Previous work showed that mRNA degradation is coordinated with transcription in yeast, and in several genes the control of mRNA degradation was linked to promoter elements through two different mechanisms. Here we show at the genomic scale that the coordination of transcription and mRNA degradation is promoter-dependent in yeast and is also observed in humans.cis-regulatory sequences between two yeast species affects both transcription and mRNA degradation and suggest that while some cis-regulatory elements control either transcription or degradation, multiple other elements enhance both processes. Second, we show that adjacent yeast genes that share a promoter (through divergent orientation) have increased similarity in their patterns of mRNA degradation, providing independent evidence for the promoter-mediated coupling of transcription to mRNA degradation. Finally, analysis of the differences in mRNA degradation rates between mammalian cell types or mammalian species suggests a similar coordination between transcription and mRNA degradation in humans.We first demonstrate that swapping upstream Our results extend previous studies and suggest a pervasive promoter-mediated coordination between transcription and mRNA degradation in yeast. The diverse genes and regulatory elements associated with this coordination suggest that it is generated by a global mechanism of gene regulation and modulated by gene-specific mechanisms. The observation of a similar coupling in mammals raises the possibility that coupling of transcription and mRNA degradation may reflect an evolutionarily conserved phenomenon in gene regulation. Gene expression patterns are regulated by a multitude of processes, and while these processes have traditionally been studied in isolation, numerous instances of cross-talk between them have been revealed in recent years -5. TheseCoupling of transcriptional induction with enhanced mRNA degradation generates a peaked response that is an important feature of dynamic biological systems that must respond quickly but transiently as they adapt to environmental changes ,13,14. Htrans and through cis mutations, as determined from analysis of interspecific hybrids [trans mutations presumably involves changes in the activity of protein complexes that regulate both transcription and degradation, such as Rpb4/7 and Ccr4-Not [cis-mutations was observed at more than a hundred genes, but the mechanism of cis-coupling mutations is unknown. We speculated that promoter cis mutations could affect the recruitment of transcription complexes that not only regulate transcription but also prime the newly transcribed mRNA for future regulation in the cytoplasm [cis-coupled genes we discovered were underrepresented with Rap1 targets and with cell-cycle periodic genes (not shown). This suggests the existence of additional unknown coupling mechanism(s) through cis-regulatory sequences, which may involve promoter, UTR or open reading frame elements.Evolutionary changes in transcription and mRNA degradation were coupled both through  hybrids . CouplinCcr4-Not . Couplinytoplasm . Interesytoplasm ,18. Yet ytoplasm  since thcis-dependent coupling by swapping upstream cis-regulatory sequences (promoters and 5' UTRs) between Saccharomyces cerevisiae and Saccharomyces paradoxus and measuring the effect of swapping on mRNA levels and mRNA degradation. We further demonstrate the role of promoters in controlling mRNA degradation by showing that yeast genes that share a promoter tend to have similar mRNA degradation profiles. Based on these results we propose that a general promoter-mediated mechanism couples transcription to mRNA degradation of a large fraction of yeast genes and that this mechanism may be modulated by various cis and trans elements. Finally, we present evidence for a similar coupling in mammalians through analysis of differences in mRNA degradation between different cell types or species.Here, we validate our previous prediction of cis-dependent changes in gene expression for hundreds of genes, and a cis-dependent coupling of transcription and mRNA degradation. To validate these predictions and to examine whether the relevant cis-acting mutations may reside at the promoter or 5' UTRs, we generated S. cerevisiae strains in which upstream cis-regulatory sequences (200 to 700 bp of the sequence immediately upstream to the start codon of the gene of interest) have been replaced by the orthologous sequence from S. paradoxus, which are approximately 80% identical  of strains with the wild type (WT) and with the swapped (Swapped) regulatory sequences of these genes, and measured the mRNA log2-ratio (Swapped/WT) at four time points  reflects the difference in steady-state mRNA levels, while the change in that ratio along the time course reflects differences in mRNA degradation rates: if the swapped sequences increase mRNA degradation rates, then the mRNA log2-ratio (Swapped/WT) should decrease linearly across the time course . Strikingly, swapping affected both the steady-state mRNA levels and the rates of mRNA degradation for most genes examined (7 of 9), and in all of these cases the change in mRNA levels was opposite to that expected from the change in mRNA degradation: increased degradation was associated with increased (rather than decreased) steady state mRNA levels, and vice versa. This indicates that changes in mRNA degradation were accompanied by changes in transcription that had an opposite effect on mRNA levels, and that the changes in transcription were larger and thus determined the direction of change in mRNA levels. Four of these genes were chosen for validation , and their results were reproduced in additional experiments with eight time points  the swapped strains also had an effect on mRNA degradation, suggesting that these effects could be due to the selection marker instead of the swapped regulatory region. Indeed, analysis of control strains (with the marker but without swapping the regulatory sequences) indicated that, for some genes, the selection marker affected both transcription and mRNA degradation  of a relatively small sequence element, and comparison of these strains uncovers the effect of mutations in this sequence only.Two genes were selected for a more detailed analysis . For each of the two genes, we generated a series of sequential s Figure . Each paWe first analyzed six swap strains of MRI1 and found that they are divided into two clusters that differ from the control strain Figure . The firIn contrast, the second cluster showed an additional decrease of approximately two-fold in mRNA degradation and a more subtle increase in mRNA levels (compared to the first cluster). The increase in mRNA levels is expected from the decreased rates of mRNA degradation, suggesting that mRNA degradation is the primary effect and that transcription was either unaffected or only mildly affected by the additional swapped sequences. This cluster includes two strains with swapped segments of 225 and 300 bp, indicating that the causal sequences that distinguish this cluster from the first cluster are in the 197 to 225 bp region. This region is also upstream of the mapped transcription start site) and therefore reflects a promoter element that controls mRNA degradation.P < 0.05; see Materials and methods) and were consistent with two independent experiments in which we swapped the containing segment 360 to 414 and observed a similar effect  and hence neighboring gene pairs, with divergent orientation  Figure , might sSharing of upstream intergenic regions among divergent gene pairs might not indicate shared promoters, especially when the size of the intergenic region is relatively large . MoreoveWe also examined the similarity in mRNA degradation as deduced from the more recent and completely different technology of metabolic labeling , in whicTo add more support to the idea that shared promoter regulation results in coordination of post-transcriptional regulation of mRNA degradation, we examined pairs of genes that are not adjacent on the genome but share multiple promoter-associated regulators. For this we used physical measurements of protein-DNA interactions  and tookIn summary, although these analyses are correlative, they are based on patterns of hundreds of genes. Together they suggest that promoter sharing may contribute to the similarity in mRNA degradation profiles among neighboring genes, and provide support to the notion that promoters regulate mRNA degradation. Note that correlated mRNA degradation is observed not only for divergent but also, more weakly, for convergent and tandem gene pairs. This