC cells . It is pal genes , genes nal genes  and tendal genes . ImportaES cells , supportfl/flMed13/13l ERT2-Cre ES cells) (fl/flMed13/13l ES cells with OHT resulted in a loss of MED13 and MED13L protein (MED13/13L KO) and reduced levels of the other subunits of the CDK-kinase module  . We chosS cells) . Treatmee module . Importae module  but did e module . We nexte module . Importae module , we obsee module . TogetheΔCXXCFbxl19 cells display impaired gene activation in our in vitro differentiation model, we asked whether the inability of FBXL19 to bind and function at CpG islands could also affect mouse development. In order to address this, we generated ΔCXXCFbxl19 mutant mice by crossing fl/flFbxl19 animals with animals constitutively expressing Cre recombinase. From these crosses, we failed to obtain any viable ΔCXXCFbxl19 homozygous offspring, indicating that removal of the ZF-CxxC domain of FBXL19 leads to embryonic lethality   . At 9.5 um (dpc)  the embrHere, we discover that FBXL19 recognizes CpG islands throughout the genome in a ZF-CxxC-dependent manner . Unlike Previously, another F-box protein, FBW7, was shown to function as an E3 ubiquitin ligase substrate selector for the MED13/13L subunit of CDK-Mediator and to regulate its stability through proteasomal degradation . By contIndividual Mediator subunits have been shown to interact with DNA-binding transcription factors that recruit the Mediator complex to specific DNA sequences in regulatory elements and support gene expression . In partHistorically, CDK-Mediator has been associated with gene repression . TherefoOur finding that FBXL19 can target CDK8 to gene promoters and that this is required to prime the expression of developmental genes during ES cell differentiation is conceptually reminiscent of recent work in yeast and human systems which suggested that CDK8 is required for appropriate re-induction of inducible genes following an initial activation stimulus . In thisfl/flFbxl19 ES cells were treated with 800 nM 4-hydroxytamoxifen (Sigma) for 96 hr in order to delete the ZF-CxxC domain. For RA differentiation of ES cells, 2.5 × 104 cells/cm2 were allowed to attach to gelatinised dishes (~12 hr) and treated with 1 µM retinoic acid (Sigma-Aldrich) in EC-10 medium  for 72 hr. For embryonic body differentiation, 2 × 106 cells were plated on non-adhesive 10 cm dishes in EC-10 medium and cultured for the indicated days. For generation of stable cell lines, E14 ES cells were transfected using Lipofectamine 2000 (Thermo Fisher scientific) following manufacturer’s instructions. Stably transfected cells were selected for 10 days using 1 μg/ml puromycin and individual clones were isolated and expanded in the presence of puromycin to maintain the transgene expression. TOT2N E14 cells used for TetR targeting experiments were previously described (Mouse ES cells were cultured on gelatine-coated dishes in DMEM (Thermo Fisher scientific) supplemented with 15% fetal bovine serum (BioSera), L-Glutamine, beta-mercaptoethanol, non-essential amino acids, penicillin/streptomycin (Thermo Fisher scientific) and 10 ng/mL leukemia-inhibitory factor. escribed . 293 T cFbxl19 genomic region. Recombination was carried out by Gateway system (Life Technologies). One of the loxP sequences was inserted at mm9 chr7:134,891,537, and FRT flanked PGK-neo was inserted at mm9 chr7:134,892,000 together with an additional loxP sequence. The targeting construct was electroporated into M1 ES cells to obtain targeted insertion. Clones of targeted ES cells were aggregated with eight-cell embryos to generate the targeted mouse line. The fl/flFbxl19 line was generated by removal of the PGK-neo marker gene by mating the targeted mice with mice expressing FLP recombinase. These fl/flFbxl19 mice were further mated with mice harboring the ROSA26-CreErt2 locus to generate Fbxl19fl/fl:ROSA26-CreErt2+/- mice, from which the fl/flFbxl19 ES cells used in this study were derived.The targeting construct was generated from C57BL6/J mouse genomic sequence spanning mm9 chromosome 7: 134,888,487–134,895,358 containing the exons 1 to 6 of Med13/fl/fl13l ES cell line, we inserted loxP sites downstream of exons 7 and upstream of exons 8 of the Med13 and Med13l genes. The targeting constructs for the insertion of each loxP site were designed to have 150 bp homology arms flanking the loxP site and to carry a mutated PAM sequence to prevent retargeting by the Cas9 enzyme. The targeting constructs were purchased from GeneArt Gene Synthesis (Thermo Fisher scientific). The pSpCas9(BB)−2A-Puro(PX459)-V2.0 vector was obtained by Addgene (#62988). sgRNAs were designed to specifically target the desired genomic region for each loxP insertion (http://crispor.tefor.net/crispor.py) and were cloned into the Cas9 vector as previously described (ROSA26 locus (ROSA26:ERT2-Cre) were used. First, the downstream loxP sites for Med13 and Med13l were targeted. ROSA26:ERT2-Cre ES cells were transiently co-transfected with 1 μg of each Cas9-sgRNA plasmid and 3.5 μg of each targeting construct using Lipofectamine 3000 (Thermo Fischer Scientific). Successfully transfected ES cells were selected for 48 hr with 1 μg/ml puromycin. Individual clones were screened by genotyping PCR to identify correctly targeted homozygous clones. A clone homozygous for both Med13 and Med13l LoxP1 sites was then used to target the upstream loxP sites using the same transfection protocol and screening strategy. Correct loxP targeting was verified by sequencing of the genomic region surround the loxP sites.In order to generate a conditional escribed . ES cellFbxl19  was PCR amplified and inserted into a pCAG-IRES-FS2 vector ) and incubated for 10 min at 4°C with slight agitation. After centrifugation, the cell pellet was resuspended in 3x PV Buffer A containing 0.1% NP-40 and incubated for 10 min at 4°C with slight agitation. Nuclei were recovered by centrifugation and the soluble nuclear fraction was extracted for 1 hr at 4°C with slight agitation using 1x PV Buffer C . Protein concentration was measured using Bradford assay.Cells were harvested by scraping in PBS at 4°C, resuspended in 10x pellet volume (PV) of Buffer A . Samples were incubated with the respective antibodies and 25U benzonase nuclease overnight at 4°C. Protein A agarose beads (RepliGen) were blocked for 1 hr at 4°C in Buffer BC150 containing 1% fish skin gelatine (Sigma) and 0.2 mg/ml BSA (NEB). The blocked beads were added to the samples and incubated for 4 hr at 4°C. Washes were performed using BC150 containing 0.02% NP-40. The beads were resuspended in 2x SDS loading buffer and boiled for 5 min to elute the immunoprecipitated complexes.Purification of FBXL19-FS2 was performed using StrepTactin resin (IBA) as previously described with the exception of the wash buffer used  . BetweenSamples from FBXL19-FS2 affinity purifications were subjected to in-solution trypsin digestion and mass spectrometry analysis was performed as described previously . Two bioAntibodies used for IP were rabbit anti-MED12 , rabbit anti-CDK8 , rabbit anti-HA . A polyclonal antibody against FBXL19 was prepared in-house by rabbit immunisation  with a recombinant peptide encoding for amino acids 137–336 of mouse FBXL19 protein. The FBXL19 peptide antigen was coupled to Affigel 10 resin (BioRad) and the antibody was affinity-purified and concentrated.Antibodies used for Western blot analysis were mouse anti-Flag M2 , mouse anti-SKP1 , goat anti-CDK8 , rabbit anti-MED12 , rabbit anti-MED13L , rabbit anti-MED13 , rabbit anti-MED1, , rabbit anti-MED26 , rabbit anti-T7 , rabbit anti-TBP , rabbit anti-RNF20 , rabbit anti-ubiquityl-Histone H2B (Lys120) , and mouse anti-ubiquityl-Histone H2B (Lys120) .flFbxl19fl/ ES cell line in which the endogenous Fbxl19 gene is tagged with a 3xT7-2xStrepII tag by CRISPR/Cas9 knock-in (Fbxl19 gene (http://crispr.mit.edu/) and cloned into the Cas9 vector as previously described (fl/flFbxl19 ES cells were transiently co-transfected with 1 μg of Cas9-sgRNA plasmid and 3.5 μg of targeting construct using Lipofectamine 3000 (ThermoFischer Scientific). Successfully transfected ES cells were selected for 48 hr with 1 μg/ml puromycin. Individual clones were screened by Western blot and genotyping PCR to identify homozygous targeted clones.As the FBXL19 antibody failed to work reliably for Western blot analysis, we generated an knock-in . This alescribed . Fbxl19fg for 20 min at 4°C. 200 µg chromatin diluted in ChIP dilution buffer , 150 mM NaCl) was used per IP. Chromatin was precleared with protein A Dynabeads blocked with 0.2 mg/ml BSA and 50 µg/ml yeast tRNA and incubated with the respective antibodies overnight at 4°C. Antibody-bound chromatin was purified using blocked protein A Dynabeads for 3 hr at 4°C. ChIP washes were performed as described previously (3) and reversed cross-linked overnight at 65°C with 200 mM NaCl and RNase A (Sigma). The reverse cross-linked samples were treated with 20 μg/ml Proteinase K and purified using ChIP DNA Clean and Concentrator kit (Zymo research).Chromatin immunoprecipitation was performed as described previously  with slieviously . ChIP DNThe antibodies used for ChIP experiments were rabbit anti-FS2 , rabbit All ChIP-seq experiments were carried out in biological duplicates. ChIP-seq libraries for FS2-FBXL19 ChIP were prepared using the NEBNext Fast DNA fragmentation and library preparation kit for Ion Torrent  following manufacturer’s instructions. Briefly, 30–40 ng ChIP or input DNA material was used. Libraries were size-selected for 250 bp fragments using 2% E-gel SizeSelect gel (Thermo scientific) and amplified with 6 PCR cycles or used without PCR amplification. Templates were generated with Ion PI Template OT2 200 kit v3 and Ion PI Sequencing 200 kit v3, or with Ion PI IC 200 kit (Thermo scientrific). Libraries were sequenced on the Ion Proton Sequencer using Ion PI chips v2 (Thermo scientific).ChIP-seq libraries for CDK8 ChIP were prepared using the NEBNext Ultra DNA Library Prep Kit, and sequenced as 40 bp paired-end reads on Illumina NextSeq500 platform using NextSeq 500/550 (75 cycles).Total RNA was isolated using TRIzol reagent (Thermo scientific) following manufacturer’s instructions and cDNA was synthesized from 400 ng RNA using random primers and ImProm-II Reverse Transcription system kit (Promega). RT-qPCR was performed using SensiMix SYBR mix (Bioline). Idh1 and Atp6IP1 genes were used as house-keeping controls.For labeling of nascent RNA transcripts, cells were grown in 15 cm culture dishes. Labeling was performed for 20 min at 37°C by the addition of 50 mM 4-thiouridine  to the culture medium. After the incubation, the medium was removed and RNA was isolated using TRIzol reagent (Thermo scientific) following manufacturer’s instructions. The total RNA was treated with TURBO DNA-free kit  in order to remove contaminating genomic DNA. 300 µg RNA was biotinylated using 600 µg Biotin-HPDP  in Biotinylation buffer . The reaction was carried out for one and half hour on a rotor at room temperature. Unincorporated Biotin-HPDP was removed by chloroform/isoamylalcohol  wash followed by isopropanol precipitation of the biotinylated RNA. Labeled biotinylated RNA was isolated using µMacs Streptavidin Kit  following manufacturer’s instructions and purified using RNeasy MinElute Cleanup kit (Qiagen). The quality of the RNA was confirmed using the RNA pico Bioanalyser kit (Agilent).Up to 1 µg purified 4sU-labelled RNA was used to prepare libraries for 4sU-RNA-seq. Ribosomal RNA was removed using NEBNext rRNA Depletion Kit and libraries were prepared using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB) following manufacturer’s instructions. Library quality was assessed using the High-sensitivity DS DNA Bioanalyser kit (Agilent) and the concentration was measured by quantitative PCR using KAPA Library quantification standards for Illumina (KAPA Biosystems). All 4sU RNA-seq experiments were carried out in biological triplicates. 