Rhodobacter sphaeroides harbors a large operon for iron-sulfur cluster assembly comprising the iscRS and suf genes. IscR (iron-sulfur cluster regulator) is an iron-dependent regulator of isc-suf genes and other genes with a role in iron metabolism. We applied reporter gene fusions to identify promoters of the isc-suf operon and studied their activity alone or in combination under different conditions. Gel-retardation assays showed the binding of regulatory proteins to individual promoters. Our results demonstrated that several promoters in a sense and antisense direction influenced isc-suf expression and the binding of the IscR, Irr, and OxyR regulatory proteins to individual promoters. These findings demonstrated a complex regulatory network of several promoters and regulatory proteins that helped to adjust iron-sulfur cluster assembly to changing conditions in Rhodobacter sphaeroides.A multitude of biological functions relies on iron-sulfur clusters. The formation of photosynthetic complexes goes along with an additional demand for iron-sulfur clusters for bacteriochlorophyll synthesis and photosynthetic electron transport. However, photooxidative stress leads to the destruction of iron-sulfur clusters, and the released iron promotes the formation of further reactive oxygen species. A balanced regulation of iron-sulfur cluster synthesis is required to guarantee the supply of this cofactor, on the one hand, but also to limit stress, on the other hand. The phototrophic alpha-proteobacterium  This repression is released under conditions unfavorable for Fe-S maturation of IscR [suf operon expression [E. coli. Induction of the isc and suf operons by apo-IscR occurs under iron-limiting conditions and oxidative stress [ferric uptake regulator) [iscS, leading to the degradation of the 3’ part of the iscRSUA-hscBA-fdx operon mRNA. The 5´ part of the polycistronic mRNA, which contains iscR, is stabilized and translated [suf expression.Fe-S cluster biogenesis systems have to respond to environmental stimuli to maintain and repair the pool of Fe-S proteins under changing environmental conditions. The current understanding of the regulation of genes for Fe-S cluster assembly is mostly limited to the model organism pression ,10,12. Tc system ,14. In Eunctions . Holo-Is of IscR . Thus, Ipression  in E. coe stress ,10,11. Agulator) . RhyB baanslated . This faisc and suf operons by stress signals also involves the regulators Fur, OxyR, and RhyB. The iron-sensing regulator protein Fur binds to the suf promoter as Fur-Fe2+ represses the suf genes [E. coli and many other bacteria, also acts as an activator of the suf operon [2O2) was shown to inactivate the E. coli Isc system and to activate the suf operon through OxyR [Regulation of the uf genes . OxyR, af operon . It was f operon . Hydrogeugh OxyR .1 complex for photosynthetic electron transport. On the other hand, photooxidative stress caused by the formation of ROS by the light excitation of bacteriochlorophyll destroys Fe-S clusters, which in turn leads to a further increase of ROS levels by the Fenton reaction. Latifi and co-workers suggested that elevated levels of ROS upon iron starvation [R. sphaeroides [E. coli.Phototrophic bacteria have a special need for the regulation of the Fe-S cluster assembly. Fe-S clusters are required for some components involved in photosynthesis like enzymes for bacteriochlorophyll synthesis (magnesium chelatase and the dark-operative protochlorophyllide oxidoreductase) or the cytochrome bcarvation , as alsoaeroides , might bR. sphaeroides is a facultative photosynthetic bacterium, which can use a variety of metabolic pathways for ATP production. At high oxygen tension, aerobic respiration generates ATP. When the oxygen tension in the environment drops, the synthesis of photosynthetic complexes is induced, while aerobic respiration still takes place. Under anaerobic conditions in the light, anoxygenic photosynthesis generates ATP, while under dark conditions in the presence of a suitable electron acceptor, anaerobic respiration is performed. Oxygen is a major regulatory factor for the formation of photosynthetic complexes, and several proteins involved in oxygen-mediated gene regulation have been identified [R. sphaeroides loses its purple color due to the loss of photosynthetic complexes and is no longer able to grow phototrophically [entified ,22. Undephically . Since bR. sphaeroides serves as a model organism to elucidate the response of photosynthetic bacteria to singlet oxygen [R. sphaeroides is involved in both iron and manganese homeostasis [iron response regulator) protein was shown to affect genes of the iron metabolism [isc/suf genes under iron limitation [R. sphaeroides showed different responses under oxic or anoxic conditions. For some of these genes, including isc-suf genes, induction under iron limitation under oxic conditions was mediated by the OxyR protein [R. sphaeroides harbors a large operon comprising isc and suf genes for iron-sulfur cluster assembly. Like in E. coli, the product of the first gene, IscR, functions as an iron-dependent repressor of the isc genes [isc-suf operon expression to changing environmental conditions, we identified promoters responsible for isc-suf expression and quantified their activities under different oxygen concentrations in response to iron limitation and oxidative stress. This study resulted in a complex model for isc-suf gene regulation in a phototrophic alpha-proteobacterium.t oxygen ,27,28,29t oxygen ,23,30,31 protein . R. sphaE. coli strains were cultivated in Standard I medium at 37 °C, either in liquid culture by shaking at 180 rpm or on a solid growth medium, which contained 1.6% (w/v) agar. Depending on the cultivated strain, the antibiotics kanamycin (25 μg mL−1), ampicillin (200 µg mL−1), or tetracycline (20 μg mL−1) were added to the liquid and solid growth media.Bacterial strains are listed in R. sphaeroides strains were cultivated in 50 mL Erlenmeyer flasks containing 40 mL malate minimal medium [660 of 0.4–0.6. Antibiotics were added to the liquid and solid growth media depending on the cultivated strain at the following concentrations: spectinomycin (10 μg mL−1), kanamycin (25 μg mL−1), gentamicin (25 μg mL−1), tetracycline (2 μg mL−1). For generating oxidative stress, the cultures of R. sphaeroides wild type or mutants were grown in iron-repleted malate minimal medium and treated with 1 mM  hydrogen peroxide or 100 μM  tertiary butyl-alcohol (tBOOH) for 7 min or 30 min at an OD660 of 0.5. After 0, 7, and 30 min, cells were harvested and used for ß-galactosidase measurements.l medium  with conl medium ,23 and cl medium ,23. CellR. sphaeroides strain 2.4.1ΔRSP_3341 was generated by transferring the suicide plasmid pPHU2.4.1ΔRSP_3341::Sp (RirA homolog 1) into R. sphaeroides 2.4.1, and screening for the insertion of the spectinomycin resistance cassette into the chromosome by homologous recombination. Parts of the rirA gene (RSP_3341) of R. sphaeroides, together with upstream and downstream sequences, were amplified by using oligonucleotides 3341up_f/3341up_r and 3341dn_f/3341dn_r. The amplified PCR fragments were cloned into the KpnI-BamHI and BamHI-HindIII sites of the suicide plasmid pPHU281 [ΔRSP_3341. A 2.2 kb BamHI fragment containing the spectinomycin cassette from pHP45Ω [BamHI site of pPHU2.4.1ΔRSP_3341 to generate pPHU2.4.1ΔRSP_3341::Sp. This plasmid was transferred into E. coli strain S17-1 and diparentally conjugated into R. sphaeroides 2.4.1 wild-type strain. Conjugants were selected on malate minimal salt agar plates containing spectinomycin. By insertion of the spectinomycin cassette, 438 bp of R. sphaeroides rirA gene RSP_3341 was deleted. R. sphaeroides strain 2.4.1ΔRSP_2888 was generated by transferring the suicide plasmid pPHU2.4.1ΔRSP_2888::Km (RirA homolog 2) into R. sphaeroides 2.4.1, and screening for insertion of the kanamycin resistance cassette into the chromosome by homologous recombination. Parts of the rirA gene (RSP_2888) of R. sphaeroides, together with upstream and downstream sequences, were amplified by using oligonucleotides 2888up_f/2888up_r and 2888dn_f/2888dn_r. The amplified PCR fragments were cloned into the KpnI-BamHI and BamHI-HindIII sites of the suicide plasmid pPHU281 [ΔRSP_2888. A 2.2 kb BamHI fragment containing the kanamycin cassette from pHP45Ω [BamHI site of pPHU2.4.1ΔRSP_2888 to generate pPHU2.4.1ΔRSP_2888::Sp. This plasmid was transferred into E. coli strain S17-1 and diparentally conjugated into R. sphaeroides 2.4.1 wild-type strain. The conjugants were selected on malate minimal salt agar plates containing kanamycin. pPHU281 , generat pPHU281 , generatisc-suf operon, respectively, were amplified by PCR with primers listed in E. coli JM109. After confirming the correct sequence, the promoter fragment was cut from the sequenced cloning vector by XbaI/PstI or SpeI/Crf9 and subsequently ligated into the transcriptional lacZ fusion vector pBBR1-MCS3-LacZ [XbaI/PstI sites of the transcriptional lacZ fusion vector pBBR1-MCS5-LacZ, which harbors a gentamicin cassette and can be transferred into R. sphaeroides together with plasmid pRK4352 [According to the dRNA-seq data, fragments with lengths ranging from 98 bp to 1777 bp containing one of the putative five different single promoters or combined promoters of the CS3-LacZ . For tesisc-suf operon were constructed by inverse PCR. All primers used are listed in DpnI and then transferred into JM109. Subsequently, the mutated promoter fragments were cut from the cloning vectors with XbaI/PstI and ligated into pBBR-MCS3-LacZ [Mutations in promoters of the CS3-LacZ .Overexpression of RNA antisense to promoter P2 and sense promoter 2 fusion plasmids.E. coli JM109. After sequencing, the cloned fragment was cut out by XbaI-BamHI and subsequently ligated into vector pRK4352, which contains the strong 16S promoter [To test whether the antisense mRNA affects the activity of sense promoter, a DNA fragment antisense to P2 was amplified by PCR with primers asP2_fwd/rev listed in The ß-galactosidase activity of transcriptional fusions was measured by the hydrolysis of O-nitrophenyl- ß-D-galactopyranoside (ONPG)  and expressed as Miller Units, as described in .RNA isolation, TEX (Terminator EXonuclease) treatment, library construction and sequencing, read mapping, and transcription start site prediction have been described in detail in . The RNAR. sphaeroides cells were harvested by centrifugation when an OD660 of 0.5 was reached. Total RNA for quantitative RT-PCR was isolated by using the peqGOLDTriFast kit , as described by the manufacturer. Remaining traces of DNA were removed by the TURBO DNaseI . To further confirm the absence of DNA, PCR was performed targeting gloB (RSP_0799) with the primers listed in rpoZ encoding the ω-subunit of RNA polymerase of R. sphaeroides was used to normalize the mRNA expression levels [Twenty milliliters of n levels  accordinn levels . PrimersThe synteny analysis was performed by applying the EDGAR software .Purification of the IscR, Irr, and OxyR proteins and electrophoretic mobility shift assays with dsDNA and protein.E. coli M15 (pREP4/pQE2.4.1oxyR) was used for the overexpression of His-tagged OxyR protein, and the purification of OxyR was performed, as described earlier [E. coli M15 (pREP4/pQE2.4.1irr) is described in [E. coli M15 (pREP4/pQE2.4.1iscR) in [isc-suf operon was determined by an electrophoretic mobility shift assay, as described previously [isc-suf operon were produced by PCR. Primers used in the PCR are listed in 32PATP labeled DNA fragment , salmon sperm DNA (1 μg), and