might indicate that some regulatory elements could regulate transcription and mRNA degradation from the terminator region, or alternatively that genomic proximity might enable sharing of transcription and degradation factors that are recruited by regulatory elements of one gene and then co-regulate the neighboring gene.While the coordination of transcription and mRNA degradation has been studied primarily in yeast ,17,18,29et al. [We first examined the differences in mRNA degradation rates between induced pluripotent stem (iPS) cells and the human foreskin fibroblasts (HFFs) they were derived from, as recently measured by Neff et al. . If chanet al. ,12 and set al. to changes in mRNA levels as defined by the average of four other (independent) studies that each compared pluripotent to fibroblast human cells [P < 0.001; Figure S9 in Additional file We next performed two control analyses  and in newly transcribed mRNA (Δnew), both reflecting log2-ratios of mouse versus human. Δtotal and Δnew were highly correlated . To understand the connection between changes in mRNA degradation and changes in transcription, we considered three models, and the expected distributions of Δtotal and Δnew according to these models are shown for genes with increased (red) or decreased (green) mRNA degradation, in mouse compared to human , but a significant effect of mRNA degradation on total mRNA levels . If differences in mRNA degradation primarily reflect measurement errors in Δnew and Δtotal (Model II), then we expect moderate shifts in the distributions of both Δnew and Δtotal , as observed in comparison of unrelated (non-orthologous) genes , which predicts an opposite shift. Consistent with this possibility, we observe a remarkable shift in the distribution of Δnew. The shift in Δnew is much larger than the shift in Δtotal, which can only be explained by the third model. These results indicate that, on average, differences in mRNA degradation are compensated for by transcriptional effects and may be explained by a combination of the above models - in some genes transcriptional effects are independent of (and do not compensate) the changes in mRNA degradation, while in other genes transcriptional effects over-compensate for the changes in mRNA degradation and generate an opposite change of total mRNA levels.Interestingly, comparison of human-mouse orthologs shows that the distributions of d Figure . As bothcis-dependent evolutionary changes in mRNA levels and in mRNA degradation of hundreds of yeast genes. In the first part of this work we have validated these predictions and demonstrated that the relevant mutations often reside at the swapped upstream regulatory regions containing promoters and 5' UTRs. We further analyzed the contribution of different upstream segments to the divergence of two genes and identified promoter and UTR elements that affect transcription and mRNA degradation. Promoter alterations could influence the recruitment of RNA-binding factors, such as Rpb4/7, that would shuttle with the mRNA to the cytoplasm and affect their degradation and/or translation. Other evidence for this model comes from our observation that gene pairs that share a promoter tend to have similar patterns of mRNA degradation across the yeast genome. This suggests that promoter-mediated effects on mRNA degradation often act in both directions of a bidirectional promoter.We previously predicted (using interspecific hybrids) et al. [ACT1, PRPL30) also had independent effects on the mRNA degradation rate of their construct. Second, our analysis of YLR326W identified distinct cis-regulatory elements that affected both transcription and mRNA degradation. Third, we found that genes in which transcription and mRNA degradation were previously shown to be coupled by cis-mutations were also coupled through the insertion of a selection marker. It would therefore be interesting to examine if Rpb4/7 and Ccr4-Not interact differently with mRNAs of coupled versus uncoupled genes.Recent studies identified two mechanisms by which promoter elements affect mRNA degradation ,18 but, Finally, we analyzed differences in mRNA degradation between mammalian cell-types or species and provide evidence for a global coordination between transcription and mRNA degradation. Importantly, the analyses of mammalian cells, as well as the analyses of divergent yeast gene pairs, were both performed on two different datasets of mRNA degradation rates, with one that used transcriptional arrest and another that used metabolic labeling. These completely different technologies produce distinct estimates of mRNA degradation rates, yet in our analyses they show consistent results, further supporting our conclusions. Our observation of similar transcription-degradation coupling in yeast and in mammals may suggest that coupling is an evolutionarily conserved phenomenon. Given the conservation of the protein complexes Rpb4/7 and Ccr4-Not, it is tempting to speculate that similar mechanisms govern this coupling in yeast and in mammals and therefore that a promoter-mediated coupling exists also in mammals. Consistent with this possibility, recent work in a human embryonic cell line identified several mRNA-binding transcription regulators, as well as an overall enrichment of interactions between mRNA-binding proteins and transcriptional regulators, demonstrating the many potential links between transcription and post-transcriptional regulation . AlternaOur results demonstrate, at the genomic scale, that transcription and mRNA degradation are coordinated in yeast as well as in humans. In yeast, detailed experimental and computational analysis further indicates that this coupling is driven by promoters and cannot be accounted for by two recently described mechanisms, suggesting that multiple mechanisms influence the coupling of transcription and mRNA degradation. Additional work would be needed to decipher the relevant mechanisms and their implications.cis-dependent differences in mRNA levels and consistency of these cis-effects among several microarray experiments [cis-regulatory motifs.We first selected 40 genes for swapping experiments based on a high degree of inter-species eriments . The fireriments  and was eriments . Of thesS. cerevisiae BY4741 was compared to BY4741 strains in which we replaced the promoter and 5' UTR segment of an individual gene with the orthologous segment from S. paradoxus CBS432  and for two of these genes (YLR326W and MRI1) we generated a series of strains where different portions of the upstream regulatory regions were replaced, thus generating chimeric intergenic regions composed of partial S. paradoxus and partial S. cerevisiae segments  and an upstream selection marker.In the first set of 17 genes, including all 9 genes for which we examined mRNA degradation, the S. cerevisiae regulatory region but instead inserted the construct downstream of the S. cerevisiae regulatory region (directly upstream of the start codon), thereby maintaining the S. cerevisiae regulatory region next to the adjacent gene but also inserting the S. paradoxus regulatory region next to the gene being studied. For these genes we did not generate control strains. For measurements of mRNA degradation rates, the swap and control S. cerevisiae strains were mated with a wild-type S. paradoxus (CBS432) in order to allow comparison of the S. cerevisiae allele (whose regulatory sequences are swapped) with the orthologous S. paradoxus allele, and also because transcriptional arrest experiments appeared to be more efficient in diploid strains (not shown).A caveat of the above swapping approach is that the swapped region might contain an element (promoter or terminator) that regulates the adjacent upstream gene (not the one being studied), and these elements are not only swapped but are also being separated from the adjacent gene by the selection marker. To avoid this potential effect, in the second set of 17 genes we did not replace the Real time PCR was performed for the WT and swap strains with duplicates or triplicates. We compared the mRNA levels of the genes whose regulatory sequences were swapped to reference genes that are not expected to be affected by the swapping and thus provide a normalization factor for comparison between the WT and the swap strains. The effect of swapping was thus quantified as:cis-regulatory segment (rather than absolute mRNA degradation rates) and hence are less dependent on the choice of reference gene as long as the reference gene is not directly affected by the swapped regulatory segment. Nonetheless, for the validation experiments . These