4sU RNA-seq libraries were sequenced as 80 paired-end reads on Illumina NextSeq500 platform using NextSeq 500/550 High Output Kit v2 (150 cycles).Sequencing reads were aligned to the mouse genome (mm10) using bowtie2  with thePeak calling was performed using the MACS2 function with the ‘-broad’ option using the biological replicates with matched input . Peaks mDifferential expression analysis was performed using the DESeq2 package , versionChIP-seq and RNA-seq data from the present study are available to download at GSE98756. Previously published studies used for analysis include mouse ES cell BioCAP , Kdm2B  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by four peer reviewers, one of whom is a member of our Board of Reviewing Editor and the evaluation has been overseen by a Senior Editor.Thank you for submitting your work entitled "FBXL19 recruits CDK8-Mediator to CpG islands and primes developmental genes for activation during lineage commitment" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this letter to crystallize our concerns going forward. The work presented was deemed important and interesting but key issues reduced enthusiasm for the manuscript considerably. Given that these revisions are substantial and likely to take a significant amount of time, we are currently declining the present version of the paper. That said, if you feel you can successfully address all the concerns raised, we will be open to consider a revised submission, which would be treated as a de novo submission but sent to the same editors and, if possible, referees for re-assessment.Summary:The reviewers considered the manuscript to be strong, reporting a thorough characterization of FBXL19, the least-well studied of three proteins that contain a ZF-CxxC domain. The authors demonstrate that over-expressed FBXL19 accumulates in the nucleus, and that a majority of its genome-wide binding is at sites associated with CpG islands. Affinity purification of FS2-tagged FBXL19 indicated a physical association with CDK8-containing Mediator complex, and a TetR-FBXL19 fusion protein was able to recruit CDK8 to a chromosomally-integrated artificial TetO array, suggesting that FBXL19 can recruit CDK8 to chromatin. Conditional deletion of the ZF-CxxC domain of FBXL19 led to reduction of CDK8 binding at CpG islands in ES cells. Many of these sites are associated with silent developmental genes that are no longer activated during ES cell differentiation in the FBXL19 mutants. Finally, deletion of the ZF-CxxC domain was shown to be embryonic lethal in mice. Together, these findings suggest that FBXL19 may prime developmental genes for subsequent activation during differentiation, possibly involving recruitment of CDK8. Overall, this represents an interesting finding and potentially a novel role for CDK8. However, concerns about the degree to which this represents a significant advance over previous FBXL19 studies, some missing controls, and a lack of a direct test of CDK8 requirement for expression of these genes resulted in lessened enthusiasm for this manuscript.The reviewers discussed these concerns and summarized their critiques into the following points.Essential revisions:1) A key conclusion of the manuscript is that CDK8-Mediator is required for induction of developmental genes regulated by FBXL19, but this is actually not proven in the manuscript. The authors should deplete CDK8  and test for the impact on induction of FBXL19-dependent genes.2) Throughout the paper, the authors refer to CDK8-Mediator. Given that there are two paralog kinases associated with the Mediator complex, CDK8 and CDK19, the authors should test for the presence of CDK19 and clarify whether the interaction is exclusive to CDK8-containing complexes or not. In Figure 2A, are the CDK8 peptides identified unique to CDK8 or could they map to CDK19 as well? In Figure 2C, please add western blot for CDK19. As for point #1 above, is CDK19 required for control of FBXL19-regulated genes? Additionally, the authors should demonstrate that in the "EV" control lane, at the similar molecular weight, these candidate hits didn't show up in the cut gel, or at least with significantly less numbers of peptides.3) If the ZF-CxxC mediates the interaction between FBXL19 and chromatin, then which functional domain(s) on FBXL19 bind to CDK8-Mediator complex? Lee et al., claimed that the F-box is critical for the interaction between FBXL19 and RNF20. Does the same protein structure bind to CDK8?4) Most of the findings are based on overexpression of FBXL19 – what is the level relative to endogenous FBXL19? High-level over-expression could drive nuclear accumulation and other observations . Authors should provide data demonstrating the degree of overexpression produced in the key experiments.5) FBXL19 binding to CpG islands and its requirement in ES differentiation has been described previously: (https://www.ncbi.nlm.nih.gov/pubmed/28453857) "Our genome-wide target mapping unveils the preferential occupancy of Fbxl19 on CpG island-containing promoters […]" "Moreover, we reveal that Fbxl19 is critical for proper differentiation of ES cells […]" With respect to this, the authors should clarify the novelty of their results presented in the current manuscript.6) The conclusion that FBXL19 does not appear to regulate CDK8-Mediator levels is not fully supported by the data. Figure 2—figure supplement 1H needs to be repeated so that CDK8 bands are not over-exposed as there does appear to be a change in amount/mobility when overexpressed -/+ MG132. Furthermore, there seems to be a noticeable increase of MED12 protein upon FBXL19 overexpression, which was even more obvious when cells were treated with MG132.7) In Figure 4, CDK8 recruitment has only been tested for the FBXL19 ZF-CxxC deletion -is this recapitulated for depletion of the whole protein? Given that Figure 4B appears to show a decrease in the levels of the deletion protein this is an important point to verify.8) The authors need to show that OHT treatment of the delCxxC ES line leads to reduced binding of FBXL19 in conditions used for CDK8 ChIPseq -several genes would be ok -these same genes should be used to validate CDK8 CHIPseq signal.9) Supplemental tables with differential gene expression results should be provided -or at least working gene lists used to reach the conclusions in the manuscript. Full GO enrichment results should be included as supplementary tables so that specific gene lists can be examined.10) Various figure panels are missing statistical tests: Figure 4C needs statistical tests, e.g. KS/Wilcoxon -See Figure 4E; Figure 4G needs statistical tests; Figure 4H and 4I need boxplots and need statistical tests eg KS/Wilcoxon as for E/F; Figure 4H-J should include FBXL19-bound groups; Figure 6B,E,F need statistical tests.11) It is very nice that the authors showed that genome-wide chromatin binding of FBXL19 was completely lost when the ZF-CxxC domain was deleted or mutated. To further confirm that it is indeed this ZF-CxxC domain mediating interaction with the non-methylated CpG islands, some controls are necessary. For example, in Figure 1—figure supplement 1D, the authors showed that almost all of the FBXL19 binding sites are overlapped with NMIs locations, with only 93 exceptions. Then are the signals at these 93 sites also changed in the FBXL19 mutant ChIPSeq data? When the cell DNA methylation levels are modulated, will the binding pattern of FBXL19 changed accordingly?[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors and the evaluation has been overseen by James Manley as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "FBXL19 recruits CDK-Mediator to CpG islands of developmental genes priming them for activation during lineage commitment" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The revised manuscript by Dimitrova et al., is an excellent report describing the characterization of FBXL19, a member of the ZF-CxxC domain-containing family of proteins, and its role in recruiting CDK-Mediator to developmental genes in stem cells.As for other members of this family, the authors show that FBXL19 recognizes unmethylated CpGs in a ZF-CxxC-dependent manner. Unlike the lysine demethylases KDM2A and KDM2B in this protein family, FBXL19 lacks a JMJ-C domain. Instead, the authors report that FBXL19 interacts with the CDK-Mediator complex. The authors then embark on an experimental tour-de-force to demonstrate that FBXL19 is required for recruitment of CDK-Mediator to a subset of genes containing CpG islands in ES cells, mostly genes involved in lineage commitment. Interestingly, these genes are transcribed at low levels in ES cells when bound by FBXL19 and CDK-Mediator, but their induction is compromised in the absence of FBXL19. This indicates that FBXL19-CDK8-Mediator is required to 'prime' these genes in ES cells and their subsequent induction upon differentiation stimuli. Finally, the authors demonstrate that FBXL19 depletion is embryonic lethal. Overall, the manuscript is very well written, and the data is of excellent quality.This is a new submission of a paper that was originally reviewed by the same referees and found to be lacking data to support some of the key conclusions. The Senior Editor opened up the possibility of a new submission without a precise timeline and the authors have taken the opportunity to address the major concerns raised in the original review. The referees are now supportive of publication of a revised manuscript addressing the following issues:Essential revisions:1) In AP-MS , what is the background value of emPAI? Are there any other FBXL19-interacting proteins that show significantly high levels of emPAI in the list? How significant are the components of CDK-Mediator complex represented in all the hits?2) In Figure 3, the authors demonstrated that FBXL19 was able to recruit CDK-Mediator complex, such as CDK8 and MED12, to artificially designed chromatin regions. What about CXXC-domain or F-box deletion mutants in this assay?3) In Figure 4B, it is not very clear which band(s) are exactly indicated as