Saccharomyces cerevisiae. We started from foreign  core promoters as templates. They are, usually, unfunctional in yeast but can be activated by extending them with a short sequence, from the CYC1 promoter, containing various transcription start sites (TSSs). Transcription was modulated by mutating the TATA box composition and varying its distance from the TSS. We found that gene expression is maximized when the TATA box has the form TATAAAA or TATATAA and lies between 30 and 70 nucleotides upstream of the TSS. Core promoters were turned into stronger promoters via the addition of a short UAS. In particular, the 40 nt bipartite UAS from the GPD promoter can enhance protein synthesis considerably when placed 150 nt upstream of the TATA box. Overall, we extended the pool of S. cerevisiae promoters with 59 new samples, the strongest overcoming the native TEF2 promoter.Promoters are fundamental components of synthetic gene circuits. They are DNA segments where transcription initiation takes place. New constitutive and regulated promoters are constantly engineered in order to meet the requirements for protein and RNA expression into different genetic networks. In this work, we constructed and optimized new synthetic constitutive promoters for the yeast  Saccharomyces cerevisiae, the baker’s yeast, was the first eukaryote whose genome was completely sequenced [S. cerevisiae has been used in brewing, winemaking, and baking [S. cerevisiae has become a widely adopted chassis for genetic circuits due to its short growth cycle, simplicity of large-scale cultivation, and, above all, the easiness of genomic manipulation [S. cerevisiae cells into factories for the production of valuable chemicals such as drug precursors [equenced . Traditid baking . More repulation . These pecursors  and natuecursors .S. cerevisiae RNA polymerase II-dependent promoters are characterized by four main elements: The upstream activating sequence (UAS), the TATA box, the transcription start site (TSS), and the 5′UTR (untranslated region). Each of the first three elements can be present in multiple copies [The promoter is the place on the DNA where transcription starts. Hence, it represents a fundamental component of synthetic gene circuits. A set of e copies . The core copies .S. cerevisiae, such as the enhancers in higher eukaryotes, are bound by activator proteins that recruit the promoter RNA polymerase II together with the general transcription factor [The UASs in n factor . UASs arn factor .A consensus sequence for the yeast TATA box has been proposed by Basehoar et al.  as TATAWS. cerevisiae play an important role into synthetic gene circuits since they guarantee a relatively stable protein (or RNA) expression level under different conditions [S. cerevisiae cells and could also undergo homologous recombination with their original copy in the yeast genome.Constitutive endogenous promoters in nditions . Howevernditions  or buildnditions ,20. Thesp”) has been reported to be working in S. cerevisiae, as well [S. cerevisiae for transcription activation [To achieve orthogonality, either fully synthetic or foreign promoters shall be adopted. Viral promoters, for instance, are strongly active in mammalian cells and the cytomegalovirus promoter  and studied how to modulate its strength by: (1) Using different TATA boxes obtained by mutating, in each position, the strong TATAAAA sequence; (2) varying the distance between a selected, moderately strong, TATA box (TATATAT) and the TSS; (3) removing nucleosomes from the promoter sequence; (4) placing UASs (of variable length) upstream of the core promoter; (5) varying the distance between the UASs and the TATA box. The best results from each of these analyses were used together to improve the transcription initiation rate of both the strongest and the weakest core promoter in our initial library.S. cerevisiae genome, we found out important rules for designing and placing the sequences of both TATA boxes and UASs in order to modulate gene expression in yeast. Thus, our promoters and, more in general, our results can be used to fine-tune the working of synthetic transcriptional networks and metabolic pathways in S. cerevisiae.In addition to building, overall, 59 new yeast synthetic promoters that shared a minimal homologous region with the S. cerevisiae, by two viral promoters commonly used in mammalian cells—pCMV [S. pombe NMT1 promoter (no message in thiamine) [S. cerevisiae  and, therefore, represent a potential genomic burden that might have a negative effect on the promoter transcription efficiency. Both versions of pSV40 were unfunctional, whereas pCMVfull and the reduced CMV and NMT1 promoters turned out to be very weak, by expressing only 4.7, 1.5, and 2.3% of the fluorescence driven by the strong GPD promoter  . As for oter see .S. cerevisiae. Core promoters retain only the elements strictly necessary for RNA polymerase II binding without the help of any activator proteins. Therefore, we reasoned that the main hurdle to gene expression by foreign core promoters was the short distance between the TATA box and the TSS. At least 40 nt between a TATA box and a TSS are considered to be necessary to allow transcription initiation. It should be noted, though, that Redden et al. [As mentioned in the Introduction, foreign promoters are usually not activated in n et al.  showed tn et al. , 21 nt  minimal core promoters starting with a TATA box. Hence, with the only exception of pCaMV35Score (see below), from the promoters published in [CYC1 promoter, where six TSSs have been detected. In a previous work from our lab [S. cerevisiae promoters can be successfully built by placing pCYC1noTATA downstream of a TATA box-like sequence . In the extended version of pCMVcore, pCMVcore* (from now on we will indicate with a “*” any promoter extended with pCYC1noTATA) the TATA box is able to activate all six TSSs since the TATA box-TSS distance varies from 51 to 93 nt. In contrast, pSV40core* would fail to start transcription from the TSS at position 35 and 43 , whereas pNMT1core* activates only the TSS at position +1 (116 nt distant from the TATA box). As shown in CYC1core and ACT1 promoter  and a longer one also derived from the viral SV40 promoter (we will refer to it simply as pSV40) based on little changes on the sequences in  . Overalto 24 nt   we divided our 26 core promoters into eight groups according to their fluorescence expression . However, only the TATAAAG-containing promoter is significantly different from the other two promoters in Group 4. Thus, we decided to assign pSVcore*(TATAAAG) to a different group (number 3), that contains no other instances. The two strongest TATA boxes (the above-cited TATAAAA together with TATATAA), which forms Group 1, gave an average fluorescence level corresponding to about 91% of that of the constitutive ACT1 promoter. The only promoter in the second group, pSVcore*(TATATAC), is roughly as strong as the core CYC1 promoter [S. cerevisiae [Overall, we constructed 25 variants of the pSV40core*(TATAAAA) via single or multiple mutations on the seven nucleotides belonging to the TATA box. The highest mean fluorescence value was detected from TATAAAA, the lowest  from TATTAAT. By making a statistical analysis based on both one-way ANOVA and the two-sided Welch’s sion see A. We arepromoter  that retpromoter . From threvisiae .From our results, we can make interesting considerations about the effects of diverse mutations on different positions along the TATA box.As for single point mutations on TATAAAA, the least detrimental one was the change of an adenine to a thymine (T → A) at position 5, which did not significantly alter the core promoter strength.We checked all the possible three mutations at position 7. Each of them induced a more consistent decrease in fluorescence expression, the highest (68%) was due to the C → A substitution. Interestingly, this result is consistent with . In contWe made each of the possible three point mutations also at position 1. The decreases in fluorescence were significantly higher with respect to those concerning position 7. In particular, the G → T mutation at position 1 reduced fluorescence of even 84%. Single mutations at position 2, 3, and 4 affected fluorescence in a similar way to those at position 1 see . MutatioDouble and triple simultaneous mutations along TATAAAA confirmed the trend of reducing promoter strength with a fluorescence decrease ranging from 76 to 86% see . The onlLooking more in detail at the different kinds of point mutations, we saw that T → A is responsible for a strong decrease in fluorescence expression, unless it takes place  at position 5. In particular, a simultaneous T → A mutation at both positions 5 and 6 returned an 83% fluorescence repression see B, pointiIn general, a TATA box mainly contains adenine and thymine but a single cytosine or guanine can be found in natural TATA boxes. The insertion of a single “C” or “G” into TATAAAA caused a reduction in fluorescence, though without following a precise pattern. At position 1, C → T is more favorable than G → T (and even A → T), whereas at position 7, G → A is more tolerated than C → A. In contrast, C → A has a relatively low effect if carried out at position 7 of TATATAA, whereas G → A and T → A have an apparent negative influence on gene expression see .Overall, we have seen how simple modifications on the TATA box of a synthetic core promoter span a 9.3-fold dynamic range in gene expression. In our analysis, TATAAAA and TATATAA resulted in being strong signals, whereas the weakest was TATTAAT that, interestingly, does not contain any cytosine, guanine or thymine at position 6—a feature that seemed to attenuate considerably DNA transcription. Moreover, differently from what was present in the literature, we found out that a thymine and a guanine at the seventh position cause a strong decrease in gene synthesis. This might indicate that context effects due the nucleotides directly upstream and downstream of the TATA box play a so far not understood role in DNA transcription, which needs further study.After ranking the TATA box heptamer according to the fluorescence they drove from pSV40core*, we looked for the