slopes were also used to estimate the relative effect of the swapping on mRNA degradation rates  of the native S. cerevisiae gene from two previous studies [Transcription was arrested by addition of 150 μM 1,10-phenanthroline to the WT and swap hybrid strains and monitored mRNA levels at four or eight time points. Least squares linear regression was used to estimate the slope along the time-course, which reflects the effect of swapping on rates of mRNA degradation. For example, a slope of -0.05 indicates that after 20 minutes the relative mRNA level of the swap strain decreases by 2-fold : Δi,i+20 - Δi,i+20, for i = 0,20,40 and k = 1..n, where n is the number of biological repeats. The pairing of corresponding time intervals is important because the pattern of changes along the time course is not exactly linear and is often gene-specific. By comparing the same gene between different strains and restricting the comparison to corresponding intervals we can largely eliminate the variability along the time course and focus on the variability between strains .where mmRNA degradation experiments were typically performed with three technical replicates and with two biological replicates, with three exceptions: (1) YLR326W swaps of 200, 386, 398, 414, 444, 459 and 471 bp Figure  and a laet al. [2(mouse/human) for total  and nascent (Δnew) mRNA levels and centered these log2-ratios on zero. Human and mouse half-lives were taken from the supplement of Friedel et al. and genes with differential mRNA degradation rates were identified as those with at least 1.75-fold difference between the species both according to the Friedel et al. half-life estimates and according to . We filtered out genes with very low hybridization intensities and/or very large interspecies differences, although the trends shown in Figure Total and newly transcribed mRNA levels for human BL41 and murine NIH-3T3 cell lines with three replicates were taken from Gene Expression Omnibus (GSE10026). We averaged the three replicates, and compared the averages of selected probes for orthologous genes . We calMadison, WI, USA), including DNAse treatment to reduce contaminations of genomic DNA. To discriminate between mature capped mRNA and mRNA degradation products, RNA samples were dephosphorylated using FastAP thermosensitive Alkaline Phosphatase . RNA was then cleaned using RNeasy MinElute Cleanup kit . 5' Caps were then removed using Tobacco acid pyrophosphatase (TAP) enzyme (Epicenter Biotechnologies) to generate 5' monophosphate for mature intact mRNAs and RNA was cleaned using RNeasy kit. A unique RNA adaptor (UCUUUCCCUACACGACGCUCUUCCGAUCUGCGC) was ligated using T4 RNA ligase  with incubation of 1.5 hours at 22°C, and RNA was cleaned using RNeasy kit. First strand cDNA was generated from total amount of RNA ligation product, random hexamer primers and M-MLV reverse transcriptase. cDNA (2 μl) was used as template for each following PCR reaction. To estimate the length of the 5' UTR a reverse primer located downstream of the start codon and a forward primer on the adaptor (ATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTT) were used together and products were run on a 1.5% agarose gel. Reverse primer sequences: CGGTAGAAGTGCAAGTAATGG for YLR326W and for ATAAATTAACGGCTGTGGGTC. Primers were validated using an additional forward primer located on the ATG of each gene, ATGTCAGGGTTCATTAAGAGC for YLR326W and ATGTCGTTGGAAGCCATCGTC for YPR118W.RNA was extracted from WT and two mutant strains using Yeast MasterPure kit  in mRNA levels and mRNA degradation is maintained in two control analyses. Figure S10: scatter plot of interspecies differences in total mRNA levels and in nascent mRNA levels. Figure S11: human-mouse comparison of non-orthologous genes reproduces the expected behavior of the Noise model [Figure 4).Click here for file


Using RNA-seq (RNA sequencing) of ribosome-depleted RNA, we have identified 1,273 lncRNAs (long non-coding RNAs) in P493-6 human B-cells. Of these, 534 are either up- or downregulated in response to MYC overexpression. An increase in MYC occupancy near their TSS (transcription start sites) was observed for MYC-responsive lncRNAs suggesting these are direct MYC targets. MYC binds to the same TSS across several cell lines, but the number of TSS bound depends on cellular MYC levels and increases with higher MYC concentrations. Despite this concordance in promoter binding, a majority of expressed lncRNAs are specific for one cell line, suggesting a determinant role of additional, possibly differentiation-specific factors in the activity of MYC-bound lncRNA promoters. A significant fraction of the lncRNA transcripts lack polyadenylation. The RNA-seq data were confirmed on eight selected lncRNAs by NRO (nuclear run-on) and RT-qPCR (quantitative reverse transcription PCR). MYC is a basic helix-loop-helix leucine zipper (bHLHLZ) protein that controls cell proliferation, differentiation, metabolism, apoptosis, and the maintenance of pluripotency. It is a key component of a broad transcription factor network, forming heterodimers with the bHLHLZ protein MAX -4. MYC-MMYC under the control of the immunoglobulin heavy chain locus [MYC plays a critical role in human cancer , 6. In nin locus , leadingin locus , 8. The in locus -13.Studies on MYC-mediated transcriptional regulation have focused mainly on coding transcripts; specific links between MYC and non-coding RNAs (ncRNAs) have been investigated only recently -19. The P<10−3). A visualization of expression ratios by heat map  wermap Fig.  or volcamap Fig.  reveals map Fig. . This isMYC could influence the expression of lncRNA genes either by directly binding to their promoters or by regulating the expression of other transcription factors which subsequently target the non-coding genes. To test for direct regulation by MYC, we have interrogated publicly available P493-6 ChIP-seq (chromatin immunoprecipitation sequencing) data for MYC enrichment proximal to the transcription start sites (TSS) of the 1,273 detected lncRNA genes . Using a linear discriminator classifier, we found only a small number of coding transcripts that lack polyadenylation . Performing a similar analysis for the lncRNA transcripts, we found a weaker, but significant, linear relationship between ribosome-depleted and poly-A methods . Applying the discriminator established with coding transcripts, a much larger number of non-coding transcripts  were found to lack polyadenylation.Whereas the vast majority of coding genes are polyadenylated, many lncRNAs are not -59. To iDANCR, MIR17HG, SNHG15, SNHG16), (2) concordantly regulated sense-antisense pairs , and (3) discordantly regulated sense-antisense pairs . Analysis of exon-exon junction spanning split reads supported the intron-exon gene models annotated in the UCSC genome browser hg19 for all eight lncRNA genes. Parallel nuclear run-on (NRO) and steady-state gene expression analysis by RT-qPCR (quantitative reverse transcription PCR) were performed on P493-6 cultures in the presence and absence of doxycycline. Given the precedent for sense gene regulation by proximal antisense transcripts, we also investigated expression of neighboring or overlapping transcripts where applicable. Using this strategy, RNA-seq expression data were validated at both the transcriptional and steady-state level for all eight lncRNAs. A detailed example of two of these validated genes is presented in Fig. P=0.0027) was observed between NRO and RT-qPCR expression ratios indicating that these lncRNAs are predominantly regulated at the level of transcription.To validate the RNA-seq data, we selected eight representative lncRNAs Table  based onDANCR and SNHG16) whereas three transcripts were enriched in the nucleus . The remaining transcripts were evenly distributed between nucleus and cytoplasm (P=0.023) was observed between transcript abundance and the cytoplasmic:nuclear distribution ratio such that the more abundant lncRNAs tended to be enriched in the cytoplasm. All eight of the validated lncRNAs could be amplified from oligo dT primed cDNA libraries suggesting that these transcripts are polyadenylated, and that polyadenylation is unlikely to be a determinant of sub-cellular localization for this subset of transcripts.The properties of lncRNAs can provide clues as to their function. For example, lncRNAs which execute their functions at the transcriptional level are expected to be enriched in the nucleus. To investigate the sub-cellular localization of the eight representative lncRNAs, P493-6 cells were separated into nuclear and cytoplasmic fractions and lncRNA levels assessed