full-length FBXL19 and which one(s) are CXXC deletion? Why are there double bands? Did the author use T7 antibody to detect the endogenously introduced protein?4) In Figure 4C, what proportions of each group of CDK8 binding sites overlap with FBXL19? In theory, the author expects to see that CDK8 sites showing binding intensity changes upon tamoxifen treatment would tend to overlap more with FBXL19.5) In Figure 4—figure supplement 1C, please use a Venn Diagram to show directly the number of peaks of CDK8, FBXI19 and NMI overlapping with each other. This would be more straightforward.6 In Figure 5, please perform the same GSEA or functional annotation analysis on genes that are associated with unchanged CDK8 binding sites upon FBXL19 CXXC-domain deletion.7) In Figure 6B, what is the result for genes that are associated with increased CDK8 binding signals in FBXL19 CXXC-domain deletion mutant upon RA-induced differentiation?8) In Figure 6C, even though the authors claimed that those upregulated genes (889) in FBXL19 CXXC-domain deletion mutant upon RA treatment were not involved in developmental processes, it is still interesting to find that significantly more genes were activated upon CXXC-domain deletion. What explanation can the authors provide for this finding?9) In Figure 7, the authors claim that FBXL19 relies on CDK-mediator complex to activate the specific set of developmental genes. Is it possible to test this hypothesis in the FBXL19 CXXC-deletion system they established in Figure 6? In wild-type ESC, depletion of MED13 will have similar effects as FBXL19 CXXC deletion, whereas in FBXL19 mutant, the effects would be much alleviated?10) In Figure 8, why did heterozygous deletion of FBXL19 CXXC-domain show even better developmental status than the wild type?11) The authors demonstrated that the CXXC-domain mediates the binding of FBXL19 at NMIs, where F-Box mediates its interaction with the CDK-Mediator complex. What about CDK8 chromatin binding or gene expression patterns upon F-Box deletion of FBXL19? Will the authors see the same results as what they found in CXXC-domain deletion mutant? [Editors’ note: the author responses to the first round of peer review follow.]Essential revisions:1) A key conclusion of the manuscript is that CDK8-Mediator is required for induction of developmental genes regulated by FBXL19, but this is actually not proven in the manuscript. The authors should deplete CDK8  and test for the impact on induction of FBXL19-dependent genes.We agree with the reviewers that testing the impact that removal of CDK-Mediator has on the induction of FBXL19 target genes is an important experiment. To disrupt CDK-Mediator we have developed a new cell system to remove MED13 and its closely related paralogue MED13L (both of which are expressed in mouse ES cells) in an inducible manner. We chose to delete both MED13 and MED13L because they are thought to play an interchangeable role in physically linking the CDK subunits to the core Mediator and to be required to form an intact CDK-Mediator complex . To achieve this, we exploited gene targeting to insert loxP sites around essential exons in both MED13 and MED13L. We also engineered these cells to express a tamoxifen inducible form of Cre recombinase allowing inducible deletion of MED13/13L . In new Figure 7, we demonstrate that the addition of tamoxifen leads to complete loss of MED13/13L protein. Furthermore, we show by ChIP that CDK8 no longer occupies its target sites. We then examined FBXL19 target genes after loss of CDK-Mediator binding and found that they are no longer appropriately induced during differentiation , in keeping with a requirement for CDK-Mediator in FBXL19dependent gene activation. We thank the reviewers for suggesting this important experiment as it now robustly supports and strengthens our key argument that FBXL19 recruits the CDK-Mediator complex to the CpG islands of inactive developmental genes to prime them for appropriate activation during differentiation. We have now edited the text throughout the manuscript to reflect these important new observations.2)Throughout the paper, the authors refer to CDK8-Mediator. Given that there are two paralog kinases associated with the Mediator complex, CDK8 and CDK19, the authors should test for the presence of CDK19 and clarify whether the interaction is exclusive to CDK8-containing complexes or not. In Figure 2A, are the CDK8 peptides identified unique to CDK8 or could they map to CDK19 as well? In Figure 2C, please add western blot for CDK19. As for point #1 above, is CDK19 required for control of FBXL19-regulated genes?As indicated by the reviewer, CDK8 has a highly similar paralogue, CDK19. As suggested, we have now examined our mass spectrometry results in more detail to determine if we find any peptides that also map to CDK19. From this analysis, we have identified five different peptides covering CDK8/CDK19. Four are common to both CDK8 and CDK19, and one is unique for CDK8 . In our ‘CDK8 and its paralogue CDK19 share 77% amino acid identity (89% similarity)  and four out of the five peptides identified by mass spectrometry were common between the two proteins (data not shown). Therefore, it is likely that FBXL19 is able to interact with both CDK8- and CDK19- containing Mediator complexes.’Furthermore, because the FBXL19-associated Mediator complex could contain either CDK8 or CDK19, we have edited the text throughout the manuscript to replace ‘CDK8-Mediator’ with ‘CDK-Mediator’. In future work it will be interesting to understand in more detail if CDK8- and CDK19-containing Mediator complexes have redundant or unique roles in gene activation during differentiation.Additionally, the authors should demonstrate that in the "EV" control lane, at the similar molecular weight, these candidate hits didn't show up in the cut gel, or at least with significantly less numbers of peptides.We carried out mass spectrometry analysis on affinity-purified FBXL19 by in-solution digestion followed by LC-MS. This was to overcome the limitations inherent to cutting bands from SDS-PAGE gels and the isolation of tryptic peptides from gel slices. Importantly, we only considered a protein to be an FBXL19 interactor in our mass spectrometry analysis if it was absent from matched EV control purifications analysed using the same in-solution approach. It would have been beneficial to have described this approach in more detail in the initial submission, so we have now expanded our description of these experiments in the revised Material and Methods. Furthermore, we validated that CDK-Mediator components were not found in the ‘EV’ control purifications by Western blot analysis .3) If the ZF-CxxC mediates the interaction between FBXL19 and chromatin, then which functional domain(s) on FBXL19 bind to CDK8-Mediator complex? Lee et al. claimed that the F-box is critical for the interaction between FBXL19 and RNF20. Does the same protein structure bind to CDK8?We thank the reviewers for this suggestion and agree this is an important point. To address this, we performed transient overexpression of FBXL19 followed by co-immunoprecipitation comparing wildtype FBXL19 with versions of FBXL19 in which individual domains had been removed. We found that deletion of the F-box largely disrupted the interaction with CDK-Mediator, while removing the ZF-CxxC domain had little effect on this interaction. We have included these new observations in the revised manuscript . We also attempted to determine whether the LRR domain of FBXL19 was required for this interaction but unfortunately removing this portion of the protein seemed to render it unstable (unpublished observations) limiting our capacity to draw any meaningful interpretation.4) Most of the findings are based on overexpression of FBXL19 – what is the level relative to endogenous FBXL19? High-level over-expression could drive nuclear accumulation and other observations . Authors should provide data demonstrating the degree of overexpression produced in the key experiments.We have examined FBXL19 transcript levels in the transgene lines and they are roughly eight times that of wild type . DespiteGiven that we know FBXL19 is in the nucleus and interacts with CDK-Mediator, tethering experiments in Figure 3 were used to test if de novo targeting of FBLX19 to naïve chromatin would result in recruitment of CDK-Mediator. This showed that FBXL19 binding was sufficient to recruit CDK-Mediator , in agreement with our observations that when the capacity of FBXL19 to bind CpG island chromatin is disrupted, CDK8 is no longer appropriately recruited to a subset of CpG island-associated target genes .5) FBXL19 binding to CpG islands and its requirement in ES differentiation has been described previously: (https://www.ncbi.nlm.nih.gov/pubmed/28453857) "Our genome-wide target mapping unveils the preferential occupancy of Fbxl19 on CpG island-containing promoters […]" "Moreover, we reveal that Fbxl19 is critical for proper differentiation of ES cells […]" With respect to this, the authors should clarify the novelty of their results presented in the current manuscript.During the preparation of our manuscript, the manuscript cited by the reviewer was published in NAR. It reported that FBXL19 occupied CpG islands and appeared to play a role in cellular differentiation. However, we would like to point out that, although our observations were similar with respect to these two points, the main discoveries resulting from our careful and systematic study are completely distinct from that of the published study. In direct contrast to the published work, we find no evidence for any connection between FBXL19 and RNF20 using unbiased biochemistry and mass spectrometry . Furthermore, using our conditional genetic ablation strategies we do not observe any effect of FBXL19 loss on H2BK120ub1 . Therefore, we are unable to reproduce the central findings of the published study. In our opinion this severely limits and brings into question the validity and, therefore, the novelty of the published work. Our observations likely differ from those of the NAR study because we use unbiased as opposed to candidate-based (guess work) biochemistry and conditional knockout strategies as opposed to stable knockdown cell lines that are prone to indirect and secondary effects due to the long-term culture necessary for their generation. Nevertheless, we agree with the reviewer that the wording of the indicated sentences could have been more inclusive and contrasting of the published work and have clarified this in the revised manuscript .We would also like to take this opportunity to clarify the novelty of our study and the importance of its central conclusions. (1) By using unbiased biochemical approaches, we discover a link between FBXL19 and CDK-Mediator in mouse ES cells. (2) Using conditional genetic ablation, de novo targeting strategies, and genomics, we go on to show that FBLX19 can recruit CDK-Mediator to chromatin and the CpG island promoters of silent developmental