optimal distance between the TATA box and the TSS to maximize gene expression.A TATA box is believed to activate only TSSs that are placed from 40 up to 120 nt downstream of it . HoweverFor this new analysis, we selected the configuration of pSV40* that contained the moderately weak TATATAT box (roughly 25% as strong as TATAAAA).We constructed nine variants of pSV40*(TATATAT) by varying the distance between the TATA box and TSS from −129 to +12 nt, being −90 nt the original one . As shown in S. cerevisiae due to a non-optimal spacing between the TSS and TATA box. In contrast, promoters where TATATAT was −29, −39, −49 or −69 nt far from the TSS showed up to a ~2.5-fold increase in the fluorescence level of the original pSV40* (where TATATAT is 90 nt upstream of the TSS). The variant whose distance was −39 nt showed a lower mean fluorescence level. However, it was not significantly different in statistical terms from the other three. On the whole, our analysis shows that the region where the TATA box should lie in order to achieve maximal transcription goes roughly from position −30 to −70 with respect to the TSS.Too short or too long distances  determined low fluorescence expression, which confirmed the initial conjecture about the inoperativeness of foreign promoters in As a further attempt to improve the strength of our new synthetic promoters, we tried to remove possible nucleosomes from their sequences ,39. In tS. cerevisiae, UASs should lie at a proper distance from the TATA box, i.e., between 100 and 1400 nt [After finalizing the construction and the investigation of our core promoters, we built on them novel hybrid promoters by placing an UAS upstream of their TATA box. UASs recruit activators of different strengths and, therefore, can increase considerably the transcription initiation rate of a promoter. Moreover, in  1400 nt . For thi 1400 nt .GPD(long) (532 nt) [TEF1(long) (203 nt) [GPD(40nt) [TEF1(RAP1bs) [TEF2(59nt) [FEC built by Redden et al. [Initially, we explored the effect of different UASs on DNA transcription from pSV40*(TATATAT−49) again. We chose the long UAS-plus-spacer sequence UAS(532 nt)  and UASTF1(long) 3 nt [20,(203 nt) . In addiPD(40nt) ; the 20 (RAP1bs) ; and theF2(59nt) . Finallyn et al. . All theTEF2(59nt) (1.65-fold increase), the best one UASGPD(long) (2.45-fold increase). However, in statistical terms, UASGPD(long), UASGPD(40nt), UASTEF1(long), UASTEF1(RAP1bs), and UASFEC did not show any significant difference, pointing out that the length of synthetic promoters can be shortened remarkably by considering only endogenous activator binding sites as UASs (see Each UAS contributed to an increase in the fluorescence expressed by pSV40*(TATATAT-49). The least performant one was UASUASs see .GPD(40nt) 150 nt upstream of the pSV40*-45t, which resulted in a 1.91-fold increase in the promoter strength  to the TATA box.FEC and a core promoter was enough to achieve a very high fluorescence level. This short distance permitted, as well, obtaining minimal synthetic promoters. We placed, therefore, UASGPD(40nt) just 30 nt upstream of the TATA box of pSV40*(TATATAT-49). Compared to the bare pSV40*(TATATAT-49), the increase in fluorescence was modest (1.36-fold), whereas a 150 nt spacer guaranteed a 2.33-fold enhancement. Therefore, a too-short distance between UAS and TATA box cannot be regarded as a universally valid strategy to enhance the promoter strength strongly  150 nt upstream of the TATA box.In the previous analyses, we found that the activity of SV40*-based promoters can be improved using the heptamer TATAAAA (or TATATAA) as a TATA box, setting the distance between the TATA box and the TSS to 49 nt, and placing the short UASGPD(40nt) and the TATA box to 30 nt could give a considerable enhancement in gene expression.According to our initial measurements, MLPcore* and pTKcore* were the strongest and the weakest synthetic core promoters obtained by extending their native sequences with pCYC1noTATA. In order to see if our findings had general validity, we modified both MLPcore* and pTKcore*, where necessary, with the same changes that were effective on pSV40*. Moreover, we checked if a reduction, on both promoters, of the distance between UASGPD(40nt) either 30 or 150 nt upstream of the TATA box. In the former case, no significant difference was detected with respect to the original MLPcore*, whereas the latter provided a 1.77-fold increase in fluorescence expression. Moreover, the fluorescence intensity expressed by UASGPD(40nt)−150nt-MLPcore* exceeded that of the strong yeast constitutive TEF2 promoter . Thus, we constructed two variants of this promoter simply by adding UASoter see  such thaGPD(40nt) from the TATA box induced a huge improvement in the promoter performance: 17.54- and even 27.45-fold, respectively  is very different from the optimal one and had to be replaced. In this case, both configurations with 30 and 150 nt distancing UASvely see B. TherefS. cerevisiae synthetic promoters based on foreign template sequences coming from viral and eukaryotic cells.In this work, we constructed a large library of overall 59 new Foreign promoters, in yeast, are either unfunctional (such as pSV40) or poorly performant  due to the absence of the activators necessary to recruit RNA polymerase II and the short distance between the TATA box and the TSS.S. cerevisiae by extending them with an 87 nt long sequence that included the whole yeast CYC1 promoter 5′UTR, where at least six TSSs are present. This guaranteed a sufficient distance between the TATA box of the foreign promoter and some TSSs of CYC1 promoter to initiate transcription. We managed to make foreign promoters work in S. cerevisiae [Our experiments permitted us to confirm—as already pointed out in —that therevisiae . Moreoverevisiae . By carrGPD, TEF1, and TEF2 promoters can highly enhance promoter activity, especially when placed 150 nt upstream of the TATA box. In contrast, a much lower distance (30 nt) does not appear to be always effective.These initial experiments allowed the construction of 47 core promoters. We turned some of them into stronger synthetic promoters with the addition of an UAS. Importantly, we showed how short natural UASs from the constitutive TEF2 promoter. Additionally, the usage of a foreign template lowers the risk of homologous recombination though both UAS and 5′UTR are taken from endogenous promoters and, therefore, prevent full orthogonality.Overall, most of the core promoters in our library are relatively weak with three of them showing a moderate strength. The addition of a (short) UAS allowed the construction of stronger promoters, one of them overcoming the strong yeast constitutive Developing synthetic leaders and UASs represent the next step to realize libraries of synthetic orthogonal promoters spanning a wider dynamic range in gene expression that will be beneficial for many diverse applications of Synthetic Biology.The backbone for all plasmids constructed in this work was the yeast integrative shuttle-vector pRSII406  . The plaCYC1 terminator (CYC1t) [Every transcription unit realized in this work is composed of a synthetic promoter followed by the HIS tag, the yeast enhanced green fluorescent protein (yEGFP) , and the (CYC1t) . All theEach plasmid containing a novel transcription unit was assembled via the Gibson assembly method . To thisE. coli competent cells  were transformed with all the DNA fragments mixed in equimolar amount (30 s heat-shock at 42 °C) and grown overnight at 37 °C in LB (Luria-Bertani) plates  supplied with ampicillin. Plasmid extraction from bacterial cells was carried out using standard methods [ methods . All plaS. cerevisiae strain CEN.PK2-1C  kindly provided by Yuan Yingjin . Plasmids (5 μg) were linearized with the restriction enzyme StuI (NEB-R0187V) that cuts inside the URA3 marker. Genomic integration was carried out according to the lithium-acetate protocol, as described in [Each of our new plasmids was integrated into the genome of the yeast ribed in . TransfoTM Flow Cytometer . The FACS machine set-up was checked at the beginning and end of each experiment using fluorescent beads (BD FACSuiteTM CS&T Research Beads—650621). Measurements were considered as reliable only when the relative difference between the initial and final value of the peaks of the beads was not higher than 5%. Each strain was measured, at least, in three independent experiments . During each experiment, 30,000 events were recorded.Yeast cells were initially grown in a synthetic complete medium (SDC) at 30 °C, 240 RPM, for 18 h. Then, cells were 1:40 diluted. Green fluorescence was measured with a BD FACSVerset-test. In contrast, one-way ANOVA was adopted to analyze groups of more than two strains .Data from the flow cytometer was analyzed with the flowcore R-Bioconductor package . The mea