binding buffer, as described elsewhere [w/v) polyacrylamide gel in 0.5×TBE buffer , and the electrophoresis was performed at 130–180 V for 2–5 h at room temperature or at 4 °C. Irr and IscR were isolated and tested under non-reducing conditions. OxyR was purified and tested under reducing and non-reducing conditions with the same outcome. earlier . Isolatiribed in , and theiscR) in . Bindingeviously ,38. DNA lsewhere ,42. AfteR. sphaeroides harbored a cluster of genes, with isc and suf homologs  and aerobic anoxygenic phototrophs (AAPs). There was some variation in regard to the non-annotated open reading frames. The non-phototrophs Ruegeria and Paracoccus also showed a similar synteny of isc and suf genes, and the gene for the IscR homolog of Paracoccus denitrificans was, however, positioned at a different chromosomal locus. A similar synteny of iscS and suf genes was also found in Rhizobiales  as in R. sphaeroides.homologs . RNA-seqn scheme . The othteraceae . The synzobiales ; howeverisc-suf expression in R. sphaeroides [iscR and suf genes suggested that not all isc-suf genes were under the control of a single promoter and the identical regulatory elements.Previous studies addressed the role of proteins with established roles in iron-dependent regulation in aeroides ,31,36,37R. sphaeroides promoters share little sequence identity [iscR. Another promoter (P3) was predicted within iscS and initiated transcripts spanning the suf genes. Furthermore, two promoters on the opposite DNA strand might lead to transcripts that were partially antisense to isc-suf transcripts. P4 was represented by a very low number of reads and led to a transcript mostly antisense to the iscR mRNA. P5 represented the promoter for transcription of RSP_0444. In previous studies, RSP_0444 showed only small differences in expression in the various strains or response to iron-limitation or H2O2 [isc-suf genes initiating at P1 or P2. The dRNA-seq data did not hint to further promoters for isc-suf transcription [Most identity , which midentity . This anidentity . While T or H2O2 ,31,36,37R. sphaeroides have a TTG around position –35 and an A at position –10/–11 relative to the TSS [iscR gene [iscR gene was experimentally verified previously [iscS (P3), an A residue at position –10 and a TTG around position –34 were present. Putative promoter P4, which seemed to be very weak based on the read number detected in RNA-seq, had a TTG around position –35, and the same was true for the putative promoter P5.Many promoters in  the TSS . For thescR gene . The preeviously . For thelacZ gene. Fragments containing 60–148 nt upstream and 34–63 nt downstream of the TSS, as determined by dRNA-seq, were cloned in front of the lacZ gene of plasmid pBBR1-MCS3-lacZ [R. sphaeroides 2.4.1 wild type by conjugation, and the ß-galactosidase activity was determined for exponentially growing cultures. Since the oxygen levels influence the formation of photosynthetic complexes and consequently the demand for Fe-S clusters in R. sphaeroides, the cultures were either incubated under aerobic or microaerobic conditions. Under the latter conditions, the formation of photosynthetic complexes was strongly increased. The activity determined for the different promoter fusions showed marked differences . Likewise, the activity of P5 was dependent on the length of the upstream region: a shorter upstream region (88 nt) resulted in higher promoter activity than a longer upstream region (112 nt). We describe below that the OxyR protein binds to the P5 upstream region and represses P5 activity. Only for promoter P3, the activity was significantly (increase >1.5-fold and p < 0.01) but only slightly higher (slightly more than 1.5-fold) in aerobic conditions compared to microaerobic growth.In order to confirm the activity of the predicted promoters, we constructed transcriptional fusions to the ferences . While Pisc-suf operon transcription and that the antisense transcripts initiating at P4 and P5 may also influence isc-suf operon transcription. To test this hypothesis, we applied the same primers as used for the single promoter fusions to also construct reporter plasmids that harbor combinations of the different promoters . The additional presence of the strong P4 promoter led to a strong increase of the activity compared to the P25-lacZ fusion, when 98 nt long upstream region was present (P254–98) but not with only 60 nt of the upstream region (P254–60).Considering the arrangement of the different promoters on the chromosome, it is conceivable that both P1, P2, as well as P3, contribute to romoters . Our resromoters . When P1lacZ (P12543), the ß-galactosidase level was lower than for P254 and similar to P12 under microaerobic conditions  activity, we used different sized downstream fragments of P2 fused to lacZ. As shown in lacZ might contain other elements, which could affect the lacZ transcript level. For determining the influence of P5, the P2 downstream DNA fragment fused to lacZ was extended by only 80 nt (P25–88) or by 104 nt (P25–112). For testing a putative additional effect by P4, the P25–112 fragment was extended by 237 nt (P254–60) or by 275 nt (P254–98)  on P2 (main promoter for P254–98) . To veriP254–98) A. There  plasmid B. As shoWe applied the same strategy to test the effect of P4 on P2 activity. However, changing the TTG at the putative –35 of P4 only slightly decreased ß-galactosidase activity in the P254–98 construct. Consequently, the resulting construct mutP254–98 would still increase the activity of the P25 fusion D. As alrisc-suf genes is affected by oxidative stress [R. sphaeroides faces in the presence of pigments, oxygen, and light. None of the promoters P1, P2, P3, P4, P5, or any combination of promoters showed marked changes in activity upon addition of hydrogen peroxide  with similarity to the consensus Irr-box  was present about 50 nt upstream of the TSS of P3, which is a similar distance to the TSS as observed for the confirmed Irr target ccpA [P3 activity was affected by IscR, both under iron repletion and iron depletion C. Lack oget ccpA . A gel rDifferences in activity between wild type and the tested mutants for antisense promoter P4 were small (≤1.5-fold) and/or statistically not significant for the reporter construct with 98 nt upstream of the TSS D and alsThe activity of antisense promoter P5 was strongly affected by the OxyR protein when 112 nt upstream of the TSS was present. The activity in the mutant was increased by a factor of 3-4 under both iron repletion and iron depletion E. OxyR discR gene and may, therefore, increase IscR levels in the cell, it could not be tested in conditions lacking IscR. The repressing effect of Irr under iron depletion was more pronounced when all promoters were present than in the P3-fusion alone . We constructed knock out strains of R. sphaeroides lacking either RSP_2888 or RSP_3341 or both genes together. As seen in isc-suf operon showed very similar activities in the wild type and the double mutant, and also the effect of iron depletion on promoter activity was very similar in both strains. We concluded that the RirA homologs of R. sphaeroides had no major role in the iron-dependent regulation of the isc-suf operon. The double mutant showed identical growth curves as the wild type in iron repletion and iron depletion  might guarantee the high expression of the proteins required for the assembly of iron-sulfur clusters. IscR has a regulatory function and may not be required in a high amount. IscS is a cysteine desulfurase that is required for mobilization of sulfur from L-cysteine. A higher expression of iscS might not be required, since, with SufS, another cysteine desulfurase was encoded by the isc-suf operon downstream of P3. This arrangement with two cysteine desulfurases was conserved among Rhodobacteraceae and also found in Rhizobiaceae  shows a higher affinity to Type 1 sites than apo-IscR, while both IscR forms bind with similar affinity to Type 2 sites [Thermincola potens [R. sphaeroides.P2 was the main promoter for transcription of  2 sites . Interesa potens . Our in iscR, we could not exclude that elevated levels of IscR influence activity of the P3 promoter in this strain. Higher IscR levels could increase the effect of iron in the irr deletion strain (compare 5C to 5F). The P3 promoter was the only promoter that showed a significant but small effect in the mutant lacking Fur/Mur, indicating an activating function of Fur/Mur under iron depletion. Johnston and co-workers suggested that iron-dependent regulation in alpha-proteobacteria mainly occurs by regulators different from Fur [Rhizobia and the Fur homolog of R. sphaeroides were shown to affect the expression of the sit operon in an Mn2+-dependent manner and were consequently named Mur [fur/mur gene resulted in stronger expression of many iron-dependent genes in R. sphaeroides [isc-suf operon, while an almost perfect Mur box is located between the –10 and –35 region of the sitA promoter. Fur/Mur is required for a strong induction (about 50-fold) of sitA expression in response to Mn2+ limitation [isc-suf operon, does not include direct binding but is rather mediated indirectly.Despite the presence of an Irr box around the P2 promoter, we detected only a small effect of Irr on P2 (1.8-fold) and did not detect binding of Irr to the P2 region in vitro. A small effect of Irr on P1 activity (1.5-fold) was observed, but no binding of Irr to the promoter region in vitro was observed. Our results implied that Irr did not make a major contribution to P1 and P2 regulation under the tested conditions and suggested that the influence of Irr might be indirect. There was also an influence of Irr on P3 that was even more pronounced when all upstream promoters were present, and binding of Irr to P3 was demonstrated. Since the P12543 reporter also carries a complete copy of from Fur . Some Fuamed Mur ,56,57,58aeroides , suggestmitation . It is lisc and suf genes was reported previously [suf genes, which might also be indirect. Since P5 is the promoter for RSP_0444, OxyR could also affect the expression of RSP_0444, but previous microarray studies revealed hydrogen peroxide- and OxyR-independent expression of RSP_0444 [R. sphaeroides [While none of the sense promoters was influenced by the OxyR protein, antisense promoter P5 was strongly repressed by this regulator, under iron-replete and iron deplete conditions, and binding of OxyR to the P5 upstream region was confirmed. The repressing effect of OxyR on the P5 promoter required 112 nt of the upstream region and was not present with only 88 nt of the upstream region E. An effeviously ,41,45. Sisc-suf expression and also indicates the influence of the P1, P5, and the P4 promoter region on the activity of P2, which is the main promoter for iscRS expression but also regulates other genes of iron metabolism. Our study revealed IscR as the main regulator for iron- and tBOOH-dependent expression of the isc-suf operon. IscR (under iron repletion and iron depletion) and Irr (under iron depletion) also repressed P3 activity by direct binding. The presence of the upstream region, including P1, allowed higher transcription rates of isc-suf than P2 alone, and both antisense promoters stimulated P2 activity. The influence of Fur/Mur was observed for P3, but the effects were small and most likely indirect. Thus, multiple promoters and multiple regulators were involved in adjusting isc-suf expression to environmental conditions, and by regulating iscR expression indirectly, other genes of the iron metabolism that were controlled by IscR were also affected.