genes in ES cells. (3) In the absence of FBXL19, these genes are no longer appropriately activated upon differentiation, providing an explanation for why normal differentiation cannot be achieved in the absence of FBXL19. (4) In the revised study, based on the reviewer’s suggestions (point 1), we now demonstrate that FBXL19 target genes also rely on CDKMediator for their activation, validating our biochemistry and genomics. (5) Finally, we discover that loss of FBXL19 leads to abnormal development and early embryonic lethality, in agreement with the inability of cells to appropriately regulate gene expression during development.Therefore, our discoveries are novel in describing a completely new modality by which FBXL19 and CpG islands prime genes for future expression. Furthermore, we provide compelling new evidence that CDKMediator can be recruited to gene promoters independently of classical transcription factors and transcription itself to prime CpG island-associated genes for activation. These new discoveries force us, and the field, to think differently about how CpG islands integrate and regulate gene expression, while also provide new insight into the poorly understood mechanisms by which CDK-Mediator is targeted to genes and functions to control gene expression. We believe these important points are now more clearly framed in the revised manuscript.6) The conclusion that FBXL19 does not appear to regulate CDK8-Mediator levels is not fully supported by the data. Figure 2—figure supplement 2H needs to be repeated so that CDK8 bands are not over-exposed as there does appear to be a change in amount/mobility when overexpressed -/+ MG132. Furthermore, there seems to be a noticeable increase of MED12 protein upon FBXL19 overexpression, which was even more obvious when cells were treated with MG132.We have repeated these experiments based on the reviewer’s suggestions. We again failed to observe any significant difference in CDK8 protein levels following FBXL19 over-expression at lower exposure levels. We agree it appeared that MED12 levels may have increased slightly when FBXL19 was overexpressed in original Figure 2—figure supplement 1G. However, we believe this is an artefact resulting from inefficient transfer on the left had side of the gel. MED12 is a large protein (250KDa) and we previously have encountered difficulties efficiently transferring MED12 to membrane for Western blot analysis. We have now optimised our transfer of large proteins and fail to see any significant or reproducible alterations in MED12 levels . MED12 is known to be regulated by the proteasome in ES cells . In agreement with this, we observe an increase in MED12 levels after MG132 treatment in both FBXL19 overexpressing cells and in the control cells transfected with an empty vector. This effect is therefore not related to FBXL19. Finally, we have also included in the revised manuscript a Western blot analysis of MED13  and again we observe no change in protein levels following FBLX19 overexpression . We conclude that FBXL19 does not regulate CDK module protein levels.7) In Figure 4, CDK8 recruitment has only been tested for the FBXL19 ZF-CxxC deletion -is this recapitulated for depletion of the whole protein? Given that Figure 4B appears to show a decrease in the levels of the deletion protein this is an important point to verify.We reasoned that conditionally removing the ZF-CxxC domain would be the most surgical way of disrupting binding of FBXL19 to CpG islands, without interfering other potential functions of FBXL19. We agree that deletion of the ZF-CxxC domain appears to cause some reduction in FBXL19 protein levels. We speculate that FBXL19 may be more rapidly degraded if not bound to chromatin (something we have observed for other ZF-CxxC domain containing proteins). Importantly, CDK8 proteins levels are unaffected in the ΔCxxC-FBXL19 cells . We have now carried out a biochemical purification of ΔCxxC-FBXL19 and shown that it still interacts with CDK8-Mediator . Given that ΔCxxC-FBXL19 does not bind to CpG islands  yet still interacts with CDK8, we conclude that reductions in CDK8 recruitment at FBXL19 targets genes is most likely explained by the loss of FBXL19 binding to CpG islands. Nevertheless, we now draw attention to the reduced protein levels of the ZF-CxxC deleted protein as follows (subsection “FBXL19 is required for appropriate CDK8 occupancy at a subset of CpG island-associated promoters”):‘Following removal of the ZF-CxxC domain of FBXL19, we observed some reductions in FBXL19 protein levels , but importantly CDK8 levels were unaffected .’8) The authors need to show that OHT treatment of the delCxxC ES line leads to reduced binding of FBXL19 in conditions used for CDK8 ChIPseq -several genes would be ok -these same genes should be used to validate CDK8 CHIPseq signal.All commercially available antibodies for FBXL19 that we have tried fail to ChIP FBXL19 and our exhaustive attempts to generate our own FBXL19-specific antibody that works for ChIP have been unsuccessful (two separate epitopes and 4 rabbits). This has precluded us from carrying out the proposed experiment. However, we do observe a uniform loss of FBXL19 binding when the ZF-CxxC domain is removed in epitope-tagged transgene experiments  suggesting that FBXL19 binding to CpG islands relies on an intact ZF-CxxC domain.9) Supplemental tables with differential gene expression results should be provided -or at least working gene lists used to reach the conclusions in the manuscript. Full GO enrichment results should be included as supplementary tables so that specific gene lists can be examined.This information is now included as Supplementary file 1.10) Various figure panels are missing statistical tests: Figure 4C needs statistical tests, e.g. KS/Wilcoxon -See Figure 4E; Figure 4G needs statistical tests; Figure 4H and 4I need boxplots and need statistical tests eg KS/Wilcoxon as for E/F;We have now included the necessary statistical tests and plots as requested by the reviewer.Figure 4H-J should include FBXL19-bound groups;ΔCXXC ES cells with FBXL19 peaks (519/783) while there is only a small overlap between FBXL19 binding and sites with increased CDK8 binding (90/379). We have now included this information in Figure 4—figure supplement 1I.As suggested by the reviewer, we divided all CDK8 peaks into those which have FBXL19 (FBXL19+) and those that do not have FBXL19 (Fbxl19-). We then examined FBXL19, BioCAP and CDK8 enrichment in these groups . As observed when we analysed all CDK8 peaks , we found that CDK8 peaks that are bound by FBXL19 have increased levels of FBXL19, CDK8, and BioCAP signal when compared to sites not bound by FBXL19. Furthermore, sites that rely on FBXL19 for CDK8 binding have broader peaks of FBXL19 enrichment. This is consistent with the fact that these are large CpG islands with broad BioCAP signal . The majority peaks that show reduction of CDK8 binding in FBXL19Figure 6B,E, F need statistical tests.We have now included the necessary statistical tests.11) It is very nice that the authors showed that genome-wide chromatin binding of FBXL19 was completely lost when the ZF-CxxC domain was deleted or mutated. To further confirm that it is indeed this ZF-CxxC domain mediating interaction with the non-methylated CpG islands, some controls are necessary. For example, in Figure 1—figure supplement 1D, the authors showed that almost all of the FBXL19 binding sites are overlapped with NMIs locations, with only 93 exceptions. Then are the signals at these 93 sites also changed in the FBXL19 mutant ChIPSeq data? When the cell DNA methylation levels are modulated, will the binding pattern of FBXL19 changed accordingly?We have now compared the enrichment of WT FBXL19 and ΔCxxC FBXL19 at these 93 sites , which cFurthermore, to illustrate the relationship between FBXL19 binding and CpG-rich non-methylated DNA, we have examined BioCAP and FBXL19 signal across all ATAC-seq peaks in the mouse ES cell genome . ATAC-seWe have not been able to examine FBXL19 binding in cells with altered DNA methylation levels as this would require recreation of a DNMT1 (or DNMT3A/B) null situation in our transgene containing cell line . However, when we have previously examined DNA methylation-deficient cells and the behaviour of the closely related KDM2A/B proteins, which have nearly identical CpG island binding patterns to FBXL19 , we demonstrated that KDM2A/B occupancy is completely dependent on recognition of CpG-rich non-methylated DNA .[Editors' note: the author responses to the re-review follow.]Essential revisions:1) In AP-MS , what is the background value of emPAI? Are there any other FBXL19-interacting proteins that show significantly high levels of emPAI in the list? How significant are the components of CDK-Mediator complex represented in all the hits?To clarify these points, we have now included a table in the supplementary information  detailing the AP-MS results. This includes the biological replicate FS2-FBXL19 purifications and matched empty vector (EV) controls. We limited analysis of our AP-MS data to proteins that were uniquely identified in the FBXL19 purifications (i.e. not the EV control). We have included a column highlighting the proteins that were identified in this screen and their associated emPAI scores in Supplementary file 1. Importantly, Mediator components feature heavily in this table, which is what prompted us to examine CDK-Mediator as a possible interaction of functional relevance. We have yet to validate whether other proteins in this table are bona fide interactors and, therefore, their relationship with FBXL19 and its function is not known.2) In Figure 3, the authors demonstrated that FBXL19 was able to recruit CDK-Mediator complex, such as CDK8 and MED12, to artificially designed chromatin regions. What about CXXC-domain or F-box deletion mutants in this assay?In these experiments we purposely used a version of FBXL19 in which the CxxC domain has a single substitution mutation (K49A) that prevents CpG island binding. We designed the experiment in this way to prevent the TetR-fusion protein from binding to CpG islands throughout the genome. This allowed us to focus our analysis on the artificial nucleation site. Although we have not tested a version of FBXL19 with its CxxC domain deleted, we believe that it would function in a very similar way to the K49A mutant as both proteins are deficient in CpG island binding and we have shown that ΔCxxC-FBXL19 retains its interaction with CDK-Mediator .In all of our artificial tethering experiments we generate clonal stable cell lines expressing the TetR-fusion proteins to ensure uniform expression within lines and comparable expression across test lines. Despite exhaustive efforts, we have been unable to generate stable cell lines expressing FBXL19 lacking the F-box. We are unsure why this is the case but speculate that FBXL19 without the F-box may be unstable or lead to a dominant negative influence that affects cell growth (negative