The mechanism involves transcriptional read-through from the flanking promoter. This conclusion is based on the following. Read-through driven by a heterologous enhancer is sufficient to repress, even when homie is in place. Furthermore, when the flanking promoter is turned around, repression is minimal. Transcriptional read-through that does not produce anti-sense RNA can still repress expression, ruling out RNAi as the mechanism in this case. Thus, transcriptional interference, caused by enhancer capture and read-through when the insulator is removed, represses eve promoter-driven expression. We also show that enhancer-promoter specificity and processivity of transcription can have decisive effects on the consequences of insulator removal. First, a core heat shock 70 promoter that is not activated well by eve enhancers did not cause read-through sufficient to repress the eve promoter. Second, these transcripts are less processive than those initiated at the P-promoter, measured by how far they extend through the eve locus, and so are less disruptive. These results highlight the importance of considering transcriptional read-through when assessing the effects of insulators on gene expression.Several distinct activities and functions have been described for chromatin insulators, which separate genes along chromosomes into functional units. Here, we describe a novel mechanism of functional separation whereby an insulator prevents gene repression. When the  per se. When the insulator homie is deleted from the end of a transgenic eve locus, a flanking transposable element promoter is activated by eve enhancers, causing repression of the eve promoter. The mechanism involves transcriptional read-through from the flanking promoter, which disrupts normal eve enhancer-promoter activities. When the flanking promoter is turned around, repression of eve is minimal. Thus, transcriptional interference, caused by enhancer capture and read-through when the insulator is removed, represses the eve promoter. These results show a novel role for transcriptional read-through in the effects of insulators on gene expression.Several distinct activities and functions have been described for chromatin insulators, which are regulatory DNA elements that separate genes along chromosomes into functional units. Here, we describe how insulators can prevent repression of one gene by preventing inappropriate transcription of another gene, without blocking read-through of transcription  Recent studies have shown that insulators are multi-functional elements, performing several important tasks . They blcis-regulatory domains in the bithorax complex (BX-C) have been well studied [Fab-7 boundary separates the iab-6 and iab-7 cis-regulatory elements of the Abdominal-B (Abd-B) gene that control phenotypes of parasegments (PSs) 11 and 12, respectively. Mutations that remove Fab-7 cause transformation of PS11 to PS12, explained by ectopic activation of iab-7 in PS11 due to a loss of enhancer blocking activity in early embryos. Later studies found that Fab-7 is also required for insulating the repressive effects of a Polycomb Response Element (PRE) that prevents inappropriate activation of Abd-B in specific tissues [In Drosophila, insulators (a.k.a. boundaries) that separate adjacent  studied –25. For  tissues ,27. Geno tissues .abdominal-A (abd-A) in segment A1 [Insulators are also involved in defining TADs  ,29,30. Agment A1 ,33. Simigment A1 ,35. Whilgment A1 ,37, CTCFgment A1 . Anothergment A1 , while mgment A1 . Despitescs insulator [iab-8 regulatory domain of the BX-C and reads through several insulators, is required for repression of abd-A in parts of the nervous system, so that disrupting it causes defects [Roles for nuclear RNAs in regulating chromatin structure and gene expression have been uncovered in recent years –43. Trannsulator  have bee defects .eve expression, so that even minor changes cause developmental defects, making manipulations of the endogenous locus problematic [eve locus and the 5’ end of the 3’-neighboring gene, TER94. In this pseudo-locus, the eve protein coding region is replaced by that of lacZ, while that of TER94  [eve insulator homie from the pseudo-locus reduced eve-lacZ activity, particularly that of enhancers located 3’ of the transcription unit [eve locus, causes severe, lethal disruptions in segmentation, illustrating the value of using the pseudo-locus to study eve regulation [Segmentation is very sensitive to the levels of blematic ,49. In oblematic , which ced gene) –52 is reed gene) , this psed gene) . Removinion unit ,55. Thision unit . Early sion unit –59, and gulation ,49.eve locus, homie and nhomie [eve enhancer activity by a flanking P-element promoter [eve enhancers in a pattern that matches the pattern of repression of eve-lacZ. We further show that the repression is primarily attributable to transcriptional interference by read-through, with a minor contribution from promoter competition. The imbalance is not prevented by the insertion of multiple poly-adenylation (polyA) signals from the α-tubulin gene [SV40 [heat shock 70 (hsp70) locus [eve-lacZ expression. These studies add to our knowledge of the mechanisms underlying disruptions of gene expression seen when insulators are removed, or their functions compromised.Here, we focus on the two insulators that flank the d nhomie ,12,19. Wpromoter –63. The lin gene  and SV40ne [SV40 , because0) locus  does notnhomie [eve-lacZ, making the pseudo-locus identical to the endogenous eve locus including the 5’ end of TER94, except for the replacement of protein coding sequences . However, when nhomie was removed in this N5 orientation, both early stripe 3 at stage 5  and the later 7-stripe expression at stage 7 . In contrast, in the H5 orientation where removal of homie had a dramatic effect, removing nhomie had little effect . We tested the same pseudo-locus transgenes inserted at other P-element-attP sites, and they showed the same tendencies, but with even stronger repressive effects when the insulator at the “5’P” end of the locus was removed , which contains a P-element promoter [eve-lacZ enhancer activity. To begin to test this, we quantified transcripts in the eve regulatory regions using RT-qPCR. First, we found that transcripts were barely detectable throughout the endogenous eve locus . With the wild-type pseudo-locus, slightly higher transcript levels were seen (yw”). However, without homie (homie”), transcript levels were increased. Consistent with our hypothesis, deletion of nhomie in the H5 orientation showed similar levels of transcripts to wild-type (nhomie"). This is potentially attributable to a blocking activity of insulators, since nhomie in the H5 orientation is not between the eve enhancers and P-element promoter . The ratio of Δhomie or Δnhomie transcripts to wild-type showed a maximum near the 5’P-element end, and tended to decrease throughout the locus , along with faint stripes 2, 3, and 7. Importantly, this expression pattern correlates with the pattern of repression of eve-lacZ . In order to test whether this non-coding RNA expression is unidirectional, consistent with its coming from the P-element promoter, we used probes that recognize the opposite strand. With these probes, no detectable expression pattern was observed in any of the enhancer regions .What is activating the P-element promoter? The nhancers . In a pr removed . It is pnhomie R). Weak stripe 1 expression has been shown to be driven by sequences upstream of the eve start site [eve expression are driven by enhancers upstream of the eve promoter. This expression pattern can be seen throughout the locus, suggesting that the transcripts detected are continuous, long non-coding RNAs R, (2+7)R, (4+6)R, (1+5)R). We did not observe any expression pattern with probes that recognize the opposite strand , although there are faint stripes with “late” probe (nhomie), similar to the ones seen in the H5 orientation (described above).In the N5 orientation, weak expression in the pattern of both early stripes and the 7 late stripes was observed downstream of the P-promoter, even in the presence of art site , in addiart site . Therefoe” probe , Δnhomiehomie") using RT-qPCR. We produced cDNA using specific primers to late or EL enhancer regions, which anneal to transcripts coming from the direction of the P-element promoter , although we detected some stripe imbalances when both homie and nhomie were deleted . However, these imbalances were more variable and less severe than those seen in eve rescue constructs lacking the insulators were inserted in random locations throughout the genome [eve enhancers is the main cause of the repression of eve-lacZ expression in the absence of an intervening insulator.In order to confirm that the 5’P-element end is the source of the transcriptional read-through, we used a MiMIC line , which de genome ,55. Thereve-lacZ repression when the intervening insulator is removed. What are the mechanisms behind this? It is possible that the read-through itself disrupts eve enhancer activity, as read-through from both P-element [eve-lacZ , stripes 1, 4, 5, and 6 were observed in the enhancer regions .The above experiments implicate the flanking P-element promoter in causing -element  and UAS--element . In the  used in  that shoromosome , "wt". Ts intact , “Pwt”. s intact , “PΔ”, h present , “PinvΔ” present , except promoter , consisteve-lacZ is caused by transcriptional read-through, then lacZ RNA would be reduced in PΔ, but not in PinvΔ. If promoter competition is the cause, then lacZ RNA would be reduced in both PinvΔ and PΔ. In fact, lacZ RNA is reduced in PΔ, but not in PinvΔ. Therefore, eve-lacZ repression requires transcriptional read-through, while promoter competition alone is insufficient. This is the case even though when read-through is occurring, the enhancers driving the read-through expression are themselves subject to the read-through. We also cannot rule out a contribution to eve-lacZ repression from read-through of the eve promoter (in addition to the enhancers), which could disrupt either initiation itself, or E-P communication. It is also possible that promoter competition plays a role, even though it is not sufficient by itself to cause significant repression . When homie was removed, stripes in the ventral region became stronger, and the same stripes were also visible in the lateral and dorsal region, consistent with the removal of homie’s enhancer blocking activity (eve-lacZ was repressed wherever these transcripts were present (homie still not present), NEE-driven transcript expression was somewhat reduced, but the pattern of expression was the same as with the original orientation , showing that the “potentially competing” promoters are sharing NEE enhancer activity in the absence of homie. Interestingly, when there is read-through of the eve promoter, lacZ is not expressed in the NEE pattern, suggesting that read-through of the eve promoter region is sufficient for repression, since the NEE itself is not experiencing read-through (rhomboid NEE) causes disruption of eve expression, indicating that read-through is sufficient for repression. When homie is present, read-through transcription is driven primarily by the NEE, and eve-lacZ is repressed only where there is read-through. This shows that homie blocks only E-P interactions, not read-through transcription itself, at least of those transcripts initiated by the P-element promoter .An intervening insulator blocks communication between the cer, NEE , was addactivity . Again,  