by RT-qPCR. Two transcripts were predominantly cytoplasmic (ytoplasm . Interes1/2) calculated (MYC (fast turnover: t1/2=30 minutes) and GAPDH (slow turnover: t1/2=215 minutes) were also measured for comparison. The eight lncRNAs showed transcript stabilities that extended from half-lives of 132 minutes (DANCR) to 25 minutes (MIR17HG). These results demonstrate that the eight validated lncRNAs have mRNA-like stability properties and are unlikely to be PROMPTs (despite some being promoter-overlapping) or a consequence of transcriptional noise.Determination of transcript stability can be informative with respect to function as one class of lncRNAs (the PROMPTs or PROMoter uPstream Transcripts) is known to be highly unstable . Equallylculated , Table 1Recent work has suggested that MYC acts as an amplifier of global transcriptional output , 11. TheThe functions of most lncRNAs are currently unknown. There are, however, broad criteria that define various categories of lncRNAs and that are relevant to function. These include antisense RNAs likely to control their sense counterparts and nuclear versus cytoplasmic localization that could serve as broad indicator of function. Transcriptional activities initiated from bidirectional promoters can be divided into concordant and discordant with implied consequences on function.DANCR, MIR17HG, SNHG15 and SNHG16 act as precursors of small RNA species including miRNAs and snoRNAs. These function in post-transcriptional gene regulation and rRNA editing/maturation respectively.Eight lncRNAs that were upregulated in high MYC expression conditions were validated by RT-qPCR. MYC binding was enriched at the promoters of these genes when MYC expression was high, and regulation at the transcriptional level confirmed by NRO. This set of representative MYC-regulated lncRNAs exhibited diverse properties in terms of their genomic organization, sub-cellular localization, abundance, and transcript stability Table  althoughMIR17HG, USP2-AS1, and DANCR; the roles of the remaining five are currently not known. MIR17HG encodes the miR-17-92 cluster. This miRNA cluster is upregulated by MYC  was added to the media to a final concentration of 0.1 μg/mL.P493-6 cells suspensions were maintained in RPMI 1640 (Life Technologies) containing 10% tetracycline-free FBS (Gemini) and supplemented with 2 mM L-glutamine, 100 U penicillin, 0.1 mg/mL streptomycin (Sigma-Aldrich) at 37°C and 5% COBoth ribosome-depleted and poly-A-enriched RNA-seq +/− doxycycline P493-6 were previously published and were obtained from NCBI SRA accession numbers: SRR1313741, SRR1313742, SRR1313743, SRR1313735, SRR1313736, SRR1313737, SRR567561, SRR567562 . U87MG  and resuspended in Nuclei Storage Buffer . NRO transcription was performed by adding 2x transcription buffer  supplemented with 1 mM ATP, 1 mM CTP, 1 mM GTP, 0.5 mM UTP, 0.5 mM BrUTP and 100 U RNaseOUT and incubating for 30 minutes at 30°C. A spike-in control RNA (Renilla luciferase mRNA prepared by T7 in vitro transcription in the presence of BrUTP) was added to act as an external reference for data normalization that was independent of global changes in nuclear RNA content. NRO reactions were terminated by immediately lysing the nuclei and extracting RNA using the Maxwell-16 System RNA LEV kit (Promega). Genomic DNA was removed by a DNase I treatment step included in the extraction protocol. ~500 ng of NRO-RNA per sample were immunoprecipitated using mouse monoclonal anti-BrdU antibodies (Santa Cruz Biotechnology) and Protein G Dynabeads (Life Technologies). Beads were blocked with blocking buffer  and NRO-RNA samples incubated with the beads for 30 minutes at room temperature on a rotating platform. Subsequently, the beads were washed three times with PBST (supplemented with 8 U/ml RNaseOUT). Immunoprecipitated NRO-RNAs were extracted using TRIzol Reagent (Life Technologies) according to manufacturer's instructions and RT-qPCR performed.Treated P493-6 cultures were counted using a Countess Automated Cell Counter (Life Technologies) and 3.6×10free kit (Life Technologies) according to manufacturer's instructions.P493-6 nuclear and cytoplasmic RNA samples were prepared using the Protein and RNA Isolation System (PARIS) kit (Life Technologies) and genomic DNA removed using the TURBO DNA-P493-6 cultures were treated with 2 μg/mL Actinomycin D and then harvested at 30, 60, 120 and 240 minutes later. RNA was extracted and transcript levels assessed by RT-qPCR. Transcript half-lives were calculated using the formula:RPLP0 and RPL10 . For NRO-RT-qPCR, data were normalized to a bromouridylated spike-in control transcript (RLuc) added at the NRO phase in order to provide an external reference transcript independent of MYC-induced changes in global transcriptional activity. miRNA data were normalized to RNU6 (#001973) expression.RNA samples were reverse-transcribed using the High Capacity cDNA Synthesis kit (Life Technologies) according to manufacturer's instructions (using a random priming or oligo dT-priming strategy as appropriate). qPCR was performed on either a Mastercycler ep RealPlex 2 (Eppendorf) or LightCycler 96 (Roche) real-time PCR instrument. Reactions were prepared using KAPA SYBR FAST qPCR Master Mix (KAPA Biosystems) and universal cycling conditions (95°C for 3 minutes followed by 40 cycles of 95°C for 3 seconds and 60°C for 30 seconds). Reaction specificity was confirmed by melt curve analysis. All primer sequences are listed in


Escherichia coli approximately 25% of genes comprise pairs of topologically linked divergently transcribed units. Given that transcriptional complex formation at each promoter in the pair induces topological changes and is itself sensitive to DNA structural perturbations, study of the functional anatomy in such areas requires special approaches. Here we suggested the dual-colour promoter probe vector which may become an ideal tool for divergent transcription profiling. The vector was used to characterize the specific genomic region nearby appY with multiple bidirectional promoters predicted in silico. Only three promoters of this region were shown to be engaged in the transcription initiation resulting in the expression of reporter genes. RNA product transcribed in antisense direction is suggested as a novel RNA. Nalidixin-induced topological modulation differentially affected transcription in sense and antisense directions thus exemplifying anticooperative mode in the response to topological alterations.Mosaic pattern of transcription in alternating directions is a common feature of prokaryotic and eukaryotic genomes which rationality and origin remain enigmatic. In  It is already well established that global and local variations in superhelical density can affect the rate and efficiency of gene expression in bacteria1thE.coli (http://regulondb.ccg.unam.mx/)E.coli genes could be subjected to complex regulation mediated by superposition of global and local changes in superhelicity defined as supercoiling-dependent transcriptional couplingData regarding the effects of DNA topology on transcription were accumulated since early 80E.coli. Topological drift in these regions in response to multiple functional interactions with proteins of transcription machinery is expected with the highest probability.The role of divergent transcription, its topological consequences and dependence on overall DNA structure seems to become more generalized if the data of computational predictions12Salmonella typhimurium activated in topA mutant remained functional if this mutation was suppressedFor eukaryotes the role of divergent promoters in coordinated gene expression and transcription noise suppression was extensively discussed over the last decade14E.coli we constructed a promoterless reporter vector divergently expressing genes of two colour proteins EGFP (green) and mCherry (red). In order to address how changes in DNA superhelical density may affect divergent transcription, we treated cells with low concentrations of gyrase inhibitor nalidixic acid and monitored activities of two overlapping promoters for fepA and fes genes and promoters of appY-associated “promoter island”. We found that both promoter regions responded to the decrease in negative supercoiling and in both cases changes in the relative activity of divergent promoters were registered, but the scale of effect was greater in the region with multiple promoter-like signals, which assumes its higher topological flexibility. Given that “promoter islands” are more bent and overtwisted than other genomic regions tested so farTo study the relative activity of oppositely oriented