Unlike promoters, enhancers are not necessarily adjacent to target genes and can exert their functions regardless of enhancer orientations, positions and spatial segregations from target genes. Thus, for a long time, the question as to how enhancers act in a temporal and spatial manner attracted considerable attention. The recent discovery that enhancers are also abundantly transcribed raises interesting questions about the exact roles of enhancer RNA (eRNA) in gene regulation. In this review, we highlight the process of enhancer transcription and the diverse features of eRNA. We review eRNA functions, which include enhancer-promoter looping, chromatin modifying, and transcription regulating. As eRNA are transcribed from active enhancers, they exhibit tissue and lineage specificity, and serve as markers of cell state and function. Finally, we discuss the unique relationship between eRNA and super enhancers in phase separation wherein eRNA may contribute significantly to cell fate decisions.Enhancers are  Enhancers are short regulatory elements of accessible DNA that help establish the transcriptional program of cells by increasing transcription of target genes. They are bound by transcription factors, co-regulators, and RNA polymerase II (RNAP II). Enhancers are flanked by histones with permissive chromatin markers, including H3K4 methylation (H3K4me) and H3K27 acetylation H3K27ac). While promoters direct gene transcription in a position- and orientation-dependent manner, enhancers traditionally function independently of their position and orientation with respect to their target gene, as they can loop over long genomic ranges to engage distant promoters . DespiteA surprising discovery regarding enhancers was made in 2010, when it was shown that enhancer regions are actively transcribed . The proWhile enhancers are critical to all cell types, eRNA abundance varies dramatically across tissues. The FANTOM5 project identified immune cells, neural tissues, and hepatocytes among those with the highest abundance of cell-specific enhancers, a higher ratio of enhancers/gene, and high enhancer transcription . Indeed,In this review, we outline current understanding of eRNA production, structure, and classification, and discuss recent findings concerning eRNAs in gene regulation. Finally, we discuss super-enhancers, their relationship with eRNA, and the potential roles of eRNA in super-enhancer formation and structure, as well as their roles in phase separation and cell fate decisions.cis loci  recruitment of transcription factors and coactivators necessary for enhancer formation and gene activation, (2) histone modifications at the active enhancer loci, and (3) eRNA transcription, elongation, and processing .de novo nucleosome remodeling precedes enhancer activation and eRNA transcription  among enhancer-derived long intergenic non-coding RNAs (e-lincRNAs), which plays roles in RNA structure, stability, and processing  RNA-pulldown, RNA immunoprecipitation (RIP), and RIP-qPCR approaches, that eRNAs interacted with the RAD21 and SMC3 subunits of the cohesin complex. Knockdown of eRNAs by siRNA or locked nucleic acid (LNA) resulted in decreased recruitment of cohesin to those enhancers as determined by RAD21 ChIP, thus providing one of the early demonstrations of a functional role for eRNA. A later study found that RNA is able to stabilize some enhancer-promoter loops. RNAse H1 digestion of RNA prevents RAD21 association with chromatin, thus implicating RNA-RAD21 interactions in formation and stabilization of promoter-enhancer loops  in vertebral interneurons. 4C-seq and ChIP-seq (cohesin subunits SMC1 and SMC3) analyses between Evf2+ and Evf2− knockout mouse ganglionic eminences demonstrated that Evf2 regulates cohesin positioning to direct long-range UCE interactions  region that helps regulate enhancer interactions in ractions . Additiontiation .Several subsequent studies have shown decreases in promoter-enhancer chromatin looping with knockdown of the respective eRNA . In addiIn contrast, other studies have found that enhancer-promoter architecture was maintained in spite of eRNA knockdown or treatment with the RNAP II inhibitor flavopiridol to prevent eRNA transcription . In anotMost eRNAs are chromatin-associated, as opposed to free in the nucleoplasm or the cytoplasm . It may The CBP and p300 histone acetyltransferases have been shown to interact with eRNAs , and eRNEnhancer RNA not only increases H3K27ac but also decreases transcriptionally repressive H3K27me3 levels . This main vivo in rat amygdala as LNA knockdown of Arc eRNA lead to decreased Arc expression and increased NELF-E occupancy at the Arc promoter . A line is drawn from the highest ranked enhancer to the lowest (B). That line is then slid along the curve until it is tangential to the curve (C). All regions ranked above this point are considered super enhancers, while those below are defined as typical enhancers (D).Super enhancers are bioinformatically defined by having an exceptionally high concentration of enhancer markers such as cofactors, Mediator, and H3K27ac . TraditiSequencing of nascent RNA can be used to identify and discriminate between typical and super enhancers, similar to how ChIP-seq data has traditionally been used to identify typical and super enhancers  Henriqu. AccumulRecent studies have shown that super enhancers exist as liquid-liquid phase separated biomolecular condensates  Figure . The act6A methylations are present. The m6A marks serve as binding platforms for YTHDF proteins and arranges their IDRs to induce phase separation (6A modification is enriched in eRNA (RNA in general is known to act as a scaffold and directly contribute to the formation of many phase-separated structures. For example, competition for mRNA drives the formation and dissolution of P granules, while the lncRNA NEAT1 provides a scaffold for the formation of paraspeckles . Similarparation . Interes in eRNA . eRNA is in eRNA . Protein in eRNA . Thus, PIn addition to contributing to PSE formation, eRNA may also serve as a potential client and contribute to the composition, biophysical characteristics, and maturation of PSEs. For example, HPSE eRNAs interact with and recruit both hnRNPU and P300 to super enhancers . Other hThe high production of eRNA by PSEs, the pervasive interactions between eRNA and PSE components, and the critical role of RNAs in biomolecular condensate biology all suggest a critical relationship between eRNA and PSEs. These ideas were captured in the discovery of eRNPs as essential contributors to MegaTrans enhancers, and analogous complexes await discovery in super enhancers or other PSEs. Accordingly, outstanding questions regarding the formation, regulation, maintenance, dissolution, and longevity of super enhancers may find answers in eRNAs and their impacts on super enhancers as biomolecular condensates. Thus, the impact of eRNA on PSEs is a potentially significant biological role for eRNA. Future research will shed light on the role of eRNA as scaffold or client in PSEs and the impact such roles have on enhancer biology.per se is able to identify in vivo enhancers that are “caught in the act” of regulating mRNA/gene expression and does so independently of the local chromatin state or transcription factor binding (Because of their biological roles in gene transcription, eRNAs serve as markers of active enhancers. eRNA transcription  binding . For exa binding . The abi binding . eRNA ex binding  and can  binding . As ther binding . Accordi binding . The the binding . eRNA al binding . We antiEnhancer RNAs are products of active enhancers and correlate with mRNA transcription of target genes but with unique expression profiles. Although broadly transcribed, eRNAs are a diverse array of RNAs with heterogeneous structure, length, and post-transcriptional modifications. Recent studies showing decreased gene transcription when eRNA is knocked down suggests that eRNAs have important roles in gene regulation and cellular biology. Mechanisms of action of eRNA include enhancer-promoter looping, chromatin modification, and regulation of transcriptional machinery. Importantly, the emergence of PSE such as super enhancers opens new possibilities for a role of eRNA as mediators of phase separation. The recent discovery that phase separation may be the means through which the activation domains of many transcription factors function, in addition to phase separation of MegaTrans enhancers, raises questions about the ubiquity of phase separation at enhancers and whether typical enhancers also utilize phase separation as a means to regulate gene expression. If so, eRNA-mediated phase separation may be the unified model by which various structures and forms of eRNA mediate looping, recruitment, and other biological functions. Future studies looking at eRNA should address whether the eRNA is transcribed from a phase-separated enhancer and if so, whether such eRNA is required for phase separation. Such studies promise to further elucidate the role of eRNA in the control of cell functions and fate decisions.PA researched, developed, wrote the review, and created figures. AW and XL edited, developed, and provided direction to the review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


How TATA-less promoters such as those within CpG islands (CGI) control gene expression is still a subject of active research. Here, we have identified the “CGCG element”, a ten-base pair motif with a consensus sequence of TCTCGCGAGA present in a group of promoter-associated CGI-enriched in ribosomal protein and housekeeping genes. This element is evolutionarily conserved in vertebrates, found in DNase-accessible regions and employs RNA Pol II to activate gene expression. Through analysis of capped-nascent transcripts and supporting evidence from reporter assays, we demonstrate that this element activates bidirectional transcription through divergent start sites. Methylation of this element abrogates the associated promoter activity. When coincident with a TATA-box, directional transcription remains CGCG-dependent. Because the CGCG element is sufficient to drive transcription, we propose that its unmethylated form functions as a heretofore undescribed promoter element of a group of TATA-less CGI-associated promoters. Gene expression is one of the most critical, yet enigmatic, biological processes that defines cellular and organismal identity, and that mediates cellular response to internal and external stimuli . ImportaThe best-known DNA element that mediates the initiation of transcription of protein-coding genes is the TATA box with the consensus sequence TATAA . This elVertebrate genomes contain short GC-rich sequences that are typically less than 1 kb long, termed CpG islands (CGIs) , 14. TheIn this study, we analyzed DNase-accessible CGIs in the K562 cell line and found an enriched motif with the consensus sequence of TCTCGCGAGA, which we termed the “CGCG element” due to the characteristic invariable core sequence. This element confers transcriptional activity independent of other transcriptional activator sequences. Promoter sequences related to the CGCG element have been reported previously for several individual genes, but their functional significance was never explored –25. A geα-amanitin treatment experiment, HEK293T cells were transfected with SV40 promoter-driven firefly luciferase reporter (pGL2-pro), or a construct containing a copy of TCTCGCGAGA. 24h post-transfection, cells were treated with 5 μg/ml α-amanitin (Santa Cruz Biotechnologies) as described [Human embryonic kidney 293T and mouse NMuMG cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) media supplemented with 10% fetal bovine serum, penicillin and streptomycin at 37°C and 5% CO2. For the escribed  or with DENR promoter were synthesized as double stranded oligonucleotides (IDT DNA) and cloned into the BglII and MluI restriction sites of a luciferase reporter construct that lacks promoter sequences . 