present . When th-through . In summhsp70 core promoter [NEE (NEE-hspGFP). With homie in its normal place in the pseudo-locus, GFP transcripts are expressed in a similar pattern to the transcripts detected in the comparable construct with the P-element promoter, NEE-Pwt , consistent with the loss of enhancer blocking activity. However, NEE-driven expression is almost undetectable in the eve regulatory region, with only very faint eve stripes seen on the ventral side . When this NEE-hspGFP cassette is turned around to read away from eve-lacZ, NEE-driven expression of GFP transcripts, as well as strong ventral eve stripes with weak dorsal/lateral stripes, are seen (GFP and 24B). Consistent with read-through being required for repression, eve-lacZ is expressed similarly to wild-type (lacZ probe), except that some NEE-driven expression is also seen. It is likely that homie blocks this E-P communication in the “wt” construct (lacZ probe). These data suggest that the hsp70 core promoter may not respond as well to the eve enhancers as does the P-element promoter, at least at distances of several kb. They also suggest that the hsp70 core promoter initiates transcription that has low processivity, relative to that initiated by the P-element promoter, when driven by the same enhancers.To examine the effects of promoter specificity on the actions of flanking promoters in this system, we turned to the promoter , which hpromoter . To thishsp70 promoter’s response to eve enhancers is weak due to competition with the NEE, we removed the NEE, as well as the GFP coding region. Thus, this construct is equivalent to PΔ, but with the hsp70 core promoter replacing the P-element promoter . Consistent with this difference in transcription levels, eve-lacZ expression was not noticeably repressed in hspΔ, while it was repressed in PΔ. RT-qPCR analysis showed that RNA expression at the enhancer regions from the hsp70 promoter are 10–30% of those from the P-element promoter (hspΔ/PΔ). These data suggest that the hsp70 core promoter is not activated as well by the eve enhancers as is the P-promoter  were stained with probes located at different distances downstream. The promoter-proximal probe showed comparable levels of transcripts in both (GFP"). In contrast, by the time transcription reached the stripes 1+5 enhancer region, expression was much lower in NEE-hspGFPΔ than in NEE-PGFPΔ (GFP and PRE regions in NEE-hspGFPΔ and NEE-PGFPΔ (GFP and PRE primer sets). However, consistent with the levels seen in hsp70 promoter , the commonly used α-tubulin polyA signal [homie, used in Figs eve promoter, stopping transcription before it reaches the promoter would rescue all eve-lacZ expression. If read-through is inactivating only enhancers, stripes 1 and 5 would still be repressed. Unexpectedly, the transcription pattern beyond the α-tubulin polyA signal did not diminish much at the 4+6 enhancer region, indicating that the polyA signal did not effectively stop read-through . There may also be an increase in expression of the other stripes relative to that of stripe 5, but the effect remains subtle. Quantitation of transcript levels by RT-qPCR showed that introduction of the polyA signals caused lower levels from the P-promoter overall  is intact.We further show that insulators do not block read-through reported . Insteadhomie or nhomie allows a promoter outside the locus to interact with eve enhancers, and by reading toward the eve locus, capture their activity and repress the eve promoter. Similarly, a recent study showed that removing the Fub insulator in the BX-C caused collapse of the corresponding TAD boundary [A recent study showed that TADs in Drosophila usually correspond to a single locus . This suboundary . Our dateve-lacZ expression, and that homie does not block read-through. Is this an unusual case caused by a P-element promoter? Studies of the BX-C showed that long non-coding RNAs (ncRNAs) can read through insulators and affect gene regulation [iab-8 ncRNA extends for 92 kb in the posterior segments of the embryo where abd-A is expressed at a relatively low level, and is required for repression of abd-A in the central nervous system. Two repression mechanisms were suggested, a trans effect due to a microRNA produced from the iab-8 ncRNA, and a second effect acting in cis. The cis effect is most likely transcriptional interference caused by transcription through abd-A [iab-8 ncRNA extends through several abd-A regulatory modules containing early- and late-acting enhancers that are separated by insulators [iab-8 ncRNA promoter, because the intervening insulators do block enhancer-promoter interactions. Taken together, these studies suggest that gene repression by transcriptional read-through is a likely outcome when insulator function is disrupted.In this study, we showed that transcriptional read-through effectively represses gulation . For exagh abd-A . The iabsulators . Consisteve enhancers can activate a flanking P-element promoter in the absence of an intervening insulator. In particular, when homie was present, a flanking P-element promoter was not detectably activated by the eve enhancers. On the other hand, we did observe some weak stripe expression when nhomie was the intervening insulator. This expression was, as expected, much stronger when nhomie was removed (nhomie"). These data show that nhomie’s enhancer blocking activity is somewhat weaker than that of homie. More generally, our results suggest that while blocking of cis effects along chromosomes by insulators may not be absolute, inappropriate interactions become weak enough when they are present so that genes can be considered separate functional genetic units, in most cases.The in situ hybridization using probes against the opposite strand . One line of evidence for this is that the RNAs are unidirectional, since they were not detected by e strand  Figs. Ine strand . This coeve enhancers by adding polyA signals to terminate read-through transcription between enhancers. We were surprised to find that the read-through transcription was not effectively stopped even by a tandem array of 3 polyA-addition signals, one from α-tubulin and 2 from SV40. We did see evidence of weak attenuation, accompanied by correspondingly weak rescue of a downstream enhancer’s activity on the eve promoter (eve promoter (see below). Consistent with the P-element promoter initiating highly processive transcription, wild-type P-elements have two polyA signals. The first does not stop transcription completely, resulting in two sizes of transcript [What are the mechanisms of transcriptional interference by read-through? One possibility is inactivation of enhancers, since the transcriptional machinery must at least transiently displace DNA binding proteins as it passes. How strongly such displacement affects promoter activity is expected to depend on the dynamics of the E-P interactions in any given case. We attempted to see if this is the case for anscript ,72.In light of the fact that many genes have enhancers in their introns, it may seem implausible that enhancers would be inactivated by read-through. However, such an effect would only be expected to reduce the rate of transcription, since a reduced rate of transcription would result in a reduced amount of repression, and eventually a steady-state balance would be reached. Since most genes are not expressed at very high levels, such an effect is entirely plausible.NEE enhancer), so that the enhancer itself is not being read through, seems sufficient to repress eve promoter activity. Although we cannot completely rule out that the repression we observe is due to enhancer read-through, it is more likely the result of disruption of both enhancers and the eve-lacZ promoter. Thus, our data are consistent with the idea that enhancer-promoter interactions are disrupted by read-through of either interacting element.Read-through driven by a heterologous enhancer inserted upstream of the P-promoter  is downstream of the NAi Figs  and 4B. cZ e.g., , when recZ e.g., , we obsewnstream . This isbxd region of the BX-C repress Ubx expression in cis by transcriptional interference [scs insulator generates read-through transcription that can inactivate a PRE [It was reported that a P-element inserted into the BX-C caused segment A1 to A2 transformation, and this effect was ascribed to a P-promoter that produced a long ncRNA . Thus, arference . More cote a PRE . These reve locus, according to Flybase [TER94 (lncRNA:CR45324); however, this initiation site is not present in any transgenes used in eve coding region. This initiation site is not present in any of our constructs, since we replaced the eve coding region with that of lacZ. Furthermore, according to Flybase, neither of these RNAs is expressed at any of the stages used in this study. The initiation site of the 3rd ncRNA (lnc-RNA:CR46455) is present in our transgenes. It is near the 5’ end of the eve locus, within the late 7-stripe element, and is transcribed toward the eve coding region. This transcript should be recognized by our “(late)R” probe, but we did not detect any pattern with this probe (homie). RT-qPCR showed very low expression in this region in our control yw fly strain , but not in embryos without a transgene (yw). The “late” probe detects transcripts coming from downstream of the “late” enhancer. However, the pattern is not the one expected for transcripts coming from the downstream P-element promoter in Figs eve start site). Therefore, it is likely that there is another ncRNA start site downstream of the “late” stripe enhancer, but 5’ of the eve transcription initiation site. Nonetheless, this weak expression does not correlate with any of the results in this study, and so we conclude that it is unlikely to be consequential for either eve or eve-lacZ expression.There are 3 ncRNAs in the  Flybase . One is is probe , Δhomie.y strain . Therefoines wt; , Δnhomieransgene , yw. Thehsp70 promoter is used in place of the P-element promoter, read-through transcripts are weakened, as is repression. This suggests that the P-element promoter is relatively efficient at responding to eve enhancers . However, the level of transcripts from the hsp70 promoter