promoters of Bidirectional promoter probe vectors have been proposed as a useful tool for divergent transcription studying about twenty years ago. The first systems exploited genes of different enzymes with easily detectable activity, such as β-galactosidase, alkaline phosphatase or bacterial luciferase19egfp gene fused with RBS and translational terminators was transferred from the pRBS-EGFP plasmid into the pRBS-mCherry and inserted between BglII and HpaI restriction sites. Inserted fragment was generated with primers “stops-BglII” and “T7term-rev”. Finally BglII site located between two reporter genes was used to integrate “multiple cloning site” with the context given in A ribosome binding site with a context AAGAGG preceded by a series of translational terminators for all three possible ORFs was integrated between BglII and NdeI restriction sites in each plasmid. The insert was prepared by annealing of two oligonucleotides, BglII-stops-RBS and NdeI-RBS-stops , resultifepA and fes  was estimated as the most sensitive parameter. Based on these measurements, transcriptional activity of fepA promoter, driving expression of egfp, appeared to be 5.58 ± 1.66-fold greater than that of fes promoter controlling transcription of mCherry. This prevalence of fepA over fes promoter is consistent with their relative scores estimated in silico (lacZ) and alkaline phosphatase (phoA), which intrinsic properties may contribute to registered relative sensitivity. Moreover this distinction may be due to the different experimental conditions used. Thus, we grew cells in standard Luria-Bertani medium, while Hunt and colleagues2+ ions and sodium citrate or created conditions of iron starvation by the presence of 2,2′-dipyridyl. Furthermore, in our case the expression was measured after 8 h of cultivation to decrease variability and accumulate fluorescent protein, whereas in the cited work it was assayed in early exponential phase. Finally, the promoter-containing regions inserted into reporter vectors were also different. The fragment used infes and 5’-part of fepA, while we incorporated genomic region containing 20% of fepA and about 8% of fes.To test the applicability of the reporter plasmid the fragment containing the known promoters of two divergent genes  and fes , was incn silico . The samrpoB as a reference gene. Relative amount of fepA-mRNA appeared to be 2.5-fold greater than that of fes-mRNA (ΔCt(fepA) = 3.78 ± 0.7, n = 7 versus ΔCt(fes) = 5.11 ± 0.74, n = 7), which is intermediate between the estimates discussed above. Realtive promoter activities may therefore depend on experimental conditions and detecting systems used, but all measurements testified a higher activity of fepA over fes promoter.In order to obtain independent estimate of fepA and fes relative strengths we measured their activities within the native chromosome, using qRT-PCR and highly expressed appY gene. This genomic region is oversaturated by potential transcription start sites corresponding to multiple overlapping promoters  revealed a unique start at position −81 (±2 bp) (mCherry) we registered two or three RNA products, which may be initiated near positions −145, −214 and −225 (in silico (blue arrows in panel b). Thus, using reverse transcription and reporter vector we detected divergent RNA synthesis within appY-associated promoter “island” and found novel RNA products transcribed oppositely to appY. Being non-overlapping with a potential appY-mRNA these RNAs can not be considered as regulatory molecules for appY expression operating by base pairing at complementary ends. However, in the tfaX/appY intergenic region there is enough space for the novel gene which size and functional role remain to be defined.Two transcription start points for  (±2 bp) , which fand −225 . Two of appY may be initiated at positions −113appYappY in chromosomal DNA initiates at some other of multiple promoters predicted in the appY regulatory region  with unidirectional response to osmotic stress accompanied by considerable changes in DNA topology was describedfepA and fes genes is therefore usually observed phenomenon expected from general considerations.In this study quasi-unidirectional response to the treatment by nalidixin was registered only for fepA/fes pair . AlthougappY-associated promoters this explanation is not valuable. Although two divergent promoters operating in this region are partly overlapping (the distance between registered transcription start points is 64 bp), the third one is located far enough (164 bp) to allow independent response to topological changes -tracts that confer it an extremely high propensity to form stable anisotropic bendsThe plausible clue to explain the anomalous behavior of Obviously, several factors simultaneously contribute to the scale and direction of topology-mediated transcriptional response. For divergent promoters they include background superhelical density in the given chromosomal domain, sequence-dependent individual sensitivity of both counterparts to the level of supercoiling and interpromoter distance. Reporter vector constructed and tested in this study allows studying transcriptional coupling in known environment with easily changeable sequence and structural parameters.E.coli K12 MG1655 was used for promoter activity assays, while E.coli strain E.cloni was utilized for primary selection of fluorescent transformants and for amplification of plasmids containing transcriptionally active inserts. pET-28b (“Novagen”) was taken as a parental plasmid for the construction of the reporter vector. pEGFP-N3 (“Clontech”) and pmCherry-C1 (“Clontech”) were used as a source of genes of fluorescent proteins. All plasmids were isolated using QIAprep Spin Miniprep Kit(Qiagen). Oligonucleotides used for the construction of reporter plasmid are listed in plasmid Promoter Finder). PCR-generated DNA fragment of tfaX/appY genomic region (from the position −260 up to position −61 in respect to appY ATG codon) and 857 bp long fragment of fepA/fes regulatory loci (from the position +449 of fepA coding sequence up to position +88 inside the fes gene) were inserted into the reporter vector pPF1 by blunt ends ligation into EcoRV restriction site. The presence and quality of inserts were checked by direct sequencing.E.coli K12 MG1655 cells transformed by pPF1, expressing reporter genes either from appY-associated “promoter island” or from fepA/fes regulatory region. Cells were grown in 10 ml of LB medium in 45 ml tubes under aeration and either in the presence or in the absence of nalidixic acid. After 8 h of cultivation, 1.5 ml of culture was withdrawn and cells were harvested by brief centrifugation. The pellet was resuspended in 1 ml of 1xPBS (pH 7.4) and subjected to 20 s sonication on ice. After removing the debris by centrifugation, the cell lysate was diluted up to 3 ml with PBS and fluorescence was measured in 3 ml cuvette by Varian Cary Eclipse Fluorescence Spectrophotometer. The wavelengths of excitation and emission were, respectively, 484 and 507 nm for EGFP or 587 and 610 nm for mCherry.Measurements of fluorescence were carried out in sonicated extracts of http://hydra.icgeb.trieste.it/dna/index.php)https://www.pymol.org).The sequence of 200 bp DNA-fragment  was submitted to DNAtools server “model it” . The conditions of growth were identical to that ones used for fluorescence measurements. The total RNA was isolated as indicated above, dissolved in 50 μl of 1x DNAse buffer (NEB) prepared on DEPC-water and treated by 4U (2U/μl) RNAse-free DNAse (NEB) for 1 h at 37 °C. RNA concentration was measured spectrophotometrically and confirmed by electrophoresis in 5% PAAG in the presence of 8M urea. Two micrograms of DNA-free RNA were used for reverse transcription with 1 pmol of gene-specific primers. For qRT-PCR the reaction of reverse transcription was performed at 42 °C using RevertAid Reverse Transcriptase (ThermoScientific). Primers used for reverse transcription and qRT-PCR are listed in the −ΔC(t)). The difference between the threshold values measured for the gene of interest and that of housekeeping reference gene (rpoB), ΔCt, was calculated for any given biological replicate. Three technical replicates were done in every biological replicate. Data are presented as a percentage of the expression level of reference gene.Transcription start sites mapping in How to cite this article: Masulis, I. S. et al. Visualizing the activity of Escherichia coli divergent promoters and probing their dependence on superhelical density using dual-colour fluorescent reporter vector. Sci. Rep.5, 11449; doi: 10.1038/srep11449 (2015).