selection). Therefore, we have not been able to examine how the F-box deletion behaves in our tethering experiments. Given that ΔF-box-FBXL19 does not interact with CDK-Mediator in transient overexpression IP experiments , we assume that CDK8 and MED12 would not be recruited to chromatin by TetR-ΔF-box-FBXL19. However, we have been unable to directly test this possibility.3) In Figure 4B, it is not very clear which band(s) are exactly indicated as full-length FBXL19 and which one(s) are CXXC deletion? Why are there double bands? Did the author use T7 antibody to detect the endogenously introduced protein?CxxCfl/flFbxl19 cells to remove the CxxC domain, we observe a reduction in mobility for these double bands (with the top band shifting to the previous position of the bottom band). We have now clearly indicated which bands represent WT and ΔCxxC FBXL19 in Figure 4B and have described this in the figure legend.We generated the T7 knock-in FBXL19 ES cell line  in order to detect FBXL19 by Western blot. We observe ‘double bands’ for FBXL19 and speculate that this may be due to post-translational modification. The closely related KDM2A and KDM2B proteins are also extensively post-translationally modified leading to doublets when examined by Western blot. Following tamoxifen treatment of 4) In Figure 4C, what proportions of each group of CDK8 binding sites overlap with FBXL19? In theory, the author expects to see that CDK8 sites showing binding intensity changes upon tamoxifen treatment would tend to overlap more with FBXL19.The overlap of each group of CDK8 peaks with FBXL19 peaks is shown in Figure 4—figure supplement 1H. While roughly 41% of all CDK8 peaks overlap with FBXL19 peaks, the overlap of down-regulated CDK8 peaks is about 66%. This is highlighted in the text in subsection “FBXL19 is required for appropriate CDK8 occupancy at a subset of CpG island-associated promoters”. On the other hand, we observed that only 23% of up-regulated CDK8 peaks overlap with FBXL19 peaks.5) In Figure 4—figure supplement 1C, please use a Venn Diagram to show directly the number of peaks of CDK8, FBXI19 and NMI overlapping with each other. This would be more straightforward.We chose to represent CDK8 enrichment at FBXL19 peaks as a heatmap in order to circumvent the limitation of peak-calling. However, as suggested by the reviewer, we have now also included a Venn diagram that shows that 89.4% of FBXL19 peaks overlap with CDK8 peaks and NMIs  and mentioned this in the text (subsection “FBXL19 is required for appropriate CDK8 occupancy at a subset of CpG island-associated promoters”).6 In Figure 5, please perform the same GSEA or functional annotation analysis on genes that are associated with unchanged CDK8 binding sites upon FBXL19 CXXC-domain deletion.We have now included the GO analysis for genes associated with unchanged CDK8 binding in Figure 5—figure supplement 1. This revealed a broad range of terms associated with basic molecular processes in line with Mediator being implicated as a key regulator of transcription. We now highlight this observation in the text in subsection “FBXL19 targets CDK8 to promoters of silent developmental genes in ES cells”.7) In Figure 6B, what is the result for genes that are associated with increased CDK8 binding signals in FBXL19 CXXC-domain deletion mutant upon RA-induced differentiation?We have compared the expression of genes associated with an increase in CDK8 binding in Figure 6—figure supplement 1E. While we observe no overall difference in the expression of these genes in ES cells, there is a slight increase upon RA-induced differentiation . This is consistent with Mediator playing a role in gene transcription.8) In Figure 6C, even though the authors claimed that those upregulated genes (889) in FBXL19 CXXC-domain deletion mutant upon RA treatment were not involved in developmental processes, it is still interesting to find that significantly more genes were activated upon CXXC-domain deletion. What explanation can the authors provide for this finding?ΔCxxCFbxl19 cells compared to wild type cells upon RA-induced differentiation and that this is potentially interesting. We have yet to find a compelling molecular explanation to link FBXL19 directly to these effects on gene expression. It remains possible that these gene expression changes are due to secondary transcriptional defects that manifest when cells attempt to execute a new expression programme in the absence of normal activation of silent, lineage commitment-associated genes when FBXL19 is not functional, or that FBXL19 plays other, as yet unidentified, roles in inhibiting gene expression. We have now added a sentence drawing attention to these points in the revised manuscript in subsection “Removing the CpG island-binding domain of FBXL19 results in a failure to induce developmental genes during ES cell differentiation”.We agree with the reviewer that a significant number of genes were more activated in the 9) In Figure 7, the authors claim that FBXL19 relies on CDK-mediator complex to activate the specific set of developmental genes. Is it possible to test this hypothesis in the FBXL19 CXXC-deletion system they established in Figure 6? In wild-type ESC, depletion of MED13 will have similar effects as FBXL19 CXXC deletion, whereas in FBXL19 mutant, the effects would be much alleviated?CxxCfl/flFbxl19 background would not lead to any further effect on gene activation. However, this would require a significant amount of time to generate and characterise a new fl/fl, Med13lfl/flMed13, and CxxCfl/flFbxl19cell line. While this is an interesting experiment, we believe that the robust and extensive series of observations in Figure 6 and Figure 7 more than adequately support our conclusion that FBXL19 target genes rely on CDK-Mediator for activation during differentiation.One could test whether inducible MED13/13L deletion in the 10) In Figure 8, why did heterozygous deletion of FBXL19 CXXC-domain show even better developmental status than the wild type?ΔCxxCFbxl19 embryos cannot be explained by such stage variations.We have not observed any significant difference between WT and heterozygous mutant embryos. It is common to have some stage variations until around 12 dpc, even within embryos of the same litter. Therefore, we often find small differences in size and developmental status among embryos regardless of genotypes. Both the WT and heterozygous mutant embryos presented in Figure 8 appear healthy with normal development for this stage. On the other hand, the defects observed in the homozygous 11) The authors demonstrated that the CXXC-domain mediates the binding of FBXL19 at NMIs, where F-Box mediates its interaction with the CDK-Mediator complex. What about CDK8 chromatin binding or gene expression patterns upon F-Box deletion of FBXL19? Will the authors see the same results as what they found in CXXC-domain deletion mutant?This is an interesting experiment and we hypothesize that disrupting the interaction between CDK-Mediator and FBXL19 via deleting the F-box domain would yield similar results to removing the CxxC domain. However, as described above in response to reviewer point 2, despite exhaustive efforts we have been unable to generate cell lines stably expressing FBXL19 with a deletion of the F-box, precluding us from addressing this point. It will be interesting in future work to see if we can engineer a cell line where we are able to remove the F-box in an inducible manner and examine the effects on CDK8 binding and induction of gene expression.


The polar DNA replication of mat1 generates an imprint on the Watson strand. The process by which the imprint is formed and maintained through the cell cycle remains unclear. To understand better the mechanism of imprint formation and stability, we characterized the recruitment of early players of mating type switching at the mat1 region. We found that the switch activating protein 1 (Sap1) is preferentially recruited inside the mat1M allele on a sequence (SS13) that enhances the imprint. The lysine specific demethylases, Lsd1/2, that control the replication fork pause at MPS1 and the formation of the imprint are specifically drafted inside of mat1, regardless of the allele. The CENP-B homolog, Abp1, is highly enriched next to mat1 but it is not required in the process. Additionally, we established the computational signature of the imprint. Using this signature, we show that both sides of the imprinted molecule are bound by Lsd1/2 and Sap1, suggesting a nucleoprotein protective structure defined as imprintosome.Genetic and molecular studies have indicated that an epigenetic imprint at Schizosaccharomyces pombe cells exist as two mating types (MTs), P (for plus) and M (for minus), that switch during cell divisions. The MT of the cell is determined by the allele (M or P) expressed at the mat1 locus on the right arm of chromosome II, 500 kb away from the centromere: mat1P in P cells and mat1M in M cells 1mat1 allele can be replaced efficiently by genetic information contained in one of the two silent donor cassettes mat2P and mat3M23mat2P and mat3M are located 17 kb and 29 kb centromere-distal to mat1, respectively 356IR-L and IR-R) and act as boundary elements 8mat2P and mat3M, CEN-H, promotes the formation of heterochromatin  on the imprinted chromatid that initiates MT switching Extensive pedigree analysis at the single-cell level demonstrated that two consecutive divisions are required to produce one switched cell among four related cousins. Fission yeast possesses a remarkable genetically programmed system initiated by a site and DNA strand-specific imprint at the mat1 is instrumental in the establishment and the strand specificity of the imprint mat1, RTS1 that blocks the fork coming in from the centromere and MPS1 located near the site of the imprint that pauses the fork coming in from the right. The pausing of the replication fork at MPS1 is a prerequisite for the formation of the imprint MPS117It was shown that the polarity of DNA replication of mat1 is either a single-strand DNA break mat1 allele and the H1 homology box 19mat1 differs by three nucleotides between the mat1P and mat1M alleles mat1M and mat1P sequences. The imprint is protected against DNA repair and remains stable throughout the entire length of the cell cycle to be transiently converted to a polar DSB during the following S-phase 17mat1P prefers mat3M, and mat1M prefers the nearby mat2P in 90% of switches. This preference is called directionality of switching 24Molecular studies have suggested that the imprint at cis-acting sequences are required for the formation and maintenance of the imprint. The SS2 and SS13 deletions are located within mat1M. SS2 is important for the pause and the imprint, whereas SS13 acts as a spacer element that allows efficient imprinting mat1M, the Msmt-0 deletion maintains efficient pausing but abolishes the imprint 181727mat1 requires SS2 28Msmt-0 deletion 3334353739SAS1, to the right of mat1Mswi2 gene (Switch gene 2) 4142 mat2/3 