dropped relative to those from the P-promoter between the position of the 3’ PRE and the RP2 neuronal enhancer (2.3 kb from the start site). We consider two possibilities for this apparent termination of transcripts from the hsp70 promoter. First, the eve locus is known to be a Polycomb domain, which is characterized by the histone methylation mark H3K27me3 [hsp to terminate more frequently than that from the P-promoter. A second possibility is that transcription from hsp is responding to a termination signal where ncRNA CR45324 ends, which is between the RP2 enhancer and the 3’ PRE. (This ncRNA is initiated within the 1st exon of TER94.) In summary, these data suggest that the P-promoter drives more processive transcription in combination with a variety of enhancers than does the hsp70 core promoter. This processivity allows RNAPII to read through eve enhancers located many kb away, when there is an intervening insulator, and even through and far beyond multiple polyA signals. It will be interesting to investigate how different promoters, activated by the same enhancers, cause differences in RNAPII processivity.Importantly, little reduction in intensity is seen between transcripts detected at different distances downstream of the P-element promoter, suggesting that read-through is processive Figs  and 4. Iwnstream . This is the NEE . For NEEpression , analysiH3K27me3 ,77. ThisC. elegans showed that the promoter can influence which mode is used [As discussed above, when polyA-addition signals were introduced downstream of the flanking P-promoter, RNAPII did not efficiently terminate transcription. Two models for how transcription termination occurs have been proposed . A recen is used . Furthereve-lacZ. This is consistent with either promoter competition or transcriptional interference as a mechanism of repression. However, when the P-promoter is turned around so that read-through is eliminated, repression is strongly reduced, showing that read-through is necessary for the repression . However, if transcription from the P-promoter reads toward the eve locus, and if that transcription interrupts stable eve E-P interactions, freeing the enhancers to interact with the P-promoter, it could facilitate enhancer capture by the P-promoter, allowing it to better compete for the enhancers. This in turn would boost P-promoter activity at the expense of eve promoter activity, repressing eve. This mechanism might also explain why the hsp70 core promoter is not activated as well by eve enhancers as is the P-promoter, since transcription from it can’t effectively release the eve E-P contacts, due to its low processivity. Additional work will be required to understand the dynamics of these interactions, and to tease out how much of the repression that we see is due to transcriptional interference caused by read-through of enhancers, how much is due to transcriptional interference caused by read-through of the eve promoter, and whether promoter competition plays a significant role. These repression mechanisms are not mutually exclusive, and all can be a consequence of insulator removal.There is an important caveat to this view, however. When the P-element promoter is turned around, its level of transcription appears to be reduced. If this is indeed the case, it suggests another interesting possibility, one that involves significant promoter competition. If we suppose that eve pseudo-locus constructs have been described previously [nhomie insulator [eve TSS was fused to the lacZ coding region. The 3’ end of the lacZ coding region was fused to DNA from +1.3 to +11.4 kb, which includes the eve polyA signal, and extends into the 3rd exon of TER94. The EGFP coding region, followed by the polyA signal of α–tubulin, was added at the 3’ end. The entire construct was placed between two oppositely oriented attB sequences [homie (from +9.3 to +9.8 kb) in these constructs by λ DNA or by heterologous insulators was described previously [nhomie deletion (described herein as Δnhomie). The heterologous insulator assays of homie in that figure) were done in this Δnhomie context. Exact end points are shown in attP sites that had previously been introduced into the Drosophila genome by P-element transgenesis [homie”) end of the locus is close to the 5’ P-element end, or the "N5 orientation", in which the 5’ (“nhomie”) end of the locus is close to the 5’ P-element end. The attP sites used are those at cytological locations 95E5, for Figs The original eviously . A modifnsulator  was usedequences . Replaceeviously . The orisgenesis . The dirnhomie), TER94-EGFP was removed, and the 3’ end was shortened to +9.8 kb, which is the 3’ end of homie. The homie sequence (from +9.3 to +9.8 kb) was then replaced by λ DNA. Modified forms of these with either “wt” or “Δ” in the name were used in the figures after P) [rhomboid neuroectodermal enhancer (NEE-P) [heat shock 70 core promoter [NEE-hspGFP) was then inserted at the 3’ end of each of these, in either orientation, as diagrammed in Figs NEE was inserted into the construct without homie to give NEE-PGFPΔ  –63, the  (NEE-P) , or the promoter  driving  used in ; an α-tuA signal  was inseA signal  plus two40 polyA  signals 40 polyA ,74. OrieRP49 was used as a reference gene, while eve was also used as a reference gene for lacZ expression in RP49 value  transgenes in the H5 orientation  transgenes in the N5 orientation  RT-qPCR quantification of total RNA  from the indicated enhancer regions (probe locations shown as red bars below the map), in the wt and Δhomie transgenic lines  The ratios of average signals (with standard deviations) from wt and Δhomie in B, and wt and Δnhomie in D are graphed. Note the general trend toward a decrease in average signal moving away from the location of the 5’ P-element end (the right side in B and the left side in D).tion see  at site tion see  at 23C4. used in  in A, or used in  in C. Av(DOCX)Click here for additional data file.S2 FigMap: Transgene construct, showing location of the 5’ P-element end, where read-through transcripts are initiated. Images: RNA in situ hybridization to embryos at stage 5 carrying Δnhomie at 23C4 (used in  used in . Probes (DOCX)Click here for additional data file.S3 FigRP49, analyzed in parallel for each sample, using RP49-specific primers). (A) cDNA from wt and Δhomie transgenes at 74A2 . (B) Same as A, except that cDNA is from Pwt and PΔ transgenes (used in cDNA primed in either the "late" or “EL” (green block arrows) enhancer regions were analyzed by qPCR using primers recognizing other enhancer regions (red bars below the map) Click here for additional data file.S4 Fig(A) Top: Map of the eve pseudo-locus. miR and miL are Minos inverted repeats. Bottom: Expression of eve-lacZ from the pseudo-locus inserted into a MiMIC site at cytological location 24B1 detected by in situ hybridization. Both orientations of insertion are shown (left and right panels) at embryonic stages 5, 7, 11, 13, (as indicated), with either nhomie alone (Δnhomie), or both nhomie and homie , deleted. (B) Top: Map of the eve pseudo-locus modified by removal of TER94-GFP. Bottom: Same as in A, using a line carrying this modified pseudo-locus.(DOCX)Click here for additional data file.S5 FigSame as (DOCX)Click here for additional data file.S6 FigMap: The wt transgene with locations of PCR products used for transcript detection shown as red bars under the map. (A) Loci used in PΔ), PinvΔ), and yw  were subjected to RT-qPCR quantification of total RNA  from the indicated enhancer regions. Averages (with standard deviations) of 4 biological samples each are graphed. Note that transcript levels are strongly increased throughout the locus in PΔ, but not in wt or PinvΔ. (B) The ratios of average signals (with standard deviations) from PΔ and wt are graphed. Note the general trend toward a decrease in the relative PΔ signal moving away from the location of the P-element promoter. (C) Similar to A, except that the set of wt, PΔ, and PinvΔ transgenes are inserted in the opposite orientation at the same MiMIC site, and averages (with standard deviations) of 5 biological samples each are graphed. (D) The ratios of average signals (with standard deviations) from PΔ and wt in C are graphed.(DOCX)Click here for additional data file.S7 FiglacZ coding region in embryos, in either wt and Δhomie (in A), or wt and Δnhomie (in B), normalized to control RNA from either RP49 or endogenous eve, as indicated. (A) Line used in (B) Line used in RP49 is not significant at the P < 0.05 level, but relative to eve it is significant at the P < 0.05 level . Normalizing to eve expression may better control for variations in the developmental stages represented in the embryo collections that were the source of the RNA.RT-qPCR quantification of total RNA from the (DOCX)Click here for additional data file.S8 Figeve-lacZ and flanking P-element promoter-driven RNA expression in stage 5 embryos from eve pseudo-loci inserted at the 24B1 MiMIC site. Orientation of inserts are the same as in homie replaced with phage λ DNA (refer to maps). Positions and orientations of the RNA probes used are shown as black and red arrows in the map. Insertion site of polyA signals is shown in the maps of B and D. Affected lacZ stripes are labeled as 4, 5, and 6 (compare the intensity of stripes 4 and 6 to stripe 5 in the same embryos).  Same as in α-tubulin polyA signal is present here in B.  Same as in NEE-PGFP cassette used in (DOCX)Click here for additional data file.S9 FigRP49 RNA) from the indicated enhancer regions (shown as red bars) in the PΔ and PpA2Δ lines used in (A) Averages (with standard deviations) of 4 biological samples each are graphed. (B) Ratios of signals (average and standard deviation) from PΔ and PpA2Δ are graphed. (C) Quantification of lacZ RNA . The increase in lacZ RNA in PpA2Δ relative to that in PΔ is significant at the P < 0.01 level .Quantification of total RNA Click here for additional data file.S10 Fig(A) End-point coordinates are given according to Flybase [eve transcription start site. (B) List of sequences and primers used in this study. Flybase  (FB2021_(DOCX)Click here for additional data file.S11 Fig(XLSX)Click here for additional data file.