Drosophila melanogaster. We identify 41 mis-expression alleles that enhance and 20 that suppress the effect of His1 depletion in vivo. Most of them are important for chromosome organization, transcriptional regulation, and cell signaling. Specifically, the reduced viability of H1-depleted animals is strongly suppressed by ubiquitous mis-expression of the ATP-dependent chromatin remodeling enzyme CHD1. Comparison of transcript profiles in H1-depleted and Chd1 null mutant larvae revealed that H1 and CHD1 have common transcriptional regulatory programs in vivo. H1 and CHD1 share roles in repression of numerous developmentally regulated and extracellular stimulus-responsive transcripts, including immunity-related and stress response-related genes. Thus, linker histone H1 participates in various regulatory programs in chromatin to alter gene expression.Chromatin structure and activity can be modified through ATP-dependent repositioning of nucleosomes and posttranslational modifications of core histone tails within nucleosome core particles and by deposition of linker histones into the oligonucleosome fiber. The linker histone H1 is essential in metazoans. It has a profound effect on organization of chromatin into higher-order structures and on recruitment of histone-modifying enzymes to chromatin. Here, we describe a genetic screen for modifiers of the lethal phenotype caused by depletion of H1 in  The genomes of all eukaryotes are packaged into a compact nucleoprotein complex called chromatin . The hisDrosophila melanogaster.Chromatin is a dynamic structure that undergoes a variety of modifications, including methylation of DNA, posttranslational modifications of histones, and local changes in compaction. H1 binding to chromatin is highly dynamic with an average residence time of minutes . Unexpectedly, in the genetic screen described here, we identified Chd1, which encodes a SNF2-related ATPase, as an enhancer of His1. Furthermore, by analyzing transcript profiles in H1-depleted and Chd1 mutant larvae, we found that these two factors share a genome-wide role in repression of numerous gene targets in vivo in Drosophila. These results suggest a mechanism for cooperative regulation of genetic activity by chromatin structural proteins (linker histone H1) and remodeling enzymes (CHD1).Unlike classical “synthetic lethal or sick” interaction screens in yeast, our strategy allows us to analyze interactions of Chd1 (1Chd1) as well as 4MpINT-H1 and 1M PpINT-H1-element insertion alleles  that encompass a UAS-driven His1 hairpin transgene and produce moderate H1 knockdown under the control of Tubulin- or Act5C-GAL4 drivers were described elsewhere  or the GAL4 driver-carrying chromosome (+Cy) were scored. The offspring were scored twice in each of the two vials at 2 d and 5 d after the beginning of eclosion, and the four numbers were combined.The recombinant 1/CyOChd1; Tub-GAL4/TM3, Sb and 1/CyOChd1; 1MpINT-H1 fly lines were constructed in a series of crosses. To examine potential genetic interactions of Chd1 and His1, virgin 1/CyOChd1; Tub-GAL4/TM3, Sb females were mated with 1/CyOChd1; 1MpINT-H1 males and reared at 18, 22, 26, or 29°. The adult progeny carrying the TM3 balancer chromosome (Sb) were discarded, and the remaining flies were scored as either Chd1 homozygotes (+Cy) or heterozygotes (Cy). An inter se cross of 1/CyOChd1 flies was performed as a control, and Chd1 homozygotes and heterozygotes were scored as above.Act5C-GAL4, 4M/SM5pINT-H1 or Act5C-GAL4/SM5 females were mated with 10 males carrying an EP insertion on the second chromosome , either homozygous or balanced heterozygous  were calculated using the two-tailed Fisher’s exact and binomial tests to reject the null hypothesis . P<0.01 was considered statistically significant.All statistical tests were performed separately for data sets of viability numbers from crosses with homozygous and heterozygous P<0.05) among genetic interactors, the NIH web-based tool DAVID  classes that are significantly enriched  and varying fold-change thresholding . The functional annotations of the resulting gene lists were performed using DAVID.The RNA expression profiling data were processed and analyzed using the GeneSpring GX11 software (Agilent Technologies) as described  database entries were analyzed by the GEO web-based tool, tabulated in Excel, cross-referenced according to the Affymetrix probe IDs or Flybase IDs and sorted by the relative fold-change and statistical significance. 5M/Tub-GAL4pINT-H1 L3 larvae at 29°. Animals that underwent reduction of expression of an unrelated gene Nau were used as a control . As a control, similar crosses were performed with 1118w; Act5C-GAL4/SM5 females. At 26°, H1 depletion resulted in a substantial decrease in the proportion of +Cy progeny . In contrast, ubiquitous GAL4 expression alone did not result in a significant decrease of viability at any temperature (P<0.01) while scoring a relatively small number of adult progeny (50–100).First, to streamline the screen, we prepared a recombinant eratures . To thisperature . TherefoEP insertion alleles on the second chromosome were substituted for the 1118w males; 433 of the EP insertions are homozygous viable, and males with a homozygous insertion were used for the crosses (CyO or SM5) and adjusted the scoring accordingly . In contrast, mis-expression of MESK2 in EP2347Act5C-GAL4/P{EP}MESK2 flies increases the proportion of +Cy adults in the progeny of the cross to ∼66% or ∼132% relative to the expected Mendelian ratio (Act5C-GAL4/P{EP} adults of ∼47% (∼94% of expected) in crosses with homozygous fathers or ∼33% (99% of expected) in crosses with heterozygous fathers.Mis-expression of individual genes alone had variable effects on viability of adult an ratio . The disAct5C-GAL4, 4M/SM5pINT-H1 mothers), the mean fraction of Act5C-GAL4, 4M/P{EP}pINT-H1 adults was ∼24% (∼48% of expected) for homozygous crosses and ∼21% for heterozygous crosses (∼63% of expected), consistent with the control numbers  and control (Act5C-GAL4) crosses and calculated P values for deviation from the expected numbers. (The “expected” numbers assume independent effects of H1 depletion and gene mis-expression on viability.) Second, we performed the Fisher’s exact test  using a representative gene as a reference. Normalized effect (NE) numbers were used as the measure of gene-specific effects and signify increased or decreased viability for Act5C-GAL4, 4M/P{EP}pINT-H1 flies normalized to viability for Act5C-GAL4/P{EP} flies and median viability of Act5C-GAL4, 4M/P{EP}pINT-H1 flies across the whole collection of second chromosome EP alleles (in lieu of the wild-type isogenic control). The OR in both the Fisher’s exact and binomial tests strongly correlated with the calculated NE for each EP allele. We defined results as statistically significant when relative viability of H1-depleted adults was affected more than 25% by the EP-driven mis-expression (NE<0.8 or NE>1.25), and when P values calculated in both the Fisher’s exact and binomial tests were less than 0.01  term analysis using the NIH web-based toll DAVID . These interactions strongly suggest that H1 plays important regulatory roles in the cognate biological processes.To classify alleles that genetically interact with EP insertions (∼15% of the identified genetic interactors) are upstream of genes that control chromatin structure and chromosome organization/metabolism. Five of them are suppressors of H1 depletion-mediated lethality. It is possible that the reduction of H1 expression can be partially compensated by ubiquitous mis-expression of EP-driven genes due to their ability to modify chromatin structure and activity. For instance, we observe a strong positive genetic interaction between His1 and E(Pc), a member of the PcG gene family. It is possible that H1 is biochemically involved in Polycomb silencing and, vice versa, Polycomb-dependent repression and modification of chromatin structure may partly compensate for the global loss of H1. Interestingly, a biochemical link has been previously proposed between H1-mediated oligonucleosome fiber compaction and Polycomb silencing: H1 can stimulate the enzymatic activity of Drosophila PRC2 complex on a dispersed oligonucleosome substrate  potentially drive mis-expression of transcription factors and other DNA-binding proteins. Interestingly, a large group of genetic modifiers of His1 [18 ] have functions in the nervous system development and/or activity, and the majority of genetic interactions between His1 and members of this group are negative. The enrichment of neuronal genes among His1 interactors suggests that H1 may play a specialized role in regulation of their chromatin structure and activity. A chromatin-dependent epigenetic mechanism is known to be essential for silencing a broad range of neuronal genes in all non-neural tissues across metazoan evolution. A common repressor element, RE1/NRSE, in their regulatory regions binds a ubiquitous (outside of the nervous system) transcription factor REST/NRSF (Tramtrack in Drosophila), which in turn