region Several mat1 region. Because the Lsd1/2 complex interacts with a cis-acting element located within the MT cassettes and promotes replication pause at mat1 but not at mat2P and mat3M, we compared the interactions of the four proteins in the wild-type strain and in two strains containing a deletion of both the mat2P and mat3M cassettes. Thus, we have established the computational signature of the imprint and propose a model in which Lsd1 and Lsd2 are exclusively recruited to the mat1 locus.To advance our understanding of the process of imprint formation and stability, we analyzed by chromatin-immunoprecipitation and whole genome sequencing (ChIP-seq) the recruitment of Abp1, Lsd1, Lsd2, and Sap1 proteins to the mat1 region 2735-S), and the length of the reads (30 nucleotides) prevented their unambiguous assignment to mat1, 2 or 3 cassettes mat1 by ChIP-qPCR It has been previously reported that Abp1, Lsd1/2 and Sap1 bind to the 90h strain . To achieve proper mapping, we constructed two reference genomes carrying either the M or P allele at mat1 . We found that both Lsd1 and Lsd2 are enriched at the mat1, 2 and 3 cassettes, whereas Sap1 was more enriched at the mat1M and mat3M cassettes than mat1P and mat2P . Additionally, Sap1 was found to be enriched at the IR-L and IR-R boundary elements 8CEN-H41mat1 in an 90h background. This observation prompted us to investigate the potential involvement of Abp1 in the early steps of MT switching.To extend the mapping of these proteins to the entire MT region, we performed ChIP-seq of Abp1, Lsd1/2 and Sap1 on the wild-type and switching proficient MPS1 activity. Unlike the situation observed at LTRs, Abp1 is not required for the pausing activity of MPS1 , consistent with Abp1 being dispensable for the formation and maintenance of the imprint mat1M cassette, and Abp1 control the expression of Swi2, a key player in the directionality process 42swi2 depends on Abp1  M and P strains with deleted donor sequences, we found that the enrichment of Abp1 at swi2 is restricted to M cells, while the enrichment at the neighboring peak is unaffected by the MT of the cells . The genome wide analysis showed that Abp1 M-specific interaction is unique for the swi2 gene.Using 2D gel analysis, we addressed the role of Abp1 in mat1, we used the mat1M ∆2-3 and mat1P ∆2-3 strains deleted for the donor sequences mat1 locus (to the right of the H1 cassette), and its enrichment is independent of the allele present at mat1 . Lsd1 and Lsd2 are enriched both in mat1M and mat1P with Lsd2 and Sap1 more enriched in mat1M . The enrichment positions of the four proteins and the repeated nature of the MT cassettes introduce an ambiguity that cannot be resolved during alignment. To pinpoint the Lsd1/2 and Sap1 binding sites to 90h wild type context , where the donor sequences are present . As expected, the highly repetitive sequences of IR-L and IR-R are no longer covered in the two 90h genomes (90hM and 90hP) . When the M allele is at mat1, we found no unique mapper reads at mat1 and mat3 . Similarly, when the P allele is at mat1, no unique mapper reads at mat1 and mat2 were found . Conversely, Abp1 is located outside of the mat1 cassette in a unique region and can thus be used for alignment. In the case of Lsd1/2, the enriched unique mappers are well maintained at mat1 (both in the M and P context) and strongly diminished to nearly background level atmat2P and mat3M loci, indicating that Lsd1/2 are preferentially recruited at mat1. Furthermore, the absence of Lsd1/2 at the two silent loci was confirmed using ChIP-qPCR . The situation for Sap1 is less clear, and its profile suggests that it is recruited to M but poorly to P. This hints that Sap1 is recruited both inside mat1M and to SAS1 outside of mat1.We next analyzed the reads that can only map to a unique position on the reference genome (unique mapper) in a in vitro and in vivo complementary approaches to further determine Sap1 binding sequences in the M allele. The gel shift experiments were achieved using 14 double-stranded DNA probes labeled in 5'. Two positive controls were included (SAS1 and Ter1) in addition to 10 regions mapping inside the M allele and 2 mapping outside . Using crude protein extracts, we observed the expected mobility shift for SAS1 and Ter1 as well as for 3 probes inside the M allele . We used purified Sap1 protein to confirm that it is responsible for the shift . Since M4, M5 and M6 are part of a region involved in the stability of the imprint (SS13) mat1M locus. The Msmt-0 deletion does not change the enrichment signal within mat1M, whereas the SS13 deletion strongly decreases the Sap1 signal, supporting the in vitro interaction result . The slight decrease on the right of the mat1M smt-0-containing deletion together with the remaining weak Sap1 signal in SS13 is attributed to the SAS1 interaction located 140 bp away from the imprint. The SS2 deletion abolishes DNA replication pausing and imprinting in vivo on two sequences (SS13 and SAS1) that are not necessary for replication fork pausing but required for imprint formation or maintenance, at least at the mat1M locus.We used mat1M ∆2-3  and mat1P ∆2-3 and 90h strains  using 130 nucleotide-long reads. 7 enriched regions were detected on the Watson strand . 6 such regions were also detected on the Crick strand . They delineate either repeated regions or regions exhibiting homology with the mitochondrial genome (Supplementary Table 1). The last enriched 5’ count is only found on the Watson strand and corresponds to the imprint site at mat1 . The imprint was described as either a single stranded break 21mat1 locus . The overall distribution of 5’ count is similar on the Watson and Crick strands, except for one nucleotide (in red) significantly over-represented on the Watson strand . The position in mat1M ∆2-3 and mat1P ∆2-3 corresponds to the imprinted position that is 3 nucleotides apart in M and P90h background that contains a mixture of mat1M and mat1P alleles . To confirm that the signal results from the imprint, we used the swi1∆ mutant strain that exhibits a low level of imprint. In this genetic background, no nucleotide shows any bias in 5' count .We next focused on the 10-kb region surrounding the mat1 . As expected, the window of one nucleotide at the site of the imprint has the lowest p-value on the Watson strand . On the Crick strand, the most significant p-value is contained within a window of 17 to 23 nucleotides facing the imprint . This corresponds to the molecular signature of a confined and persistent single-strand break upon chromatin sonication: A unique nucleotide on one strand and a larger region on the opposite strand .Because the bias in 5’ count is only visible on the imprinted strand, we conclude that sonication does not preferentially break the phosphate bond facing the nick. Therefore, we searched on the Crick strand for a region with a strong 5’ count statistical bias. Using a sliding window with different window sizes, we calculated the p-value in the 10-kb region surrounding mat cassettes, we circumvented the alignment problem by performing the experiment in the donor less strains. For Lsd1, the enrichment of the 5' count at the imprint on the Watson and Crick strands  at mat1M exhibits the molecular signature defined above . This result shows that Lsd1 interacts on both sides or directly with the imprinted chromatid. The additional spreading of the 5’ count on both DNA strands indicates that Lsd1 also interacts with the chromatid in the absence of imprint . A similar 5’ count distribution is observed for Lsd1 at mat1P . Lsd2 and Sap1 exhibit a similar pattern at mat1M as well, while their interaction is weaker at mat1P . Altogether, our results indicate that Lsd1, Lsd2 and Sap1 interact on both sides of the imprint but also with mat1 regardless of the imprint. Intriguingly, the interaction at mat1P is weaker for Lsd2 and Sap1.To determine whether Lsd1, Lsd2, and Sap1 interact with the imprinted chromatid and on the right or left of the imprint we used the molecular signature defined above. Figure 9A shows the expected distributions of enriched 5’ count upon immunoprecipitation. Since Lsd1/2 and Sap1 bind the repeated In this work, we propose a molecular definition of the imprint upon sonication and genomic sequencing. Our computational analysis demonstrates that a site- and strand-specific persistent imprint/nick can be unambiguously mapped. The computation of the 5’ count reveals a unique accumulation at the site of the imprint on the Watson strand, whereas the opposite Crick strand is mechanically broken by sonication within a window of 20 nucleotides, thus yielding a more diffuse accumulation of 5’ ends. This molecular signature is consistent with the free rotation of the Crick strand that generates a population of molecules with weakened A-T rich Watson-Crick pairing on both sides of the nick and makes the resulting single-strand in this window highly prone to breakage. However, we cannot rule out that the proteins preventing the imprint from being repaired contribute as well to the pattern of shearing of the Crick strand.mat1, but a function in replication fork pausing and imprinting was ruled out. The M-specific enrichment of Abp1 next to the Mc binding site located in the swi2 gene reinforces the role of Swi2 in directionality 42We also provide data for the chromosomal enrichment at the genome wide level of four proteins, Lsd1, Lsd2, Sap1, and Abp1. Abp1 is strongly interacting with the sequences near In mammals, Lsd1 plays an important role in gene expression by establishing and maintaining proper epigenetic marks. Several layers control the chromosomal targeting of Lsd1 for positive or negative transcription regulation. CoREST targets the Lsd1 complex to H3K4 In fission yeast, several pieces of evidence support the specific demethylation of H3K9 by Lsd1 at many promoters and boundary elements, but its role on H3K4 was not established 2929lsd1 is mutated mat1P and mat1M exhibit no sequence homology, a remarkable finding is that Lsd1 and Lsd2 are enriched in both alleles on sequences that are necessary to promote replication fork pauses at MPS1 on the chromosome, and sufficient on a plasmid system P and M sequences are present at the silent mat2P and mat3M loci, Lsd1 and Lsd2 are not detected , consistent with the absence of replication fork pause at these loci clr4 mutant) is not sufficient to trigger recruitment of Lsd1/2 at the silent mat2/3 loci (data not shown). We propose that the Phf1/Phf2 histone code readers participate to the targeting of Lsd1/2 at mat1 and probably at the other transcriptionally active loci The situation is more complicated at the mating type locus, where the methylation state of H3K9 is not involved in the early mating type switching steps. At this locus, the presence or absence of the sole H3K9 methyltransferase Clr4 does not rescue the mating type switching defect observed when mat1 in the absence of an imprint mat1 contains the M allele. When mat1 contains the P