Evolutionarily conserved kinases and phosphatases regulate RNA polymerase II (RNAPII) transcript synthesis by modifying the phosphorylation status of the carboxyl-terminal domain (CTD) of Rpb1, the largest subunit of RNAPII. Proper levels of Rpb1-CTD phosphorylation are required for RNA co-transcriptional processing and to coordinate transcription with other nuclear processes, such as chromatin remodeling and histone modification. Whether other RNAPII subunits are phosphorylated and influences their role in gene expression is still an unanswered question. Much less is known about RNAPI and RNAPIII phosphorylation, whose subunits do not contain functional CTDs. However, diverse studies have reported that several RNAPI and RNAPIII subunits are susceptible to phosphorylation. Some of these phosphorylation sites are distributed within subunits common to all three RNAPs whereas others are only shared between RNAPI and RNAPIII. This suggests that the activities of all RNAPs might be finely modulated by phosphorylation events and raises the idea of a tight coordination between the three RNAPs. Supporting this view, the transcription by all RNAPs is regulated by signaling pathways that sense different environmental cues to adapt a global RNA transcriptional response. This review focuses on how the phosphorylation of RNAPs might regulate their function and we comment on the regulation by phosphorylation of some key transcription factors in the case of RNAPI and RNAPIII. Finally, we discuss the existence of possible common mechanisms that could coordinate their activities. Saccharomyces cerevisiae, are highly conserved and their resolution has tremendously helped to understand the mechanism of transcription (S. cerevisiae and human RNAPs (The transcription of cellular RNAs is carried out by DNA-dependent RNA polymerases (RNAPs). In bacteria and archaea, only one RNAP transcribes all RNAs. In Eukarya, three RNAPs  are required for RNA transcription, except plants containing two other RNAPs (RNAPIV and -V). RNAPI synthesizes the precursor ribosomal RNA , RNAPIII produces 5S rRNA and transfer RNAs (tRNAs) and RNAPII transcribes all the protein-coding genes synthesizing messenger RNAs (mRNAs). Additionally, RNAPII and RNAPIII can synthesize other types of transcripts, such as small non-coding RNAs (ncRNAs), whose specific synthesis may differ depending on the species . Finallycription . The corcription , phosphocription . A clearcription . Unfortuan RNAPs . We discRNAPII is the best known of the eukaryotic RNA polymerases. Transcription by RNAPII is a very complex, dynamic and finely regulated process. A sophisticated network of protein–protein and protein–nucleic acid interactions is established, producing conformational and activity changes in RNAPII through the transcription cycle. Thus, a pre-initiation complex (PIC), composed basically of general transcription factors , Mediator and RNAPII, is assembled at the gene promoters, opening the DNA to initiate transcription . Other fS. cerevisiae have provided the majority of the existing knowledge about RNAPII transcription mechanisms, regulation and coordination with other cellular processes  and assembly platform ; the jaw-lobe module, made up of Rpb1 and Rpb9; the shelf module containing the foot and cleft domains of Rpb1 and the lower jaw and assembly domains of Rpb5; and the stalk module, formed by Rpb4 and Rpb7, which in the case of S. cerevisiae can be dissociated from the 10-subunit core polymerase  are also phospho-proteins  Albuque. For insModification by phosphorylation of some residues of RNAPII subunits could be important not only for the association of different factors along the transcription cycle but also for exchange of factors occupying the same or close surfaces on RNAPII. This is the case of initiation and elongation factors that compete during the transcription cycle for binding to the polymerase complex, for instance TFIIE and Spt5 . How theFinally, the high sequence conservation within the RNAPII core between yeast and humans suggests similar mechanisms of RNA synthesis. However, sequences are more divergent toward the exterior/surface residues, suggesting that biochemically distinct interfaces interact with different factors . AccordiS. cerevisiae distributed to five of the 14 subunits. Mutation of 13 of these phospho-sites indicated that most are non-essential PTMs, suggesting that they might contribute to non-essential RNAPI functions. Only one residue, Rpa190-S685, was suggested to play a role in rRNA cleavage/elongation or termination  . How eukS. cerevisiae is grown under nutrient-replete conditions, Tor1 localizes in both the cytoplasm and the nucleus. In the nucleus, Tor1 and Kog1 (the Raptor subunit in S. cerevisiae) bind to the 35S (RNAPI) and 5S (RNAPIII) promoters. However, after starvation or rapamycin treatment, they are removed from these regions, thus inhibiting transcription (HMO1 gene is known to be directly activated by Tor1 (HMO1 promoter, facilitating its transcription. After rapamycin treatment or DNA damage, Tor1 and Hmo1 are released, thus inhibiting transcription (TOR serine/threonine kinases play an essential role in controlling many aspects of living cells, such as growth, proliferation and survival in response to nutrients . Initialcription . In mammcription . This ancription  and mammcription . Whereascription , in budd by Tor1 . Hmo1 ac by Tor1 . Both Tocription . Interescription . Similarcription . It woul


Rlip76 is a multifunctional membrane protein that facilitates cancer growth, and its depletion kills cancer cells. We recently found that Rlip depletion also results in broad changes to oncogene and tumor suppressor transcription. The present studies were designed to decipher the unknown downstream signaling pathways and transcriptional regulatory mechanisms driving the effect. Building on prior findings that Rlip depletion induces broad methylomic changes, we found using bioluminescence reporter assays that depletion of Rlip also exerts transcriptional control over several cancer genes through methylation-independent changes in transcription factor-mediated activation of their promoter regions and through additional as yet unidentified mechanisms. These findings have important implications for Rlip-targeted cancer therapy.Rlip76 (Rlip) is a multifunctional membrane protein that facilitates the high metabolic rates of cancer cells through the efflux of toxic metabolites and other functions. Rlip inhibition or depletion results in broad-spectrum anti-cancer effects in vitro and in vivo. Rlip depletion effectively suppresses malignancy and causes global reversion of characteristic CpG island methylomic and transcriptomic aberrations in the p53-null mouse model of spontaneous carcinogenesis through incompletely defined signaling and transcriptomic mechanisms. The methylome and transcriptome are normally regulated by the concerted actions of several mechanisms that include chromatin remodeling, promoter methylation, transcription factor interactions, and miRNAs. The present studies investigated the interaction of Rlip depletion or inhibition with the promoter methylation and transcription of selected cancer-related genes identified as being affected by Rlip depletion in our previous studies. We constructed novel promoter CpG island/luciferase reporter plasmids that respond only to CpG methylation and transcription factors. We found that Rlip depletion regulated expression by a transcription factor-based mechanism that functioned independently of promoter CpG methylation, lipid peroxidation, and p53 status. RALBP1 gene at human genomic locus 18p11.22) has been well-established by several lines of evidence as a permissivity factor required for oncogenic transformation, cancer growth, and invasion/metastasis [RALBP1 was exceedingly rare (0.09% of patients), supporting the importance of normally functioning Rlip protein in oncogenic processes [Rlip76  mice by only heterozygous deficiency of Rlip led us to propose that a similar haploinsufficiency mechanism may exist involving Rlip, p53, and another common binding partner of both, perhaps HSF1 [Rlip inhibition or depletion, by contrast, has considerable activity against xenografted solid tumors in addition to considerable overlap with the methylomic and transcriptomic effects induced by DNMT1 inhibitors. However, Rlip inhibition has the advantage of having no known requirement for DNA replication to induce cytotoxicity. Rlip inhibition may induce its anticancer effects against solid tumors through normalization of tumor suppressor and oncogene expression, disruption of other important Rlip-mediated functions that are critical to cancer cells, or combinations of these effects. Despite the compelling case for the further development of Rlip inhibitors, owing to the impressive broad-spectrum antitumor effects and the pleiotropic functions of Rlip in cancer cells, additional information is needed to understand the mechanisms underlying the striking transcriptomic and epigenetic changes. Cells have several layers of transcriptomic regulation at their disposal including histone modifications, CpG methylation, transcription factors, microRNAs, and long non-coding RNAs. Small changes at the level of a single protein that participates in multiple protein–protein complexes can cause profound changes in the relative ratios of these complexes and disproportional phenotypic effects, referred to as haploinsufficiency phenomena, as is seen with p53-binding proteins such as MDM2 and MDM4 . Rlip haaps HSF1 . The speaps HSF1 ,33. In sIn this work, we attempted to elucidate the roles of these possible mechanisms through which Rlip depletion or inhibition may interact with CpG island methylation to regulate the expression of selected cancer-related genes identified