recruits a histone-modifying complex CoREST that can remove histone acetyl and H3K4-methyl active epigenetic marks ]. One model that would explain the modification of the H1 depletion-induced lethality by mis-expression of genes involved in stress responses is that H1 itself plays a role in tissue-specific regulation of many genes that belong to this class. Ubiquitous mis-expression of a single gene might amplify or diminish the deleterious effect of their simultaneous global de-regulation caused by the knockdown of H1 alone. Therefore, we examined the influence of strong H1 depletion on expression of immunity-related and stress response–related genes in larval salivary glands. We analyzed our previously reported microarray-based transcript profiling  genes are strongly repressed in Chd1/Chd1 null larvae  pathways through master transcription factors of NFκB-family, Dorsal, Dif, and Relish . For insegulated . TherefoChd1 suppresses H1 depletion-dependent reduction in fly viability. Thus, we expected that reduced expression of CHD1 in homozygous Chd1 null progeny of Chd1/CyO inter se crosses would enhance the semi-lethal phenotype caused by RNAi-mediated H1 depletion. To test this hypothesis, we crossed 1/CyOChd1; 1MpINT-H1 and 1/CyOChd1; Tub-GAL4/TM3, Sb parents at various temperatures and scored the relative viability of 1Chd1 homozygotes among +Sb (H1-depleted) progeny  are also affected by H1 depletion (Table S2), which represents ∼2.5 enrichment compared with the total proportion of H1-affected gene transcripts . The most significant correlation is between transcripts that are repressed by CHD1 (596) and H1 (1354): of these, 146 appear to be repressed by both CHD1 and H1, which represents ∼3.4-fold enrichment (P=2.3⋅10−41). For comparison, no significant correlation is observed between CHD1-repressed and H1-activated transcripts [29 of 596 (∼1.1-fold enrichment) P=0.32]. Thus, CHD1 and H1 exhibit an extremely strong overlap of their repressive roles in global transcriptional regulation . As expected, the transcripts co-regulated by H1 and HP1 are enriched for transposable elements and repetitive/heterochromatic sequences (P=9.7⋅10−18] (Table S4). In contrast, a similar very strong correlation is not observed between CHD1-dependent and HP1-dependent or ISWI-dependent repression. Of 596 CHD1-repressed transcripts, only 26 are also HP1-repressed , and 16 are also ISWI-repressed  (Table S2). Note that a somewhat elevated correlation between transcriptional programs of CHD1 and HP1 may represent a secondary, indirect effect of a strong link between a regulatory program of H1 with those of both CHD1 and HP1, because 15 out of 26 transcripts (57.7%) that are coordinately repressed by CHD1 and HP1 are also subject to transcriptional repression by H1  that are affected by CHD1 and H1 are not constitutively active but instead are strongly regulated in development or in response to external stimuli. It is possible that the principal mechanism of repression for these genes is at the level of elongation. In response to the stimuli under normal conditions (in wild-type), particular features of their promoters, such as cis-acting elements, may rapidly recruit factors that stimulate Pol 2 elongation, help to obviate the barrier presented by nucleosomes reassembled by CHD1, and thus de-repress transcription. Importantly, linker histones interspersed on nucleosomes within a transcription unit are expected to create an additional obstacle for the polymerase during transcriptional elongation 3-9, H1 contributes to the establishment of the silenced epigenetic state (


Sequences of the genome that are capable of silencing gene expression are thought to play a key role in gene regulation. However, very few silencer elements capable of functioning in mammalian cells have been described, and only a fraction of these have been tested for the ability to function in an autonomous fashion. We report here the characterization and functional validation of a constitutive autonomous silencer element from the human genome called T39, and the comparison of T39 to three other putative silencer elements previously described by others. Functional analysis included one assay for enhancer-blocking insulator activity and two independent assays for silencer activity, all based on stable transfection and comparison to a neutral spacer control. In erythroid K562 cells, T39 exhibited potent silencer activity, the previously described element PRE2-S5 exhibited modest silencer activity, and the two other previously described elements exhibited no silencer activity. T39 was further found to be capable of silencing three disparate promoters, of silencing gene expression in three disparate cell lines, and of functioning as a single copy in a topology-independent manner. Of the four elements analyzed, only T39 exhibits a constitutive pattern of DNase hypersensitivity and binding by CTCF. In its native location the T39 element also exhibits a unique interaction profile with a subset of distal putative regulatory elements. Taken together, these studies validate T39 as a constitutive autonomous silencer, identify T39 as a defined control for future studies of other regulatory elements such as insulators, and provide a basic chromatin profile for one highly potent silencer element. Information generated by the ENCODE project [Mammalian gene expression is regulated in  project , as well project –4. Regul project ,6). Howe project ); and el project . This isThe availability of a very limited number of autonomous mammalian silencer elements described in the scientific literature has made it difficult to identify common mechanisms of action, as well as common chromatin profiles. Advances toward indentifying and characterizing autonomous silencer elements have also been hampered by the lack of independently-validated functional assays. A question even remains as to whether silencer activity determined by transient transfection of reporter constructs can accurately predict whether a particular sequence is capable of providing silencer activity in the setting of intact chromatin. We report here the use of two functional assays based on stable transfection of K562 cells, which allow for the identification of potential silencers in the human genome. Using these assays we characterize a novel silencer element from the human genome, silencer T39, which is active in mammalian cells. We also use these stable transfection assays to functionally characterize elements previously reported to function as silencers on the basis of transient assays. Finally, we present data indicating that the T39 silencer is functional in combination with three distinct promoters and in three distinct cell lines, and that it physically interacts with other putative regulatory elements within its native locus.2.The following human cell lines were used for these studies: K562 , HEK-293 , HeLa , and HepG2 . K562 cells were cultured in Iscove's Modified Dulbecco's Medium supplemented with 10% fetal bovine serum (IMDM/FBS) and the other cell lines were cultured in Dulbecco's Modified Eagles Medium supplemented with 10% fetal bovine serum (DMEM/FBS) at 37°C, 5% COneo, transcribed by a promoter from the human γ -globin gene HBG1 and terminated with a polyadenylation sequence from the virus SV40. High level reporter gene expression is dependent on an enhancer derived from DNase I hypersensitive site 2 (HS2) of the mouse β-globin gene cluster and placed 3′ of the neo expression cassette. In order to assess both silencer and enhancer-blocking insulator activity, candidate sequences are inserted immediately 5′ of the neo expression cassette and 3′ between the neo expression cassette and HS2 enhancer. In order to assess silencer activity only, candidate sequences are inserted immediately 5′ of the neo expression cassette and 3′ of the HS2 enhancer. Candidate sequences were inserted by MultiSite Gateway exchange [6 cells using the Amaxa Biosystems Nucleofector II electroporator and Nucleofector V kit . The cells were then resuspended in 5 mL IMDM/FBS and cultured for 2 days to allow for genomic integration of the plasmid. After this, the cells were collected, counted, resuspended at a dose of 5 x105 cells in 5 mL IMDM, 20% FBS, 0.8% low-melting agarose, and 1 mg/mL of the neomycin drug analog G418 (active component), and plated in 60 mm tissue culture dishes in triplicate. These semisolid cultures were incubated another 2–3 weeks and then scored for colony formation. Inserts included the PRE2-S5, MECP2 F3, PDGFA 5′SHS, and T39 putative silencers listed in zeo as a neutral spacer control [The drug-resistant colony assay has been described previously ,8,9. Theneo colony assay, except that the cells were expanded in DMEM/FBS for 7 days without selection. The cells were then collected, washed, and analyzed by flow cytometry for reporter GFP expression. The level of expression was determined by measuring the mean fluorescence of the cells located within the GFP-positive gate. See The GFP reporter plasmids contain an expression cassette for GFP terminated