allele, only the SAS1 site remains, since no enrichment for Sap1 could be detected in the P sequences. This result further indicates that the processes required for replication fork pause and imprinting formation/protection are not identical in M and P cells. One possibility would be that Sap1 is either modified and not efficiently recognized during the immunoprecipitation or replaced by another protein in P cells.By using the molecular signature of the imprint, we provide strong evidence that the Lsd1/2 complex is interacting at the imprint site, prior and after imprint formation. The same conclusion was reached by showing that Lsd1/2 is working upstream of Swi1/3 and is still enriched at mat1 independently of Sap1 to promote replication fork pausing at MPS1 . Following replication fork restart and imprinting, Sap1 and Lsd1/2 form a chromatin structure, the imprintosome, that protects the imprint from being repaired . We propose that Lsd1/2 controls replication fork pausing at mat1 on both the imprinted and the non-imprinted chromatids, thus allowing the fork to restart in the same environment at each round of DNA replication and produce persistently and efficiently the imprint.The simplest model accounting for our results is that the Lsd1/2 complex is epigenetically recruited to S. pombe strains used in this study are listed in Supplemental Table S3. Standard media (YES) and genetic protocols for fission yeast were used The 32P dCTP and the blot analyzed on a PhosphoImager and quantified with the ImageQuant software (pause/ arc + pause in percentage) . Sequences of primers used to produce the probe are indicated in Table 2.2D gel analysis of replication intermediates was carried out as described previously E. coli was induced with 1 mM IPTG as previously described 32P with T4 polynucleotide Kinase (NEB) and incubated with its cold complementary sequence. 1 ng of the resulting double stranded probe was incubated with 5 µg of total protein extract or with 40 ng of purified Sap1-6His Production of Sap1-6His by 8 cells were used with one tenth the amount of reagents.ChIP-sequencing was performed as previously described mat1 and the WCE values. The list of primer sequences is in Supplemental Table S4. Experiments were performed 3-times and unpaired t-test were used to calculate the p values.qPCR was performed using a master mix (Eurobio Green qPCR Mix Lo-Rox) on the qTower Analytik Jena AG A6 machine. Each value is the mean of technical triplicates. The enrichment was obtained using the ∆∆Cq normalization method using the value of a pair of primers localized 3 kb away from Libraries were prepared using TruSeq ChIP Library Preparation kit following the manufacturer’s instructions (Illumina). Libraries are checked for concentration and quality on DNA chips with a Bioanalyser (Agilent). The libraries were quantified by fluorimetric measurements with the Qubit® dsDNA HS Assay Kit (ThermoFisher). 65-bp and 130-bp Single Read sequences were generated on the Hiseq2500 sequencer according to manufacturer’s instructions (Illumina). Library statistics are provided in Supplemental Table S5.S. pombe genome assembly ASM294v2.23 using Bowtie v2.1.0 Illumina reads were aligned to the ChIP peaks versus appropriate inputs (whole-cell extracts) were called using MACS v2.1.0 We used a script to extract the beginning of reads that map on the Watson or Crick strands. Enrichments of 5' count were obtained using the total library size. p-values were obtained using the negative binomial distribution fitting function in R.All scripts used for the analysis and outputs are available upon request.Click here for supplemental data file.http://microbialcell.com/researcharticles/molecular-signature-of-the-imprintosome-complex-at-the-mating-type-locus-in-fission-yeast/.All supplemental data for this article are also available online at


Transcription factors are key proteins in the regulation of gene transcription. An important step in this process is the opening of chromatin in order to make genomic regions available for transcription. Data on DNase I hypersensitivity has previously been used to label a subset of transcription factors as Pioneers, Settlers and Migrants to describe their potential role in this process. These labels represent an interesting hypothesis on gene regulation and possibly a useful approach for data analysis, and therefore we wanted to expand the set of labeled transcription factors to include as many known factors as possible. We have used a well-annotated dataset of 1175 transcription factors as input to supervised machine learning methods, using the subset with previously assigned labels as training set. We then used the final classifier to label the additional transcription factors according to their potential role as Pioneers, Settlers and Migrants. The full set of labeled transcription factors was used to investigate associated properties and functions of each class, including an analysis of interaction data for transcription factors based on DNA co-binding and protein-protein interactions. We also used the assigned labels to analyze a previously published set of gene lists associated with a time course experiment on cell differentiation.The analysis showed that the classification of transcription factors with respect to their potential role in chromatin opening largely was determined by how they bind to DNA. Each subclass of transcription factors was enriched for properties that seemed to characterize the subclass relative to its role in gene regulation, with very general functions for Pioneers, whereas Migrants to a larger extent were associated with specific processes. Further analysis showed that the expanded classification is a useful resource for analyzing other datasets on transcription factors with respect to their potential role in gene regulation. The analysis of transcription factor interaction data showed complementary differences between the subclasses, where transcription factors labeled as Pioneers often interact with other transcription factors through DNA co-binding, whereas Migrants to a larger extent use protein-protein interactions. The analysis of time course data on cell differentiation indicated a shift in the regulatory program associated with Pioneer-like transcription factors during differentiation.The expanded classification is an interesting resource for analyzing data on gene regulation, as illustrated here on transcription factor interaction data and data from a time course experiment. The potential regulatory function of transcription factors seems largely to be determined by how they bind DNA, but is also influenced by how they interact with each other through cooperativity and protein-protein interactions.The online version of this article (doi:10.1186/s12859-016-1349-2) contains supplementary material, which is available to authorized users. Cells recognize and respond to internal and external signals, often leading to changes in the transcription level of specific genes. Transcriptional regulatory systems play a key role in many biological processes, such as cell cycle progression, maintenance of intracellular metabolism, physiological balance, and cellular differentiation in developmental time courses , 2. The Most TFs bind to DNA by recognizing specific DNA sub-sequences known as transcription factor binding sites (TFBSs), and thereby they control the transcription of nearby genes through their promoters, or more distant genes through enhancers. However, it has been realized that binding of TFs to TFBSs is not enough to fully explain the regulatory program of gene expression . The setTFs can be classified according to regulatory function. Sherwood et al. (2014) introduced PIQ (protein interaction quantitation), a computational method for modeling the magnitude and shape of genome-wide DNase I hypersensitivity profiles used for identification of transcription factor binding sites . They idTFs can also be classified based on structural properties, and the most common classifications are based on the structure of their DNA-binding domains (DBDs) . In someIn a previous paper we presented a comprehensive list of properties for 1978 human TFs. We identified 1225 DNA binding TFs, based on existing annotation of Pfam domains and identification of additional Pfam DBDs. For the remaining 753 TFs we could not identify a clear DBD. Annotated properties included DBDs, protein–protein interactions, and post-translational modifications. The paper demonstrated how such a resource can be used to identify properties that are enriched in a subset of TFs . Howeverk Nearest Neighbours (kNN), and Gaussian naïve Bayes (GNB) classifiers. There are also several approaches for improving classifier performance, like boosting, where an ensemble of possibly weak classifiers as combined into a stronger classifier, and AdaBoost  if the TF had more than three of these zinc finger domains, [1 0] if it had between one to three of these domains, and as [0 0] if had none.In the initial functional classification on chromatin activity there are three main classes; Pioneers, Settlers, and Migrants. An enrichment analysis based on DAVID  indicateThe functional classification of additional human TFs is a multiclass classification problem, i.e. classification of patterns into more than two classes. Some classification algorithms are binary algorithms that can be adapted to multiclass classification, whereas other classification algorithms can handle more than two classes by design. There are general strategies for handling the problem of multiclass classification as a binary classification problem , and we Any data set that shows an unequal distribution between its classes can be considered as imbalanced. Studies have shown that for several base classifiers, a balanced data set improves the overall classification performance, compared to an imbalanced data set , 28. UsiIn this paper we used random under-sampling on training data, without replacement . Specifik Nearest Neighbors (kNN) and Gaussian Naïve Bayes (GNB) were evaluated, using the one-vs-rest strategy described above. The kNN methods need a specification of the number of neighbors, and the SVC requires parameterization of the complexity constant C and the kernel function. The number of neighbors for kNN was limited to the set {3, 5, 7, 9} [i, xj) = exp(−γ(xi − xj)2) where γ is the width of the RBF function, and polynomial K = (xi. xj)d where d is the degree. A grid search was performed to optimize the parameters of support vector machine (SVM) classifiers. For the RBF kernel, C = {2− 4, 2− 3, …, 23, 24} and γ = {2− 4, 2− 3, …, 23, 24} and for polynomial kernel C = {2− 4, 2− 3, …, 23, 24} and d = {2, 3} .Several classifiers including Random Forest (RF), Support Vector Classification (SVC) with different kernels , polynomial), 5, 7, 9} . The kNNAll machine learning methods were implemented using scikit-learn , and allWe used several common measures to evaluate the performance of the classifiers, including precision , recall (sensitivity or SN), F-score, MCC (Matthews’s correlation coefficient), and AUC (area under curve for receiver operating characteristics (ROC)) –35. The The performance of classifiers was tested using five-fold bootstrap cross-validation with ten runs (10 × 5) on all possible rebalanced TF subsets on regulatory function, and for each individual property . In eacht-test and the Wilcoxon test [t-test is a parametric one and requires that the necessary conditions for using it are true, i.e. independence, normality, and heteroscedasticity. This is not the case in the majority of experiments in machine learning [The most frequently used statistical tests to determine significant differences between two machine learning algorithms are the xon test . The t-tlearning . Thus, wlearning .After identification of the locally best classifier we evaluated the performance on each individual property using the same process as above . Again the performance measures were computed for each fold and then averaged on runs for the four classification cases separately.The final set of properties was selected using a forward best-first search on the list of properties for the four classification cases separately. We executed the runs of each cross-validation and used the average AUC to rank the properties. We started with the property giving the largest AUC, and at each step added the property (among the remaining properties) which results in the best average AUC . We alsoWe performed three analyses to illustrate how the predicted regulatory function can be used; enrichment analysis on the subsets of TFs according to regulatory function, co-localization of TFs, and function of TFs involved in a time course experiment.Subsets of TFs were analyzed for enriched properties using DAVID  and GOrip < 0.05) enrichment (or depletion) for TF-TF interactions within and between the different TF classes, using a 2 × 2 matrix for statistical analysis . Estimation of p-values on the 2 × 2 matrices was done using the fisher.test in R. Further, we used results from the ENCODE Consortium [We used data on TF-TF interactions from four different sources. Jolma et al.  used SELnsortium , where tnsortium  release nsortium , where tnsortium . Data onnsortium .p < 0.05) to functional classes. Enrichment analysis was done with a Fisher exact test, asking whether a given property was significantly enriched (or depleted) in a given set of TFs, compared to the full set of annotated TFs. The p-values were corrected for multiple testing using the Benjamini correction.Time course data were made available by the FANTOM Consortium , 45, andWe defined a training set of 457 TFs with known chromatin activity (regulatory function) according to Sherwood et al.  and a clWe first tested the performance of several classifiers using the full set of properties and the one-vs-rest strategy see  in orderWe then tested each individual property for classification by estimating the AUC score and the feature importance score, using the RF classifier. The results for the individual properties in the four classification cases are shown in Fig. p-values  and 2.3.4 (…/…/Factors with multiple dispersed zinc fingers), where the training set contains only 3.7 and 4.9% of the TFs from each class, respectively. Still the classification seems to be quite robust, in particular for 2.3.3 where the average p-value is 0.86 and margin is 0.21. This means that the classification presented here provides an important extension of the initial functional classification.Several interesting observations can be made from the classification results. It is clear that the original dataset by Sherwood et al. is highly biased, in particular with respect to TFClasses 2.3.3 (Other factors with up to three adjacent zinc fingers), with 0 cases in the training set, where 15 cases in the full set still could be classified as Pioneers (12) or positive Migrants (3) with reasonable performance . A similar example is TFClass 3.7 (Helix-turn-helix domains/ARID domain factors). However, the number of TFs in classes with no cases in the training set is very low , and in general there is a good distribution of cases in the training set. This is reflected in the classification result as several cases of high average p-values. An example is TFClass 3.5 (…/Tryptophan cluster factors), with an average p-value of 0.94 and a margin of 0.26. This indicates a quite reliable classification.The results show clearly that the regulatory function of a TF to a large extent is determined by how it binds DNA, as the main properties for successful classification are TFClass and Pfam domains. It is also clear that although TFClass is very important for the classification, it is not sufficient by itself, as other properties, in particular Pfam, provide complementary information. This is seen for example in TFClass 2.3.2  .Binding of several TFs to a regulatory region is an important process for gene activation. Interactions between TFs can take place either because the TFs tend to bind to neighboring binding sites in DNA, or because they tend to interact through protein-protein interactions, or both, and it seems likely that the relative importance of this may differ with regulatory function. We therefore used data from two different sources (SELEX and ChIP-seq) on TF-TF interaction through co-localization to DNA, and on TF-TF interaction through PPI, also from two different sources , and the overall trend is also quite clear; TF co-localization and PPI are to some extent mutually exclusive features. Whereas Pioneers and Settlers tend to be enriched for TF-TF-DNA interactions and depleted for PPI, the Migrants (and in particular positive Migrants) are enriched for PPI and depleted for TF-TF-DNA. This is consistent with what we see at a more detailed level. In the data on TF-TF-DNA interactions we see for example a clear depletion of interactions between Pioneers like ETV2 and ETV5 and positive Migrants , whereas the Settler MAX is enriched for interaction with other Settlers (log ratio 1.00). The positive Migrant TEAD4, which is known to bind non-cooperatively , is deplIn order to explain our observations we looked at three properties that may influence TF-binding to DNA; the number of DNA-binding domains in the TF, the GC-content of the binding site, and the Information Content (IC) of the binding site motif. In all three cases we split the set of TFs into two, based on a suitable cutoff value for the relevant property, and tested each functional class of TFs for enrichment or depletion. The results are shown in Table For the number of TFs with more than one DBD, we saw a very significant enrichment in Pioneers, and a clear depletion in Settlers and negative Migrants, which means that Pioneers often will have a very strong and specific binding, compared to the other TFs. With respect to GC content, the negative Migrants were strongly depleted for binding sites with high GC content, whereas the other TFs were enriched. The general enrichment is consistent with previous results  showing The results suggest that the chromatin opening index of Sherwood et al. is associated with the number of DBDs and possibly IC of the motif, whereas the chromatin dependence is associated with GC content of the binding sites. The enrichment for multiple DBDs in Pioneers and the corresponding depletion in Settlers and in particular negative migrants coincides mainly with the axis for chromatin opening index in Fig. The results from this analysis can be summarized as a scenario for how TFs may cooperate to enable gene expression. As a first step activating Pioneers can bind strongly and with high specificity (due to multiple DBDs with high IC) to nucleosome-bound regions (due to high GC) to initiate chromatin remodeling, leading to at least partly open chromatin. This is followed by Settlers, which are quite similar to Pioneers, but with fewer DBDs. They are therefore less likely to compete directly with nucleosomes for binding, at least at an individual level, but can stabilize open chromatin through cooperative binding. This process may be supported by the positive Migrants, which are somewhat similar to Settlers, although they have on average more DBDs but with lower IC, and they are more likely to interact through PPIs. This may give more general (less specific) binding in regions of open chromatin, which may contribute to stabilizing these regions in an open state. Finally, the negative Migrants have few DBDs and low GC content, which means that they can bind to more AT-rich regions as found in for example linkers , and thep < 0.05) were assigned to classes of regulatory function. The number of TFs showing significant changes in expression level at each time point are shown in Table S10  which recruits histone deacetylases and chromatin remodeling complexes to chromatin , maintaiAs already indicated, most of the observed changes are linked to down-regulation. For example, down-regulated Settlers are enriched in ubiquitination, a post-translational modification that may target proteins for degradation, possibly leading to a more rapid and efficient down-regulation of TFs than by changing the transcript level alone. For up-regulated positive Migrants there is enrichment for PPIs, supporting the observation above regarding PPIs and positive Migrants. This is possibly linked to stabilization of protein complexes involved in regulation of transcription.A couple of Pfam domains (zf-H2C2_2 and HLH) are enriched in both up-regulated and down-regulated sets. The zinc-finger domain zf-H2C2, which is often involved in sequence-specific targeting of other domains, including KRAB , is stroWe also did the same analysis over all TFs, independent of functional classification Table . This idThe results described above seem to support a general picture of these TFs that is consistent with their assumed roles. The Pioneers are rapidly regulated to modify the transcriptional program, mainly by removing repressing TFs and up-regulating activating TFs that bind in a sequence-specific manner. This process is supported by the regulation of Settlers, many of which are rapidly degraded and removed, possibly to close up the regulatory regions that are being de-activated. The up-regulated Migrants are enriched for protein-protein interactions, which may support the formation of clusters of TFs in open regulatory regions.Data on properties of transcription factors has been used as input for supervised machine learning in order to expand an experimental classification of transcription factors into functional classes associated with chromatin opening, as Pioneers, Settlers, positive and negative Migrants. The expanded classification is a useful resource for analyzing other data. Here is has been used to analyze transcription factor interaction data and data from a time course experiment. The results are consistent with the expected roles of the functional TF classes, in particular the role of Pioneers in initiating changes in gene regulation.