in previous studies as being affected by Rlip deficiency. The genes were selected on the basis of established interactions with known Rlip functions in promoting cancer cell survival or growth. Fibroblast growth factor 8 (FGF8) is a growth factor that activates the FGFR receptors and has roles in breast, prostate, and ovarian cancers ,35,36. WIn this work, we use mRNA expression and luciferase expression by novel CpG-free promoter/luciferase reporter plasmids to address several hypotheses regarding the nature of the methylomic and transcriptomic regulation previously observed following Rlip knockdown. By studying the mRNA expression of these genes, we observed the summated effects of all operative regulatory inputs that affect the transcriptome, and the promoter/luciferase reporter plasmid constructs allowed us to focus in on only methylation-based and transcription factor-based regulation, as such plasmids are insensitive to regulation by histone modifications; microRNAs; long non-coding RNAs; or other factors affecting transcript-specific mRNA splicing, base editing, or stability. Using these tools, we confirmed prior findings that Rlip depletion exerts regulatory transcriptomic influences on cancer-related genes. Our reporter results suggest that, at the 24 h time-point, this regulatory influence operates through a transcription factor-based mechanism rather than through promoter methylation, and this does not appear to depend on p53 status. These luciferase reporter results, in combination with the qRT-PCR results, also point to an additional as yet unidentified layer or layers of regulatory influence that cannot be explained by transcription factors. Additionally, our results suggest that the observed Rlip-responsiveness is not simply due to the oxidative stress of 4HNE accumulation, and is not an artifact of imbalanced cytotoxicity or clathrin-dependent endocytosis resulting from Rlip inhibition. Further studies of the transcriptomic regulatory effects of Rlip depletion are warranted, and studies on transcription factor interactions, histone remodeling, and microRNAs should be prioritized.2 and 5% CO2. MCF7 and MDA-MB-231 are representative of estrogen receptor positive (ER+) and triple negative breast cancer (TNBC), respectively. NCI-H358 is a p53-null non-small cell lung cancer (NSCLC) cell line with a Kirsten rat sarcoma viral proto-oncogene (KRAS) mutation. NCI-H520 is a NSCLC cell line with low expression of wild-type p53, which is inducible [MCF7 and MDA-MB-231 breast cancer cells were cultured in DMEM supplemented with 10% FBS and 1% Pen-Strep (Gibco). NCI-H358 (H358) lung cancer and NCI-H520 (H520) lung cancer cells were cultured in RPMI supplemented with 10% FBS and 1% Pen-Strep. Puck’s Saline A + EDTA was used to detach adherent cells and was kindly provided by the TTUHSC School of Medicine Cancer Center . Cells wnducible . These cnducible ,16. ThisRALBP1 mRNA [LRR1 and PPARA were selected for further study because of the methylomic and transcriptomic changes following Rlip knockdown by R508 and because they share a functional commonality with Rlip. Additionally, we used reduced representation bisulfite sequencing (RRBS) to examine methylomic changes in NCI-H358 and NCI-H520 lung cancer cell lines following treatment with a proprietary small molecule inhibitor of Rlip. The methylation changes were analyzed using Qiagen’s Ingenuity Pathway Analysis (IPA), and we selected PRKCA, PRKCZ, MAPK14, CREBBP, and FGF8 because they represent critical nodes in the pathways most affected by methylation changes in NCI-H358 or NCI-H520 following Rlip inhibition and because they have previously been reported to have relevance in cancer [We have previously reported global methylomic and transcriptomic changes in the livers of p53 knockout mice following Rlip depletion by R508, a phosphorothioated antisense DNA oligonucleotide targeted to the n cancer ,41,57,61RALBP1 antisense locked nucleic acid (Rlip-LNA) Exiqon GapmeRs , or scrambled control, as described previously [Cells were seeded at 70–90% confluency in six-well plates in complete medium. After overnight incubation, plates were transfected with 2.5 µg eviously . TransfeRALBP1 mRNA under control of a CMV promoter [We explored the behavior of Rlip-responsive genes after treatments to increase Rlip by protein- or plasmid-based methods and after treatment to increase oxidative stress by exposure to arachidonic acid (AA), an omega-6 polyunsaturated fatty acid precursor of 4HNE. Cells were cultured and seeded overnight in six-well plates as described above for Rlip knockdown. For Rlip overexpression, cells were transfected with 2.5 µg of a pcDNA3.1-based plasmid construct expressing promoter ,62 or wipromoter   with cOmplete™ Mini protease inhibitor cocktail , prepared in Laemmli buffer, and separated by SDS-PAGE using Bolt 4–12% Bis-Tris gels (Thermo Fisher). Protein was transferred to 0.22 µm nitrocellulose  using wet transfer for 1.5 h at 30 V. Blots were probed using commercial antibodies in Pierce Clear Milk Blocking Buffer (Thermo Fisher) in 0.1% TBST and imaged on a VersaDoc imaging system (Bio-Rad). Antibodies were diluted 1:1000 and included the following: anti-RALBP1 , anti-beta actin , HRP-linked mouse IgGκ Binding Protein , and anti-rabbit IgG HRP-linked .FGF8, PRKCA, CREBBP, MAPK14, PRKCZ, LRR1, and PPARA genes were cloned from human DNA and inserted into the pCpGL CpG-free firefly luciferase reporter plasmid graciously provided by Dr. Michael Rehli [E. coli  for selection. After restriction digestion from pGEM-T, gel purified inserts were ligated into pCpGL-basic firefly luciferase reporter plasmid and transformed into PIR1 E. coli under Zeocin (Thermo Fisher) selection to obtain the final experimental CpG island-luciferase reporter plasmid constructs. The CpG island inserts in the final pCpGL reporter plasmid constructs were validated by restriction digestion  at 15,000 to 35,000 cells per well to attain 70–90% confluency at the time of transfection. Firefly luciferase reporter plasmids and Rlip-LNA or scrambled control were transfected in 10 µL each of Opti-MEM reduced serum medium (Thermo Fisher) using Lipofectamine 3000 with p3000. The pRL-SV40 Renilla luciferase reporter plasmid (Promega) served as an internal standard and was transfected simultaneously with the firefly luciferase reporter plasmids. Owing to the considerable difference in signal intensity between the various reporter constructs and between unmethylated reporters and their methylated counterparts, all wells transfected with different constructs or different treatments were separated by one blank well to reduce the effects of light bleed on wells of lower signal intensity. Firefly and Renilla luciferase signals were analyzed on a SpectraMax ID3 microplate reader  using the Dual-Glo Luciferase Assay System (Promega) according to the manufacturer’s instructions. The responsiveness to RLIP knockdown by locked nucleic acid  relative to scrambled control was evaluated for both unmethylated (UM) and in vitro methylated (M) variants of each reporter at 24 h.MTT assay was performed in parallel to dual luciferase assays using the same cell seeding numbers and volumes, transfection conditions, and plate analysis timepoints as described for the dual luciferase methods. Briefly, additional complete medium was added to a final volume of 200 µL per well, and 40 µL of 5 mg/mL ChemCruz Thiazolyl Blue MTT reagent  in PBS was added to each well. After 2–3 h incubation at 37 °C, wells were aspirated and 50 µL DMSO was added to each well. Plates were rocked at room temp for 10 min to dissolve MTT crystals before reading on a SpectraMax Plus microplate reader (Molecular Devices).t-test with significance at p < 0.05 using Excel . Correlation analyses used Pearson’s r, performed in GraphPad Prism version 5.02 . Graphs were created using GraphPad Prism version 5.02. Predicted transcription factor binding sites were identified using PROMO hosted by the ALGGEN server [Comparisons of mean normalized mRNA and luciferase expression were done using Student’s N server ,66. PROMRALBP1-targeted locked nucleic acid (Rlip-LNA), which induces RNAse H-mediated mRNA degradation. Rlip-LNA induced 50–92% knockdown in RALBP1 mRNA at 24 h following transfection (p < 0.05) for H520. PRKCA, CREBBP, MAPK14, PRKCZ, and PPARA mRNA showed apparent decreases across the four cell lines following Rlip knockdown, although the changes for a given gene were statistically significant (p < 0.05) in only certain cell lines, as indicated by the asterisks. LRR1 showed only minor changes and no consistent response across the four cell lines. The degree of transcriptional regulation exerted by Rlip knockdown significantly correlated (p < 0.05) with the degree of Rlip depletion for PRKCA, CREBBP, and PPARA  only. FGF8, PRKCA, CREBBP, and LRR1, no change in mRNA expression was observed following exposure to L3K+P3000. Moderate deceases in mRNA were observed for MAPK14, PRKCZ, and PPARA in response to L3K+P3000. However, the direction of the regulatory effects on target expression following Rlip knockdown by LNA remained consistent, whether compared against the untreated condition, the L3K+P3000 condition, or the scrambled control condition. Thus, it appeared that no excessive perturbations in expression were induced by the L3K+P3000 transfection reagents themselves.We selected several Rlip- and cancer-related genes as described in sfection A. FGF8 msfection B, althound PPARA C. MAPK14RALBP1 mRNA (RALBP1 mRNA was increased 10-fold (MDA-MB-231) to 300-fold (H520) with the Rlip-pcDNA3.1 plasmid relative to the empty vector (EV). We also tested whether Rlip protein was detectable following transfection of Rlip-pcDNA3.1. At 24 h post-transfection, Rlip protein was increased 25-fold in attached H358 cells and 