1 μg of cloned reporter DNA along with 100 ng of a Renilla luciferase reporter construct (pRL-TK) as transfection control were transfected into HEK293T using X-tremeGENE 9 (Roche) reagent according to manufacturer’s protocol. The luciferase activities were measured 24h after transfection according to the Dual Luciferase assay protocol (Promega).One to three copies of the CGCG elements from the The bidirectional luciferase (Empty-LuBiDi) reporter was constructed by PCR amplification and subsequent cloning of the firefly luciferase gene from pGL2-Basic into the BglII site of the promoterless Renilla luciferase cassette from the pRL-Null plasmid followed by site-directed mutagenesis to remove secondary the BglII recognition site downstream of firefly luciferase poly-A site. The primer sequences used are described in For the construction of the bidirectional fluorescence reporter, pmCGFP, we first PCR amplified and cloned the h2b-mCherry fused gene (plasmid Addgene id #20972) head-to-head into a promoterless eGFP containing construct. The resulting construct (eGFP + h2b-mCherry) was then digested with AgeI to release h2b-coding fragment and self-ligated to generate the pmCGFP (eGFP + mCherry). Double stranded oligonucleotides encoding one or three copies of TCTCGCGAGA were spliced into the AgeI restriction site of this reporter.μg of purified plasmid was incubated at 37°C for 8h with 10 enzymatic units (U) of M.SssI methyltransferase (NEB) supplemented with fresh 100 μM S-adenosyl methionine (SAM) as the methyl donor. DNA was extracted using phenol-chloroform followed by ethanol precipitation. The DNA was subjected to another an additional 8h incubation with M.SssI was followed by DNA extraction. As a control, a mock reaction was also carried out lacking M.SssI enzyme. To test the methylation efficiency, 300ng of DNA was digested with 10 U of NheI and BstUI for 30 min at 37°C. Because CGCG methylation blocks BstUI cleavage, empty vector or and methylated constructs digested only by NheI enzyme producing two indistinguishable bands at 2.4 kb. However, unmethylated TCTCGCGAGA which is cut by BstUI enzyme as well as NheI produced three smaller bands.For CpG-free reporter and methylation experiments, an oligonucleotide encoding a single copy of TCTCGCGAGA was inserted into the HindIII restriction site of pCpGfree-basic-Lucia (Invivogen). 10 The sequences of inserts for each promoter fragment and related mutations are provided in the μg of LuBiDi reporter DNAs containing 0, 1, 2, 4 copies of TCTCGCGATA. Cells were lysed after 24h using TRIzol (Life Technologies), RNA was extracted using chloroform-isopropanol, and after resuspension, incubated with RNase-free DNase I (Thermo Fischer Scientific) followed by extraction by acidic-phenol chloroform protocol, precipitation using ethanol and resuspension in RNase-free water. 1 μg of the resulting purified RNA was used to prepare cDNA using M-MLV reverse transcriptase according to manufacturer’s recommended protocol (Life Technologies). Transcript levels were measured using iTaq Universal SYBR Green Supermix (Bio-Rad) on an ABI-7900 RT-PCR instrument. Transcript levels were normalized using primers for HPRT1. Primers designed to amplify the bacterially-expressed ampicillin resistance gene in the LuBiDi construct were used as a negative control to rule out plasmid contamination. Melting curve analyses for all PCR experiments were performed to validate faithful amplification of PCR products. Information on primer sequences is described in HEK 293T cells were transfected with 1 μg reporter DNA using X-tremeGENE 9 transfection reagent. 48h post-transfection, cells were treated with the cross-linking reagent formaldehyde  for 5min. Cross-linking was stopped by adding glycine solution (0.125 M) for 10min, followed by 2 washes with ice-cold PBS. The cells were lysed in buffer A , and their nuclei were isolated by centrifugation  and lysed by sonication in buffer B  using a Biorupter(Diagenode), specifically, five rounds of 10sec sonication to obtain 300-600bp range chromatin fragments. The resulting sheared chromatin was immunoprecipitated (IP) using 20 μg of antibody against Pol II  or a non-specific Isotype Mouse IgG as a mock control (Santa Cruz). The IP complexes were then bound to Protein A/G Dynabeads and washed five times using RIPA washing buffer . The DNA was extracted from the beads using buffer C  and quantified by qPCR using primers designed to amplify the promoter region of the reporter construct.ChIP was performed according to a protocol described in Lee et al. . In brieμg of pmCGFP reporter constructs containing 0, 1, 2 copies of TCTCGCGAGA along with a CMV promoter-driven Blue Fluorescent Protein expression plasmid (CMV-BFP) were transfected into HEK293T cells. Images were taken 24h post-transfection using a Nikon Eclipse TE2000-E fluorescence microscope. For live imaging, images were taken every 30min with an exposure time of 1sec immediately after reporter transfection for 24h in an incubating chamber supplied with humidity and 5% CO2. 16-bit Tiff images from individual channels were used to generate MOV files using Videomach software (http://gromada.com/videomach/). The final video was produced using Adobe Premiere CC 2017.1 DENR promoter were designed using the MIT CRISPR sgRNA design tool (http://crispr.mit.edu/). Double-stranded oligonucleotides representing sgRNAs (μg/mL) for another 72h. GFP-expressing single cells were sorted using an Aria II FACS and incubated in 96 well dishes for two weeks to form visible cellular clones. DNA was extracted from the clones using QuickExtract solution (Epibio), and successful deletions were confirmed by Sanger sequencing of PCR products. Ribbon sequences were produced using the pyRibbon software which we deposited in https://github.com/AminMahpour/pyRibbon/.Short guide RNAs (sgRNAs) to target g sgRNAs  were theμg/mL for four days.For ZBED1 knockdown, lentiviruses targeting ZBED1 or non-targeting (pLKO.1) were purchased from Sigma (MISSION shRNA). Cells were transduced with the virus according to the manufacturer’s protocol (Sigma) and transduced cells were selected for puromycin at 3μg of total proteins/lane were analyzed by SDS-PAGE followed by blotting as described Previs et al. [For immunoblots, cells were lysed in NET-N buffer  supplemented with protease inhibitors cocktail at 4°C. In all experiments, 20 s et al. . AntibodTCGAGA or completely methylated (TCTmeCGmeCGAGA). 10 μM aliquots of each duplex was bound to 100 μL streptavidin Dynabeads as recommended by the manufacturer (Invitrogen). HEK293T cells were lysed using NET-N buffer containing protease inhibitors cocktail (Sigma) and incubated on ice for 30 min. Lysates were centrifuged at 12000 RPM for 10 min to pellet cellular debris, and supernatant representing 500 μg protein was mixed with duplex-charged beads and incubated at 4°C overnight. The beads were washed five times with NET-N buffer, incubated with 50 μL Laemmli loading buffer  and boiled for 5 min to elute bound proteins. The proteins were analyzed by immunoblotting for Kaiso (Santa Cruz #D-10).Kaiso binding to CGCG elements was assessed using biotin-tagged DNA duplexes of unmodified TCTCGCGAGA, TCTCμg LuBiDi construct along with 0.5 μg pEGFP-C1 as a transfection control. RNA was extracted 72h after transfection, and the quality and purity evaluated using an Agilent 2100 Bioanalyzer, with samples with RNA integrity number (RIN) values >= 8.0 selected for further analysis. The SMARTer 5′-end RACE (Clontech) protocol was used to determine divergent TSSs from 10 μg of total RNA. Briefly, the RNA was first reverse-transcribed at 42 C for 90 min using poly-dT primers and extended beyond TSS using RT-mediated template switching that employs the SMARTer IIA oligonucleotide only when the 5′-cap is encountered. The resulting cDNA products were amplified using specific internal primers for either firefly or Renilla luciferases plus the Clontech Universal Primer Mix (UPM). A GFP primer set was used as an internal control , and sequences that overlap with K562 DNase-seq peak track extracted using Bedtools [The CpG island annotation track in the human genome (hg38) was downloaded from the UCSC genome browser  and mouse (mm9) genomes. The annotatePeaks script (Homer) was used to identify motif co-occurrence, genomic annotations, metagene, and enrichment analysis.http://hgdownload.cse.ucsc.edu/downloads.html). ENCODE accession number ENCFF867JRG was used for K562 DNase-seq data. The GRO-Cap dataset for K562 and GM12878 cell lines with GEO accession number of GSM1480321 was used to analyze nascent transcripts in promoters. Pol II ChIP-seq from K562 cell line with the accession number of ENCFF000YWS was used to determine Pol II occupancy state on CGCG elements. Heatmap plots were generated using the in-house written Wigman software (https://github.com/AminMahpour/Wigman).Processed data points for hg38 were extracted and processed using Wigman software for 50 bp upstream and downstream windows for each motif occurrence. For ENCODE WGBS (accession number ENCFF867JRG). The PhyloP and PhastCons conservation scores for hg38 assembly were downloaded from the UCSC genome browser . Data were analyzed as follows: briefly, reads were aligned uniquely to the mm9 genome allowing a maximum of two mismatches with Bowtie version 0.12.8 (-m1 -v2). Sense and divergent TSS were assigned as defined above. Start-seq heat maps depict Start-RNA reads in 10 bp bins at the indicated distances with respect to the TSS. Heatmap plots were generated using Partek Genomics Suite version 6.12.1012.Start-seq datasets from mouse bone-marrow derived macrophages were published previously and is available for download from the GEO website . A custom script was written and used to determine the number of CGCG elements in annotated coding, non-coding, uni- and bi-directional CGI promoters. Gene Ontology (GO) analysis performed using the GOrilla gene enrichment analysis platform. A list of CpG islands-associated genes was used as the background genes for enrichment analysis [b/n)/(B/N) where N is the total number of background CpG island-associated genes that have a GO term, B is the number of genes associated with a specified GO term, n is the number of genes whose promoter contain CGCG element and b is the number of genes in the intersection. Gene set interaction networks were generated and analyzed using REACTOME package v53 (http://www.reactome.org/). Networks were visualized graphically using Cytoscape software version 3.5 (http://www.cytoscape.org/).Bedtools Closest feature was used to compile a list of genes with annotated TSS less than 500 bp from CGCG elements on both plus and minus strands from the latest hg38 GTF annotation file  from the mean.All plots were generated and analyzed using GraphPad Prism version 7. Unless noted otherwise, all statistical analyses were performed using Student t-test. The following p-values are presented as *: Previous analysis suggested that roughly 50 percent of human promoters are associated with a CGI . To idenAlthough most CGI-associated promoters contain one copy of the motifs shown in DENR). The DENR promoter contains three tandem CGCG elements separated by 21 and 11 nucleotides  into a promoterless construct that encodes a firefly luciferase reporter gene (Empty pGL2-basic). The resulting constructs were then separately cotransfected along with a control reporter for Renilla luciferase driven by the HSV-1 thymidine kinase promoter (pRL-TK) into human embryonic kidney (HEK293T) cells. Motif 10, but not Motif 7, significantly activated firefly reporter gene expression . This releotides . To deteleotides .TCGAGA) and performed chromatin immunoprecipitation (ChIP) for RNA Pol II [DENR promoter containing CGCG elements , we transfected HEK293T cells with reporter constructs that contain either the consensus motif (TCTCGCGAGA) or a G→T transversion mutation  removed all three CGCG elements in one allele, and a separate 100bp deletion removed one of the CGCG elements in the other allele without affecting the remaining CGI in the promoter  to measure promoter activity using firefly and Renilla luciferase genes as reporters of directional transcription from a central control motif .AGACGCGTCT, which maintains both symmetry and CpG content. This mutation abrogated