100-fold in floating cells, and the Rlip produced by the plasmid was at the same molecular weight as the cell’s native Rlip, the band of which was detected in EV-transfected cells. In overexpressing Rlip, we expected to observe a pattern of expression changes opposite to that observed with Rlip knockdown; however, this was not observed at 24 h following the transfection of the Rlip-pcDNA3.1 plasmid  transfection of an Rlip expression plasmid and (2) addition of purified Rlip to the culture medium. Transfection of the Rlip-pcDNA3.1 plasmid resulted in considerable overexpression of both BP1 mRNA A and RliBP1 mRNA B. RALBP1 plasmid C. To val plasmid . TreatmeFGF8, PRKCA, CREBBP, MAPK14, PRKCZ, LRR1, and PPARA) into the pCpGL CpG-free luciferase reporter plasmid [In order to further study the various regulatory mechanisms contributing to transcriptomic regulation by Rlip, we cloned promoter CpG island regions upstream of these same genes . We observed no notable differences in cell kill between the reporter/LNA combinations at the 24 h timepoint, despite clear differences in the Rlip-responsiveness of the luciferase reporters . To evaluate this, we performed MTT cytotoxicity assay in parallel with dual-luciferase assay following co-transfection of a eporters .RALBP1 depletion and more robust transcriptional effects in the breast cancer cells relative to the lung cancer cells; however, this could simply be a function of the faster doubling times of the breast cancer cells, as opposed to biology specific to the cancers of origin. We found that the transcriptional regulation following Rlip depletion was not likely due to the stress of 4HNE accumulation or other effects related to arachidonic acid, except possibly in the case of FGF8. When upstream promoter CpG island regions of these same genes were inserted into luciferase reporter plasmids, the expression was also altered by Rlip knockdown, but in a different pattern. Using these reporter plasmids, we found that the expression was vastly altered by in vitro CpG methylation by M.SssI methyltransferase, with methylation being suppressive in all cases. However, 24 h Rlip-knockdown resulted in changes to reporter activity that were much smaller in magnitude. It is thus unlikely that cells were methylating the unmethylated reporter plasmids or demethylating the methylated reporter plasmids to any substantial degree within the 24 h assay window. Further, Rlip knockdown resulted in increased reporter signal from plasmids that were already fully unmethylated, thus activation by further demethylation was not possible. These luciferase reporter observations point toward a transcription factor-based mechanism of regulation by Rlip knockdown at 24 h, rather than a methylation-based mechanism. The distinctly different patterns between the luciferase reporter activity changes and the mRNA expression changes following Rlip knockdown also indicated that another layer of regulation, in addition to a transcription factor-mediated layer of regulation, is involved in the cell’s short-term transcriptomic response to Rlip knockdown, although it is unclear what this mechanism is.The results of our previous studies demonstrated that large-scale methylomic and transcriptomic changes occur following Rlip depletion through unknown intermediate regulatory steps. In the present studies, we tested the hypothesis that specific regulatory complexes involving Rlip protein may be involved. We attempted to further characterize the downstream regulatory outcomes of Rlip depletion on several cancer-related genes with functional or pathway commonality with Rlip. Using qRT-PCR, we confirmed that Rlip knockdown does in fact alter the transcription of some of these cancer-related genes. Rlip-LNA transfection resulted in more robust We have previously identified Rlip-p53 interactions in neuroblastoma  and haveCells use a variety of mechanisms to regulate expression. mRNA abundance can reflect effects based on histone remodeling, CpG methylation, transcription factors, microRNAs (miRNA), and long non-coding RNAs (lncRNA), in addition to a variety of effects involving mRNA processing , mRNA secondary structures, and modified nucleotides that can alter mRNA half-life ,69,70,71PRKCA-, FGF8-, LRR1-, and PPARA-derived CpG islands, while the CREBBP-, PRKCZ-, and MAPK14-derived CpG islands resulted in relatively little change in luciferase signal following Rlip knockdown. The reporter plasmids used for experiments were either completely unmethylated (as produced by the PIR1 E. coli strain used in cloning) or completely methylated (as expected following M.SssI methyltransferase treatment) at the time of transfection. This was verified by digestion with the methylation-sensitive restriction enzyme HpaII, which showed complete protection or complete digestion dependent on methylation status. It is unclear to what extent the DNA methyltransferase (DNMT) or ten-eleven translocation (TET) proteins can methylate or demethylate, respectively, reporter plasmids during the assay timeframe; however, given our assay design, it is neither possible for cells to further de-methylate the unmethylated constructs nor to further methylate the methylated constructs. Therefore, as Rlip inhibition tended to similarly affect the unmethylated and methylated constructs, it is unlikely that CpG methylation or demethylation was driving the observed changes in luciferase reporter output following Rlip knockdown.We have found that the expression driven by CpG island regions cloned into the pCpGL reporter vector indeed was suppressed when the plasmids were methylated, a finding that is in line with the predominant characterization of promoter methylation. We also observed that both methylated and unmethylated reporters were typically equivalently affected by Rlip knockdown. Rlip knockdown tended to upregulate the expression of both methylated and unmethylated luciferase reporters driven by A possible limitation in the interpretation of our results, in the context of previous findings on congenitally Rlip-deficient mice, lies in the very short 24 h assay duration. It is possible that evidence of methylomic regulatory changes would have arisen with longer assay durations; however, prior studies on the polyphenolic phytoalexin resveratrol in MDA-MB-231 breast cancer cells indicate that both methylation and demethylation changes occurred within 24 h of dosing, with minimal further changes observed at 48 h . AdditioDegree of knockdown is another limitation, as greater knockdown may have elicited different effects. In three of four cell lines, we attained only ~50% Rlip knockdown at 24 h; however, it is well established that ~50% depletion of Rlip by heterozygous Rlip knockout shows good anticancer activity and is even optimal, as evidenced by the phenotype and survival of heterozygous Rlip knockout mice ,31. The KRAS mutation. This consistency across cell lines is in line with the broad-spectrum anticancer activity previously reported for Rlip inhibition methods, which have demonstrated efficacy against preclinical models of melanoma; neuroblastoma; glioblastoma; and cancers of the breast, lung, colon, kidney, and prostate [Surprisingly, the expression responses as assessed by qRT-PCR were highly consistent across the four cell lines following Rlip knockdown. Similarly, the expression responses as assessed by the luciferase reporters were also highly consistent across the three cell lines tested. This argues that the observed effects may be inherent to the gene/promoter structures and are little affected by tissue of origin or by cell-line-specific differences in p53 deletion, estrogen receptor expression, or prostate ,17,18,19FGF8. Although Rlip knockdown moderately downregulated the other targets at 24 h following transfection, FGF8 expression was massively increased. This was unexpected given the oncogenic properties typically attributed to FGF8 and the cancer suppressive activities seen with Rlip knockdown. However, the FGF/FGFR family of signaling structures is quite complex, with the Ras/MAPK pathway being a prominent signal effector [FGF8 upregulation in our experiments is due to a compensatory upregulation resulting from interference with FGFR signaling. While the gene targets studied in this work are typically considered to have oncogenic activities, many of them have also been reported to mediate tumor suppressor functions [Of the gene targets studied in this work, perhaps the most interesting pattern was seen with effector ,76; Thereffector ,78. Eacheffector . Given teffector ,80. It iunctions ,80. In sThe key conclusions of this work are that Rlip inhibition clearly results in changes in the expression of the majority of these cancer-related genes in all cell lines tested, and it appears that, at the 24 h timepoint, at least two distinct mechanisms are operative. One mechanism is dependent on transcription factors, and a second mechanism is clearly operative, but cannot be explained by methylation or transcription factors. To further clarify this, future studies should prioritize the deletion of candidate Rlip-responsive transcription factor binding sites from cloned promoter inserts and the examination of how Rlip depletion affects histone acetylation and microRNA expression. Further, Rlip-responsiveness following 24 h knockdown appears to be independent of 4HNE accumulation and p53. Our findings support Rlip as a broadly important protein in determining the expression of cancer-related genes. This work, in combination with previously reported findings, supports the operation of a haploinsufficiency mechanism and the presence of at least two distinct modes of action, which elicit the oncopreventive effects of long-term Rlip depletion and the short-term chemotherapeutic anticancer effects of Rlip depletion.