the dual activation of reporters (TCG (underline represents changes) transition mutation  and an “A” insertion into CGCG  abrogated dual reporter activity  that codes for enhanced Green Fluorescent Protein (eGFP) and mCherry reporters in opposite directions Fig A in . One or CCT, AGG or ATG flanking sequences decreased relative dual reporter activity whereas variants that contain ATA or TAT showed similar activity to that of the TCTCGCGAGA motif  dataset performed on K562 cells [To determine if CGCG elements are associated with the 62 cells . GRO-cap62 cells  S3 Fig)in vivo bGene Ontology (GO) analysis showed that genes containing CGCG promoter elements produce protein-coding transcripts whose products form discernible protein-protein interacting networks . SpecifiBecause CpG dinucleotides in CGI-associated promoters are invariably unmethylated , we askein vivo or vehicle (DMSO) . DAC trein vivo.DENR protein levels  occur in annotated unidirectional promoters that drive coding or long non-coding RNAs, while 7% occur in an annotated bidirectional promoter .AGACGCGTCT). We first focused on the POLR1C/YIPF3 bidirectional promoter region, which has divergent TSS separated by 30 nucleotides that flank a single CGCG element. We inserted a promoter fragment (30bp) containing the wild-type CGCG element into the LuBiDi construct, and as a comparison, constructs were generated in which the flanking sequences (AGA and TCT) were exchanged. The WT fragment from POLR1C/YIPF3 promoter induced bidirectional expression irrespective of its orientation  . Based oentation . In contZZZ3 promoter which is similar to the DENR promoter in that it contains three CGCG elements  or on a specific nucleotide within the element in the case of the TCT motif . To map As shown in We also used 5′-RACE to determine TSS in the reporter construct containing four tandem copies of TCTCGCGAGA motif B in . As showPRDX1 promoter  sequences that account for the majority of CGCG elements do not overlap with the hDRE  was shown to positively activate metabolism-related and ribosomal protein genes through binding of ZBED1 protein , 55. TheEIF5, EIF3H, and DENR, as well as the essential translation termination factor ETF1, contain CGCG elements in their promoters. This is consistent with the current perspective that different classes of promoter elements regulate functionally distinct protein coding genes [How housekeeping genes whose products are core components of cellular processes are transcriptionally regulated is poorly understood. In this study, we found that genes whose products play a central role in translation and transcription are enriched for CGCG elements in their CGI-associated promoters. This analysis led us to identify a group of ribosomal genes whose CpG rich promoters contain one or multiple CGCG elements A in . As showAdditionally, we demonstrated that methylation of CpGs in the CGCG element could suppress promoter activity. Indeed, roughly 80 percent of CpG sites in the genome, particularly CpGs that occur outside of CGIs, are methylated . We specde novo bidirectional transcription in the absence of a core promoter element from nearby Py/Pu(+1) sites. This notion is supported by studies that show that SSTFBSs cannot initiate transcription in the absence of a core promoter element. However, similar to SSTFBSs, CGCG elements can occur in tandem copies and the copy number modulates transcription intensity. Unlike traditional core promoter elements, the CGCG element is not positioned at a fixed distance to TSSs, but it is found within a range of 20 to 70 nucleotides (with a peak of apporoximatly 50 nucleotides) upstream of TSS on either strands as was determined by the analysis of Start-seq and GRO-Cap datasets. It is also conceivable that CGCG elements modulate the induction of transcription by recruiting factors that could affect nucleosomal positioning in CGIs. Identification of the CGCG element interacting factor will likely clarify whether the CGCG element functions as a core promoter element and its role in driving transcription of housekeeping genes from CpG-rich and TATA-less promoters.In this study, we provide strong evidence that CGCG elements are evolutionarily conserved in vertebrates, functioning as an active component of CGI-associated promoters. The unmethylated form of the element may be sufficient to drive bidirectional transcription of TATA-less promoters. An interesting, yet very important question to address in future studies is whether the CGCG element functions as a core promoter element or as a sequence-specific transcription factor binding site (SSTFBS). An argument for the core promoter element characteristic of the CGCG element includes its ability to initiate local S1 FigA) ENCODE DNase-seq footprints of motif 7 and 10 for available cell lines. B) TCTCGCGAGA motif occurs within 50bp of annotated TSSs in the human and mouse genomes.(EPS)Click here for additional data file.S2 Figμm. E) CGCG element confers bidirectional expression of GFP and H2b-mCherry reporter genes in HEK293T. Time-lapse images of HEK293T cells transfected with pmCGFP-H2b (h2b-mCherry fused gene). Please note delayed H2b-mCherry signals as the fused mCherry protein is being trafficked into the nucleus. F) Images of NMuMG mouse cells transfected with a pmCGFP-H2b construct containing either 3 copies of the wild-type TCTCGCGAGA motif or 3 copies of the TCTCTCGAGA mutant motif.A) The pmCGFP bidirectional reporter structure. B) Fluorescence image 24h after transfection of HEK293T cells with pmCGFP constructs containing 0, 1 or 3 copies of the TCTCGCGAGA motif. CMV-driven BFP expression was used as an internal control. C) Immunoblots showing levels of GFP and mCherry expression 24 and 48h post transfection. D) Time-lapse imaging of HEK293T cells transfected with pmCGFP containing three copies of TCTCGCGAGA for 24h shows that both reporters are simultaneously expressed. Scale bar is 100 (EPS)Click here for additional data file.S3 Figin vivo, clearly show TSS peaks are located 50bp from the TCTCGCGAGA in both directions. The figure also includes MNase-seq plots for the two promoter element +/-1000bp. The data indicates that genomic regions containing TCTCGCGAGA and TATA-boxes are nucleosome depleted.The GRO-cap signals, which map TSS (EPS)Click here for additional data file.S4 FigAn analysis of genes that contain CGCG elements in their promoters found that most of these genes can be clustered into distinct functional groups as indicated in the figure.(EPS)Click here for additional data file.S5 FigDENR gene.A) Pulldown experiment using biotin-tagged CGCG oligonucleotides followed by immunoblotting for Kaiso or tubulin. B) Immunoblot analysis of transient expression of Kaiso in HEK293T cells. Two independent replicates (Rep1 and Rep2) were used in this experiment. C) Successful knockdown of ZBED1 does not change the DENR protein product. D) RT-PCR analysis of ZBED1 knockdown cells does not attenuate the transcript levels of the (EPS)Click here for additional data file.S6 FigA) GRO-cap signal tracks of the CpG-rich DENR promoter. B) The CGCG elements in the DENR promoter, regardless of the insert direction, activated bidirectional reporter luciferase activities. Insertion of an “A” in the center of CGCG elements eliminated the promoter activity. Data are represented as the mean of three replicates ± SD.(EPS)Click here for additional data file.S7 FigA) The sequence flanking the BglII restriction site where DNA duplexes were inserted into the promoterless LuBiDi construct. The reporter genes in either direction are shown in bold. B) The sequences of DNA fragments inserted into LuBiDi, containing flanking BglII restriction sites (boxed), used for RACE analyses.(EPS)Click here for additional data file.S8 FigA) Aligned sequences of CGCG elements and flanking regions in the promoters of ribosomal proteins genes, with the resulting consensus sequence of TCTCGCGAGA shown below. The list also indicates whether the TCT or hDRE motifs are present in a given promoter. B) Venn diagram showing the distribution of TCT and CGCG elements in human ribosomal proteins promoters.(TIF)Click here for additional data file.S1 VideoLive cells were transfected with pmCGFP reporter constructs containing none, one or three tandem copies of TCTCGCGAGA. Cells were incubated in a chamber supporting normal cellular growth and images were captured to record mCherry and GFP signals every 30 minutes. The top panel for each reporter assay shows overlapping signals. Lower panels show GFP or mCherry fluorescence channels.(MP4)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(TXT)Click here for additional data file.


CHD8) encodes a chromatin remodeling factor with among the highest de novo loss-of-function mutation rates in patients with autism spectrum disorder (ASD). However, mechanisms associated with CHD8 pathology have yet to be elucidated. We analyzed published transcriptomic data across CHD8 in vitro and in vivo knockdown and knockout models and CHD8 binding across published ChIP-seq datasets to identify convergent mechanisms of gene regulation by CHD8. Differentially expressed genes (DEGs) across models varied, but overlap was observed between downregulated genes involved in neuronal development and function, cell cycle, chromatin dynamics, and RNA processing, and between upregulated genes involved in metabolism and immune response. Considering the variability in transcriptional changes and the cells and tissues represented across ChIP-seq analysis, we found a surprisingly consistent set of high-affinity CHD8 genomic interactions. CHD8 was enriched near promoters of genes involved in basic cell functions and gene regulation. Overlap between high-affinity CHD8 targets and DEGs shows that reduced dosage of CHD8 directly relates to decreased expression of cell cycle, chromatin organization, and RNA processing genes, but only in a subset of studies. This meta-analysis verifies CHD8 as a master regulator of gene expression and reveals a consistent set of high-affinity CHD8 targets across human, mouse, and rat in vivo and in vitro studies. These conserved regulatory targets include many genes that are also implicated in ASD. Our findings suggest a model where perturbation to dosage-sensitive CHD8 genomic interactions with a highly-conserved set of regulatory targets leads to model-specific downstream transcriptional impacts.The packaging of DNA into chromatin determines the transcriptional potential of cells and is central to eukaryotic gene regulation. Case sequencing studies have revealed mutations to proteins that regulate chromatin state, known as chromatin remodeling factors, with causal roles in neurodevelopmental disorders. Chromodomain helicase DNA binding protein 8 ( CHD8) has one of the highest observed mutation rates in sporadic ASD  neurobiology, presumably through disruptions to transcriptional regulation in the developing and mature brain . The genadic ASD , and mut cohorts . In addiathology .in vitro and in mammalian cell culture  and chromatin immunoprecipitation followed by sequencing (ChIP-seq), generate comparable quantitative data enabling direct comparisons of results across models and studies.Characterizing convergent patterns of CHD8 genomic interactions and transcriptional outcomes caused by CHD8 haploinsufficiency. Across studies, we found overlapping changes in gene expression across haploinsufficiency models and a strikingly consistent set of high-affinity CHD8 interaction target genes across all binding datasets. The findings of this meta-analysis suggest evolutionarily-conserved and non-cell-type specific high-affinity genomic targets of CHD8 across human, mouse, and rat models. By disrupting these genomic interactions, or by secondary mechanisms, reduction in CHD8 expression directly and indirectly altered transcription of genes critical for neurodevelopment and previously implicated in neurodevelopmental disorders.We re-analyzed published RNA- and ChIP-seq data and built an online user interface enabling browsable comparison of gene expression changes linked to CHD8 studies were identified through a literature search with the keyword “CHD8” in PubMed and Gene Expression Omnibus (GEO) databases. Raw data from publications that featured RNA-seq or ChIP-seq analysis were downloaded from GEO with the exception of three publications that hosted raw data on DNA Data Bank of Japan (DDBJ) (Next-generation sequencing datasets generated from n (DDBJ)  and Sequn (DDBJ) . A totaln (DDBJ) . All dat2(RPKM) values were used for plotting summary heatmaps and for expression data of individual genes. Variation in sequencing depth and intra-study sample variability partially account for differences in sensitivity and power across studies and likely drive some differences observed including the total number of differentially expressed genes (DEGs). To capture an inclusive set of DEGs, DEGs were defined by uncorrected p-values < 0.05. DEG sets were used for gene set enrichment analysis for Gene Ontology via goseq  values were plotted for GO terms for data visualization. Finally, genes associated with high-affinity CHD8 binding were defined as those present in the top 2000 peaks from any ChIP-seq dataset and were intersected with the Simons Foundation Autism Research Initiative (SFARI) set of ASD risk genes.For primary analysis, we used the Gene Set Enrichment Analysis tool and the MSigDB database , moderate (FDR < 0.1), and stringent (FDR < 0.05) thresholds, though numbers of DEGs varied widely  terms using GSEA and goseq Figure . While rIn vitro models were more likely to have neuronal terms or fail to have a trend represented while in vivo models were more likely to have both, or only gene regulation associated terms, represented. There is also some indication that in vivo models of postnatal brain tended to have more enrichment of neuronal terms while models of embryonic brain were more likely to also have enrichment of terms associated with gene regulation. Overall, our results suggest that CHD8 knockdown or heterozygous knockout produces model-specific differential gene expression, with overlap present among general functional classes. Expression changes appear to be more consistent in embryonic and postnatal brain tissues of germline Chd8 haploinsufficient mice across studies.Of 36 total datasets, around 11 had synaptic or neurodevelopmental terms enriched, 7 had cell cycle, chromatin organization, and RNA processing terms enriched, 11 had a combination of both, and 7 had neither trend represented when analyzed using GSEA Figure . In addiin vivo tissue preparations and in vitro culture models from neuronal and non-neuronal cell fates to allow additional examination of tissue or cell-type specificity of CHD8 interactions. Five of the datasets were generated from bulk mouse tissue at adult . Overall correlation (Spearman’s coefficient) was compared for target genes  vs. stringent (FDR < 0.05) statistical thresholds. Future work should take this into consideration when analyzing differential gene expression data from CHD8 models. Compared to the high variability across in vitro models, the impact of germline heterozygous Chd8 mutation in mouse brain was much more consistent, with four of the five studies showing significant DEG overlap. At the level of gene set enrichment, we found global patterns of transcriptional dysregulation with downregulation of genes involved in gene regulation and neuronal development and function and upregulation of genes involved in immune signaling and metabolism.Knockdown or heterozygous mutation of CHD8 haploinsufficiency for most targets.In contrast to differences in the RNA data, the ChIP-seq results were highly consistent for high-affinity genomic interactions. Comparison across ChIP-seq experiments shows that CHD8 preferentially targets promoters, with no evidence of direct binding through a specific DNA motif. Peaks with the highest signal were constant across experiments, regardless of the model, suggesting that CHD8 preferentially interacts with promoters of a set of genes linked to cellular processes such as those involved in cell cycle, chromatin organization, and RNA processing. We found reduced transcription of these CHD8 target genes in some models, though our data also highlight widespread genomic promoter interactions for CHD8 without obviously strong transcriptional impact from While the clear concordance in high-affinity genomic CHD8 interactions suggests common regulatory functions across cell types, it remains to be examined whether the observed dysregulation of neurodevelopmental disorder-relevant neuronal genes is related to context-dependent CHD8 regulatory activity in the brain given the current cellular heterogeneity and technical challenges existing with available CHD8 ChIP-seq. Of note, we did not see enrichment among high-affinity or low-affinity targets for cell-type-specific genes in the datasets examined here, which include some cell-specific analyses. Possible explanations for changes in expression for genes that are not high-affinity CHD8 targets include secondary impacts, increased dosage sensitivity for lower-affinity genomic interaction targets, or a function of CHD8 that is not dependent on specific genomic interactions.CHD8 expression across models despite the use of common design strategies for testing the impacts of haploinsufficiency. Though we did not find an obvious correlation between CHD8 transcript levels and up- or downregulated gene expression, it seems likely that differences in experimental design, including CHD8 knockdown or knockout, contributed toward meaningful variation between models. We also noted differences in ChIP-seq datasets that suggest very different genome-wide binding patterns depending on the experiment. We note that enrichment in control libraries was present across several published datasets, which could confound CHD8-specific peak discovery. Different studies also used various CHD8 antibodies with unknown and unvalidated CHD8 specificities. Nonetheless, by examining patterns across datasets, we identified consistent patterns of enrichment suggesting that overall findings from ChIP-seq targeting CHD8 reliably identify common high-affinity interactions.We note a number of technical issues that impacted this meta-analysis, many of which are associated with variation in methods and sequencing depth. Surprisingly, we found considerable differences in CHD8 haploinsufficiency. Our results suggest that, as a chromatin remodeler, CHD8 primarily targets genes involved in cell cycle, chromatin organization, and RNA processing regardless of cell type. Therefore, as an essential gene with widespread expression across neurons and glia, homozygous loss of CHD8 would likely impact cellular viability in general while heterozygous mutation or knockdown would have subtler, more unpredictable, impacts depending on the cellular context. Such a model would explain the widespread changes in gene expression across model systems and varied reports of impact on proliferation depending on dosage. Nonetheless, given the limitations of current studies we cannot rule out the possibility of cell-type or context-dependent specificity of CHD8 function.It is clear from previous publications and this meta-analysis that CHD8 is critical for neurodevelopment. However, despite the limitations of comparing genomic datasets across variable models, our analysis challenges the simple model that cell-specific CHD8 genomic interaction patterns drive differences in the impact of CHD8 haploinsufficiency in the developing and mature brain, and (2) Does CHD8 have context-dependent function in specific stages, cell types, and regions with regard to genomic interaction patterns? Beyond addressing these two key issues, additional clarity regarding the role of CHD8 in the brain will come from studies examining molecular and biochemical properties underlying CHD8 function in the brain. As CHD8 haploinsufficiency may represent common features of haploinsufficiency of other general chromatin remodelers implicated in ASD, further characterization of CHD8 models and CHD8 genomic interactions could reveal essential functions driving pathology in neurodevelopmental disorders.Our results raise two questions that could be addressed by application of RNA-seq and ChIP-seq in the future: (1) What are the developmental stage, cell-type, and region-specific impacts of AW and AN conceived of the project. AW, KL, and AN performed analysis of RNA-seq experiments. AW, RC-P, and AN performed analysis of ChIP-seq experiments. AW and AN drafted the manuscript. All authors contributed to manuscript revision.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Moreover, the electrophoretic mobility shift assay (EMSA) and vectors containing serial deletion fragments were used to identify possible transcription factors and the DNA‐binding domain. In addition, we explored the role of three transcription factors  in the 5 promoter activity of the DRD1 gene and expression of endogenous DRD1 in the HEK293 cell line.We previously identified eight SNPs (single‐nucleotide polymorphisms) in the 5 regulatory region of the 22.1A total of nine vectors were constructed, which contained eight single SNP loci Table . The vec2.2Human neuroblastoma cell line SK‐N‐SH and human embryonic kidney cell line HEK293 were used to test the luciferase activity of the generated vectors.5 cells per well) and the pGL3 vectors (1.0 μg) were co‐transfected with Renilla luciferase (100 ng) expression vector pRL‐TK (Promega) using Lipofectamine 3000 reagent according to the manufacturer’s protocol .Both of the cells were seeded in 24‐well plates  and the pEGFP‐N1‐basic vector were purchased from Taihe Biotechnology Co. .2.4Here, 24 hours after transfection, cells were harvested. Firefly luciferase activity in the cell lysates was normalized to Renilla luciferase activity. Normalized activities of all the test clones were compared to those of the clones representing the reference haplotype. Each assay was performed in triplicate in two independent experiments.2.5Nuclear and cytoplasmic extracts were prepared from SK‐N‐SH and HEK293 cells using the Nuclear and Cytoplasmic Protein Extraction Kit  in the presence of 1% protease inhibitor . Here, 5‐biotinylated probes, unlabeled‐specific competitors, and nonspecific competitors were synthesized by Taihe Biotechnology Co. Table .For supershift, the antibodies were anti‐MEI1, anti‐SOX‐10, anti‐HOXA5, anti‐NR4A2 and anti‐ERRA .2.6http://www.gene-regulation.com/pub/programs.html).The prediction software Match was used to identify putative‐binding sites for transcription factors at specific SNPs in the promoter region of the DRD1 gene  and β‐actin  antibodies were used after being diluted with TBS‐T at a ratio of 1:1000 and 1:2000, respectively.2.9t test. P < 0.05 indicated statistically significant differences. SPSS 18.0 software  was used for statistical analysis.Data are presented as the mean (χ) ± SD. Differences between the two groups were determined using the analysis of variance (ANOVA) or independent samples using the 33.1P < 0.05; Figure In HEK293 cells, there was a significant increase in luciferase activity for clones M2, M3, M6 and M7 compared to WT with M2 demonstrating the highest activity (3.2DRD1 gene) was the binding sequence of the complex. Prediction of the transcription factor binding‐site alteration caused by the rs10078866 locus in the ACTTTGAGC oligonucleotide was performed  was detected in both HEK‐293 and SK‐N‐SH cells  could form a DNA‐protein complex in the presence of SK‐N‐SH and HEK293 nuclear extracts. In addition, the endogenous expression of DRD1 in HEK293 cells could be regulated by the CREB1, TFAP2B and SP1 transcription factors. Additional experiments are needed to better understand the function of the 5 regulatory region of the DRD1 gene in future.In this study, the C allele of the rs10078866 locus significantly increased luciferase activity in HEK293 cells, but not in SK‐N‐SH cells. This heterogeneity might be due to the differences in cell type.None.Jun Yao conceived and designed the experiments. Xue Wu and Feng‐ling Xu performed the experiments. Feng‐ling Xu, Jing‐hua Meng and Bao‐jie Wang analysed the data. Jun Yao wrote the paper. Click here for additional data file.


Faithful transcription initiation is critical for accurate gene expression, yet the mechanisms underlying specific transcription start site (TSS) selection in mammals remain unclear. Here, we show that the histone-fold domain protein NF-Y, a ubiquitously expressed transcription factor, controls the fidelity of transcription initiation at gene promoters in mouse embryonic stem cells. We report that NF-Y maintains the region upstream of TSSs in a nucleosome-depleted state while simultaneously protecting this accessible region against aberrant and/or ectopic transcription initiation. We find that loss of NF-Y binding in mammalian cells disrupts the promoter chromatin landscape, leading to nucleosomal encroachment over the canonical TSS. Importantly, this chromatin rearrangement is accompanied by upstream relocation of the transcription pre-initiation complex and ectopic transcription initiation. Further, this phenomenon generates aberrant extended transcripts that undergo translation, disrupting gene expression profiles. These results suggest NF-Y is a central player in TSS selection in metazoans and highlight the deleterious consequences of inaccurate transcription initiation. The mechanisms underlying specific TSS selection in mammals remain unclear. Here the authors show that the ubiquitously expressed transcription factor NF-Y regulate fidelity of transcription initiation at gene promoters, maintaining the region upstream of TSSs in a nucleosome-depleted state, while protecting this region from ectopic transcription initiation. Recent studies have established basic rules regarding spatial arrangements of cis-regulatory elements, ordered recruitment of general transcription factors (GTFs) for transcription pre-initiation complex (PIC) formation, and the role of chromatin in defining the promoter environment8. One key determinant of active gene promoters is the requirement for an accessible transcription initiation/start site (TSS), characterized by a nucleosome-depleted region (NDR) flanked by two well-positioned nucleosomes (the −1 and +1 nucleosomes)9.While the sequence, structure, and binding partners of gene promoters have been intensely scrutinized for nearly half a century11 and play important roles in TSS selection. However, the core promoter elements vary from one promoter to the next and can be either absent or present multiple times within a single NDR, suggesting that these elements are not the sole determinants of TSS selection. Thus, how the RNA Polymerase II (Pol II) chooses one transcription initiation site over another remains unclear. In yeast, mutational studies have identified several GTFs and other accessory factors with key roles in TSS selection18. However, despite greater specificity in TSS selection in metazoans, such accessory factors have yet to be described in higher eukaryotes.Within the NDR, several core promoter elements such as the TATA box and the initiator (Inr) element exhibit a positional bias relative to the TSS23. NF-YA, which harbors both DNA-binding and transactivation domains, makes sequence-specific DNA contacts, whereas the histone-fold domain (HFD) containing NF-YB and NF-YC interact with DNA via nonspecific HFD-DNA contacts23. The structure and DNA-binding mode of NF-YB/NF-YC HFDs are similar to those of the core histones H2A/H2B, TATA-binding protein (TBP)-associated factors (TAFs), the TBP/TATA-binding negative cofactor 2 (NC2α/β), and the CHRAC15/CHRAC17 subunits of the nucleosome remodeling complex CHRAC22.NF-Y, also known as the CCAAT-binding factor CBF, is a highly conserved and ubiquitously expressed heterotrimeric transcription factor (TF) composed of the NF-YA, NF-YB, and NF-YC subunits, all three of which are necessary for stable DNA binding of the complex25. However, the mechanisms through which NF-Y influences gene expression remain unclear. Several lines of evidence have pointed toward a possible role in the recruitment of chromatin modifiers and/or the PIC to the promoters of its target genes . Previously, we reported a critical role for NF-Y in facilitating a permissive chromatin conformation at cell type-specific distal enhancers to enable master transcription factor binding24. Here, we investigate the role NF-Y plays at gene promoters, what effect it may have on chromatin accessibility and recruitment of the transcription machinery, and how this might impact gene expression.NF-Y has an established role in gene regulation through cell type-invariant promoter-proximal bindingThrough genome-wide studies in mouse embryonic stem cells (ESCs), we find that NF-Y is essential for the maintenance of the NDR at gene promoters. Depletion of the NF-YA protein leads to the accumulation of ectopic nucleosomes over the TSS, reducing promoter accessibility. Interestingly, under these conditions, we find that the PIC can relocate to a previously NF-Y-occupied upstream site, from where it commences ectopic transcription initiation. Remarkably, the resulting ectopic transcript can create novel mRNA isoforms and, in a large number of cases, leads to abnormal translation. Overall, we establish NF-Y’s role in TSS selection and demonstrate its importance in safeguarding the integrity of the NDR at gene promoters.24. Further analysis of our NF-Y ChIP-Seq data revealed a positional bias, with nearly all of the NF-Y-binding sites located immediately upstream (median distance 94 bp) of the TSS  to knockdown (KD) the DNA-binding subunit NF-YA in ESCs  of annotated TSSs of protein-coding genesTSS Fig.  and a poTSS Fig. . To inveNF-YA KD is observed specifically at NF-Y bound promoters  digestion followed by high-throughput sequencing (MNase-Seq). Our data revealed that NF-Y-binding sites genome-wide are depleted of nucleosomes Fig. . Focusiners Fig. , rather ers Fig. , suggesters Fig. , suggest27. Consequently, upon observing the appearance of ectopic nucleosome binding within the NDR of NF-Y-bound promoters in NF-YA-depleted cells, we sought to investigate whether this outcome affects binding of the transcription machinery and thus transcription initiation. Because NF-Y is known to interact with the Pol II-recruiting TBP28 and several TBP-associated factors (TAFs)20, essential components of the TFIID complex that recruits Pol II, we decided to investigate whether TBP enrichment was also affected upon NF-YA KD. Indeed, loss of NF-Y binding led to a significant diminishment and/or upstream shift of TBP’s binding pattern , and hence the general Pol II transcription machinery, is unable to bind nucleosomal DNA9, a high-throughput sequencing method that captures capped RNA species from their 5′-ends. Start-Seq faithfully mapped the canonical TSSs in the control cells, confirming its utility in capturing transcription initiation sites  or the downstream antisense RNA (convergent transcription) exhibit no such shift -induced differentiation. Four days of RA-induced differentiation did not elicit the use of ectopic TSSs  surrounding the canonical and ectopic TSSs, as determined using Start-Seq, revealed the typical YR initiator dinucleotide11  within the ectopically transcribed region . Recent studies have shown that upstream ORFs (uORFs) can have either a favorable or a deleterious effect on downstream mRNA translation41 and suggested that the abundance of transcript is less important than the difference in translatability of the canonical versus ectopic transcript39. Thus, in situations wherein an upstream extension of the transcript leads to altered protein production, we speculate that this could be due to introduction of an uORF. To summarize, we establish here that transcripts originating from ectopic TSSs undergo translation, and highlight the variable effects this can have on the translation of the canonical ORF within these transcripts.In an attempt to evaluate whether these translated upstream open-reading frames (ORFs) generate fusion or variant forms of endogenous protein, we performed western blot analysis, using commercially available antibodies for candidate proteins, in cells depleted of NF-YA and treated with the proteasome inhibitor MG-132  within core promoters, NF-Y’s function as a regulator of gene expression has almost exclusively been studied in relation to its promoter-proximal binding. Yet, the exact mechanism by which it exerts control over gene expression remains poorly understood. Through comprehensive genome-wide studies in ESCs, we have uncovered a previously unidentified role for NF-Y in safeguarding the integrity of the NDR structure, PIC localization, and TSS selection at protein-coding genes.25. Furthermore, NF-Y’s unique DNA-binding mode, which induces an ∼80° bend in the DNA, may allow and/or promote binding of other TFs, whose recognition sequences become more accessible24. Supporting this thesis, DNase experiments have shown that NF-Y is essential for the maintenance of an accessible chromatin43. These attributes have led us and others to propose that NF-Y is a “pioneer factor”44.NF-Y can access its target DNA motif, the CCAAT box, in a heterochromatic environment46, our findings suggest steric incompatibility between NF-Y and nucleosomes. This conclusion is supported by the observation that upon NF-YA KD, nucleosomes bind within the NDRs left vacant by NF-Y, positioning them in a manner that strongly reflects DNA sequence preferences  direct PIC recruitment to the promoter region through its interactions with TBP and the TAFs, (2) prevent ectopic nucleosome binding within the NDR through its nucleosome-like structural properties and DNA-binding mode, and (3) occlude alternative transcription initiation sites to ensure correct TSS usage. The shift in TSS usage upon Ezh2; Fig. Hsp90b1; Fig. Khsrp and C7orf50; Fig. As might be expected, the usage of an ectopic, upstream TSS has variable consequences on the steady-state levels of resulting mRNAs and protein products. The strength of the ectopic initiation site along with the regulatory potential of the additional upstream mRNA sequences  undoubtedly impact transcription levels and mRNA stability. In addition, the TSS employed in NF-YA depleted cells could represent a known alternative start site , whose accurate expression is vital in most cell typesSaccharomyces pombe have shown that deletion of Php5, an NF-YC orthologue, leads to an ~250 -bp upstream shift in TSS of the gluconeogenesis gene Fbp153. Moreover, in Saccharomyces cervisiae, out of the 46 TFs studied, the binding sites for NF-YA homolog Hap2 were shown to have the biggest difference in predicted nucleosome occupancy between Hap2-bound (lower nucleosome occupancy) and non-Hap2-bound (higher occupancy) sites54. This opens the exciting possibility of NF-Y’s role in promoter chromatin organization being conserved throughout the eukaryotic kingdom.We suggest that this dual role for NF-Y may explain why promoter-proximal NF-Y binding is so well conserved across mouse and human cell types. Intriguingly, studies in NF-YA KD is that ribosomes can scan over any ectopic mRNA. Ribosomes typically initiate translation upon encountering the AUG start codon, although other codons have been shown to induce translation initiation57. In our study, we found that nearly three quarters of genes showing a TSS shift upon NF-YA KD possess at least one ATG triplet between the ectopic and the canonical TSS. Importantly, we found evidence for translation initiation from such sites  within uORFs alters the reading frame and/or protein length; alternatively, it may affect the efficiency with which ribosomes translate the rest of the transcript59.A consequence of altered TSS selection upon tes Fig. . Given tIn summary, our studies describe NF-Y’s mechanistic role at promoters, where it is necessary for both maintenance of the NDR’s structural architecture and correct positioning of the transcriptional machinery, therefore influencing TSS selection. Furthermore, our results strongly suggest that the sites of NF-Y binding and the +1 nucleosome demarcate the 5′ and 3′ boundaries, respectively, of the region available for PIC assembly, thereby directing the transcription machinery to the correct TSS while occluding alternative TSSs and other sites of sub-optimal transcription initiation. It will be interesting to explore whether other histone-fold domain proteins, with similar structural and DNA-binding properties analogous to NF-Y, may function in a similar manner.60. For experiments, ESCs were cultured on gelatin-coated plates in the M15 medium: DMEM  supplemented with 15% FBS , 10 μM 2-mercaptoethanol , 0.1 mM nonessential amino acids , 1x EmbryoMax nucleosides , 1 U/ml of ESGRO mLIF .Mouse ESCs  were maintained on gelatin -coated plates in the ESGRO complete plus clonal grade medium (Millipore), as previously describedNF-YA , NF-YB , NF-YC , non-targeting control .For transfections, ESCs were cultured in the M15 medium and transfected with 50 nM siRNA using Lipofectamine 2000  at day 0 and collected after 48 h. Gene-specific siRNAs used: 24. Briefly, mouse ESCs (1 × 107) were cross-linked with 1% formaldehyde  in the DMEM  for 10 min, and the reaction was quenched by the addition of glycine  at a final concentration of 125 mM for 5 min. Cells were washed twice with PBS, and resuspended in 1 ml of lysis buffer A . After 10 min on ice, the cells were pelleted and resuspended in 1 ml of lysis buffer B . After 10 min at room temperature, cells were sonicated in lysis buffer C  using Diagenode Bioruptor for 16 cycles  to obtain ~200–500 -bp fragments.ChIP was performed as previously described61. ChIP enrichment for a primer-set was evaluated using quantitative PCR, as percentage of input, and normalized to a negative primer set. See Supplementary Data Cell debris were pre-cleared by centrifugation at 14,000 rpm for 20 min, and 25 μg of chromatin was incubated with either NF-YA , histone H3 , RNA Pol II  or TBP  antibodies overnight at 4 °C. Protein A/G-conjugated magnetic beads  were added the next day for 2 h. Subsequent washing and reverse cross-linking were performed as previously described 24. Briefly, mouse ESCs treated with nontargeting control siRNA , or NF-YA siRNA  were collected 48 h post transfection in cold PBS. Nuclei were isolated by incubation of 107 cells for 10 min on ice with 5 ml RSB buffer , and pelleted by centrifugation at 300 g and 4 °C for 10 min Nuclei were then resuspended in 1 ml DNase reaction buffer  and counted. Additional resuspension buffer was used to generate equal concentrations of nuclei between samples.DNase I hypersensitivity experiments were performed as previously described5 cells were aliquoted into microcentrifuge tubes and incubated at 37 °C for 5 min with varying amounts of DNase . Digestion was stopped by addition of an equal volume of termination buffer . The nuclei were then incubated at 55 °C for 15 min, followed by addition of 2 μl of 20 mg/ml Proteinase K . Reaction mixtures were incubated overnight at 55 °C, followed by a phenol–chloroform extraction of the DNA. The DNA was then precipitated and resuspended in 100 μl H2O. See Supplementary Data Nuclei from 5 × 10 Sigma, T663 pH 7.24. Briefly, the total RNAs were prepared from cells using Qiazol lysis reagent , and cDNAs were generated using the iScript kit  according to the manufacturer’s instructions. Quantitative PCRs were performed on the Bio-Rad CFX-96 or CFX-384 Real-Time PCR System using the SsoFast EvaGreen supermix . Three or more biological replicates were performed for each experiment. The data are normalized to Actin, Haz, and TBP expression, and plotted as mean +/− SEM. See Supplementary Data Quantitative RT-PCR was performed as previously described24. Briefly, cell pellets, lysed in RIPA buffer  with protease inhibitors , were sonicated using Bioruptor (Diagenode) for three cycles . The lysate was boiled with SDS-PAGE sample buffer , loaded onto NuPAGE gel , and transferred using iBlot 2 Transfer Stacks . The membranes were then blocked with Odyssey blocking buffer  for 1 h at room temperature with gentle shaking. Each membrane was treated with appropriate primary and secondary  antibodies. RNA Pol II and NF-YA levels were measured using an anti-Rpb3 antibody62 and anti-NF-YA antibody , respectively, with Ran  as a loading control. The following antibodies were used in Supplementary Fig. Western blots were performed as previously described2 ; 1 mM PMSF ; 0.15 mM Spermine ; 0.5 mM Spermidine ]. 7 ml of RSB + 0.5% IGEPAL CA-630  were slowly added, and the solution was incubated on ice for 10 min Nuclei were pelleted at 300 X G for 10 min at 4 °C and resuspended in 400 μl of digest buffer , and incubated with 40 U of MNase  for 5 min 400 μl stop solution  was added and digested nuclei were incubated at 65 °C for 90 min 24 μl Tris  pH 7.6, 3 μl Proteinase K ), and 3 μl GlycoBlue  were added and digestions were incubated overnight at 65 °C. DNA was extracted by phenol–chloroform extraction, and mono-nucleosome fragments were gel-purified from fractions digested to ~70% mono-nucleosome, ~20% di-nucleosome, and ~10% tri-nucleosome sized fragments. MNase-Seq libraries were prepared using NuGen’s Ovation Ultralow Library System V2 . See Supplementary Data ES cells were cross-linked for 45 s with 1% formaldehyde  followed by glycine  quenching for 5 min. Cells were washed once with ice-cold PBS and gently resuspended in 300 μl RSB  by incubating for 15 min on ice followed by 14 strokes with a loose pestle. The dounced cells were spun for 5 min at 500x g, the supernatant (cytoplasm) was discarded, the pellet resuspended in 30 ml of swelling buffer, and spun as above. The supernatant was discarded and the nuclei pellet was resuspended in 1 ml of swelling buffer, aliquoted and stored at -80 °C. Libraries were prepared according to the TruSeq Small RNA Kit . To normalize samples, 15 synthetic capped RNAs were spiked into the Trizol preparation at a specific quantity per 106 cells, as previously described69.RNA for Start-Seq experiments were prepared from control or 70 pairs with either mate trimmed shorter than 20 nt were discarded. A single additional nucleotide was removed from the 3′ end of each read to facilitate mapping of fully overlapping pairs. Remaining pairs were filtered for rRNA and tRNA by aligning to indices containing each using Bowtie 0.12.8 , and retaining unmapped pairs. Following this, a similar alignment was performed to an index containing the sequence of spike-in RNAs only , and finally, the remaining unmapped reads were aligned to the mouse (mm9) genome utilizing the same parameters, retaining only uniquely mappable pairs.Start-Seq reads were trimmed for adapter sequence using cutadapt 1.2.1;Strand-specific bedGraph files containing the combined raw counts of short-capped RNA 5′ ends for all control replicates were generated to facilitate observed TSS calling. For all other purposes, 5′ end counts were normalized per ten million mappable reads, then based on depth-normalized counts aligning to spike-in RNAs. Spike normalization factors were determined as the slope of the linear regression of each sample’s depth-normalized spike-in read counts versus the single sample with the lowest total count. Control and NF-YA knockdown bedGraph files were generated from these spike-normalized counts by taking the mean of all replicates, genome-wide, at single-nucleotide resolution.9, based on the control Start-Seq data, using mm9 RefSeq annotations downloaded from the UCSC genome browser . Briefly, the position with the highest read count within 1000 nt of an annotated TSS, or that with the highest count within the 200 nt window of highest read density was selected, depending on proximity. When insufficient Pol II ChIP-Seq signal existed in the 501 nt window centered on the selected locus, relative to a comparable window about the annotated TSS (a ratio less than 2:3), the observed TSS was shifted to the location with the highest Start-Seq read count within 250 nt of the annotated TSS. When fewer than five reads were mapped to the selected locus, the annotated TSS was maintained. Groups of transcripts with identical observed TSS were filtered, maintaining a single representative with the shortest annotated to observed TSS distance. Groups of observed TSSs within 200 nt of one another were similarly reduced by first removing any RIKEN cDNAs, predicted genes, or observed TSSs moved to the annotation due to lack of Start-Seq reads. Following this, a single observed TSS was selected based on observed to annotated proximity. In this manner, observed TSSs were called for 24,498 transcripts; of these, 16,483 were selected based on Start-Seq data, while for 8015 the annotated position was maintained. NF-Y-bound promoters were then identified as those with an NF-YA ChIP-Seq peak intersecting the observed TSS −900 to +100 nt window.Observed TSSs were identified as previously described71. Counts were determined for all samples in 10-nt bins tiling the region −995 to +995 nt, relative to each observed TSS. Bins closer to an upstream or downstream TSS than their own were excluded, as were those in the observed TSS −25 to +24 nt region. Normalization was performed based on size factors calculated within DESeq from spike-in RNAs alone, ensuring these values were equivalent across all samples. All bins with a positive log2 fold change and adjusted p-value less than 0.1 were identified. If more than one bin associated with a single observed TSS was selected, that with the lowest adjusted p-value was retained. Within each of these bins, the position with the total Start-Seq read count across all NF-YA knockdown samples was selected as the ectopic TSS. In cases where multiple sites exist with identical counts, that closest to the observed TSS was selected.Ectopic TSSs were identified through the comparison of NF-YA knockdown Start-Seq read counts to control using DESeq6 ESCs (per sample) were treated with cycloheximide  for 1 min prior to trypsinization and cell lysis. Control and NF-YA KD cells were used as input material for the TruSeq Ribo Profile Mammalian Library Prep Kit (Illumina) following the manufacture’s protocol.Approximately 8–9 × 1070 fragments shorter than 15 nt were discarded. Read pairs were filtered for rRNA and tRNA by aligning to respective indices using Bowtie 0.12.8 , and retaining unmapped pairs. The remaining read pairs were aligned to the mouse (mm9) genome using STAR v2.6.0c72. The read counts intersecting CDSs were determined per sample. We then determined the normalization factors from the aligned counts using DESeq2 v1.18.166. Using STAR’s bedGraph output , both sequencing runs were merged using unionBedGraphs (a component of bedtools 2.25.0). We then applied the previously calculated normalization factors to the merged bedGraphs. To obtain bigwig format files, both normalized biological replicates were merged using unionBedGraphs and then converted to bigWig. To infer position of ribosomes from Ribo-Seq reads, we used the 5′ end of each fragment. We then added 12 nt in order to locate the highest peak on the A of ATG. The same process was applied to reads from total RNA-Seq for comparison, to check that RNA-Seq read positions do not exhibit a 3n periodicity like Ribo-Seq reads do. To study the coverage in shifted regions, we used a two-step process. First, we searched for all the ATGs in those regions. Then, the ATG with the highest coverage within the downstream 60 nt was selected. We finally plotted the cumulative coverage, taking the A of the selected ATG as the anchor. Genes whose cumulative Ribo-seq signal, within the shifted region, represented > 5% of total Ribo-Seq reads were removed from the phasing analysis (n = 3).The total RNA-Seq and ribosome-protected-RNA-Seq (Ribo-Seq) read pairs were trimmed with cutadapt;73 (-dna -mod zoops -nmotifs 25 -minw 6 -maxw 20 -revcomp). TRAP74 was used to search for the enrichment of 680 known TF motifs, obtained from the JASPAR database75, within the promoter sequences (defined as 200 nucleotides upstream of the TSS). Statistical significance for enrichment of sequence motifs within promoters of interest were calculated in reference to promoter regions from all mouse genes. Benjamini–Hochberg method was used for multiple-testing correction.Around observed TSSs bound by NF-YA, those not bound by NF-YA, those bound by NF-YA with an associated ectopic TSS, as well as bound and non-bound ectopic TSSs themselves, de novo motif discovery was performed in the TSS +/− 50 region using MEME26. Per-nucleotide phyloP conservation scores, based on 20 placental mammals, were downloaded from the UCSC Genome Browser. Both data sets were converted to bedGraph format using custom scripts to facilitate generation of metagene analyses and heatmaps.Per-nucleotide predicted nucleosome occupancy for the mouse (mm9) genome was obtained from the authors of a previously published studySupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2