with a polyadenylation sequence from the virus SV40. This cassette is transcribed either by the same HBG1 promoter used for the drug-resistance colony assay reporter plasmid, the promoter from cytomegelovirus (CMV), or the promoter from the human PGK gene. These reporter constructs were either used directly or with the T39 silencer element inserted either 5′ or 3' of the expression cassette. Reporter plasmids were either left circularized or were linearized with Sca I, and transfected into HEK293 cells, K-562 cells, HeLa cells, or HepG2 cells as for the http://genome.ucsc.edu).Data involving chromatin profiles, including CTCF binding, Digital DNase footprinting, DNase I hypersensitivity, and CTCF Interactions, where derived from data publically available on the UCSC Genome Browser  in the first intron of the gene ARHGAP6 . The seqA partial list of silencers from the literature appears in In theory, autonomous silencer elements should be capable of exerting their activity on different sources of promoters and within multiple cell lines. The G418-reisistant colony assay and associated reporter construct could not be easily used to assess these properties, since the HS2 enhancer does not function with all promoters, the HBG1 promoter does not function in all cell types, and not all cell types are capable of forming distinct colonies in agarose. For these reasons, we turned to the reporter construct diagramed in We noted that the degree of silencing achieved with the HBG1 promoter in this setting was far less than the 36-fold observed with the same silencer and promoter in the drug-resistant colony assay. We believe this is due to the absence of the HS2 enhancer in the GFP reporter construct, and the use of a non-erythroid cell line, resulting in a substantial reduction in the basal activity of this promoter. This in turn restricts the dynamic range available for detecting the effect of a silencer. Taking this into account, we chose the reporter construct with the highly active CMV promoter for further studies . As summThe existence of transcriptional silencers has been recognized in the literature for nearly three decades . HoweveOf the three elements we identified from the studies of others and analyzed in the drug-resistance colony assay, only PRE2-S5  exhibiteRegardless to the reasons for the discrepancies between our results and the results of others, our studies demonstrate that these two elements do not function as fully autonomous silencers.The T39 element functioned as the most potent autonomous silencer out of the four elements analyzed here. As reported here, the T39 silencer reduced the rate of G418-resistant colony formation 77-fold in the enhancer-blocking configuration and 36-fold in the silencer configuration. Although less dramatic, this element also reduced expression of the GFP reporter constructs in the context of three distinct promoters and three distinct cell lines. In addition, this silencing activity can be achieved when T39 is used as a single copy, and when the T39 element is placed either 5' or 3' of an expression cassette. From these studies we conclude the T39 element acts as a potent, constitutive, autonomous silencer.neo reporter gene exceeds a specific threshold [The T39 silencer exhibited statistically significant silencer activity in both of the drug-resistant colony assays and the GFP expression assay. However, the magnitude of this effect varied greatly between these two assay systems. Previous studies demonstrated that the drug-resistant colony assay measures the frequency of transfection events in which expression of the The T39 silencer exhibits a distinct chromatin profile, including high occupancy by CTCF and DHS formation in multiple tissues. This silencer is located in the first intron of the ARHGAP6 gene. It has been suggested that silencers located in introns could suppress transcription by blocking transcriptional elongation, by preventing recognition of intronic splice sites or by abrogating basal transcriptional apparatus assembly ,6,7. OnlIn conclusion, our studies independently validated two integration-based reporter assays for the identification of silencer elements, and use these assays to validate two autonomous silencer elements: PRE2-S5 and T39. The drug-resistant colony assay provides the greatest degree of sensitivity, while the GFP reporter assay allows for a more direct assessment of silencer activity and provides a greater degree of experimental flexibility. These studies also suggest that the T39 sequence functions as a potent, constitutive autonomous silencer in a manner that is independent of the promoter or cell line. This element can be used as a positive control for studies into other silencer elements, as well as chromatin insulators. More studies are needed to dissect the mechanisms underlying silencer function, and to link these activities to specific chromatin profiles.S1 FigThe DNase I hypersensitive site (DHS) profiles are presented for the four putative insulators in the 5 indicated cell lines. The four putative silencer elements T39, PRE2- S5, MECP2-F3, and PDGFA 5′ SH5, are described at length in the main manuscript. The five cell lines were chosen based on their use in the previous characterization of the putative insulators and the availability of publicly accessible DHS profiles. The density of DNase-seq reads are presented as vertical bars, with gray horizontal bars indicating the presence of statistically significant DHS peaks. The locations of the putative silencer sequences are indicated underneath the histograms. Each window is set at 1000 bp.(PDF)Click here for additional data file.S2 FigThe image shows the genomic organizations of a 710 kb window around the native T39 locus in K562 cells, derived from publically available data on the UCSC Genome Browser. The location of the T39 element and three surrounding genes  are indicated. Also indicated are the promoters for these genes (P). The middle two tracks show the CTCF and DHS profiles for the region. At the bottom is indicated the CTCF interactome for the region, as determined by chromatin capture technologies. Interactive regions are connected by red arches, with the intensity of the red representing the frequency of the interaction. Note that T39 interacts with a DHS/CTCF element inside the same ARHGAP6 intron, with a DHS/CTCF element at the distal end of the ARHGAP6 gene, and with a DHS/CTCF element in the intron of the adjoining MSL3 gene, but not with any of the gene promoters.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Human immunodeficiency virus type 1 is known to use the transcriptionalmachinery of the host cell for viral gene transcription, and the only viralprotein that partakes in this process is Tat, the viral trans-activator oftranscription. During acute infection, the binding of Tat to the hairpin at thebeginning of the transcribed viral RNA recruits the PTEFb complex, which inturn hyperphosphorylates RNA-polymerase II and stimulates transcriptionelongation. Along with acute infection, HIV-1 can also lead to latent infectionthat is characterized by a low level of viral transcription. During themaintenance and reversal of latency, there are no detectable amounts of Tatprotein in the cell and the mechanism of transcription activation in theabsence of Tat protein remains unclear. The latency maintenance is also aproblematic question. It seems evident that cellular proteins with a yetunknown nature or role regulate both transcriptional repression in the latentphase and its activation during transition into the lytic phase. The presentreview discusses the role of cellular proteins Ku and HMGA1 in the initiationof transcription elongation of the HIV-1 provirus. The review presents dataregarding Ku-mediated HIV-1 transcription and its dependence on the promoterstructure and the shape of viral DNA. We also describe the differentialinfluence of the HMGA1 protein on the induced and basal transcription of HIV-1.Finally, we offer possible mechanisms for Ku and HMGA1 proteins in the proviraltranscription regulation. Although the human immunodeficiency virus HIV- 1) was discovered over 30 yearsago, the fight against the HIV infection still has not been won. Highly activeantiretroviral therapy that is used to manage the HIV infection hassignificantly reduced mortality among patients with AIDS; however, interruptionof treatment inevitably results in viral reproduction and increases the viraltiter. One of the reasons for this phenomenon is the presence of cells in thehuman organism with the transcriptionally silent provirus integrated in theirgenome. The silent state, which is typical for the latent phase of the viralinfection, is characterized by the absence of full-fledged transcription fromthe viral promoter. However, without treatment, the silent provirus can beactivated and cause the development of AIDS [ was discThe hairpin structure located at the 5’-end of the synthesized mRNA andknown as TAR (trans-activation response) plays a key role in activetranscription from the HIV-1 promoter. Elongation of transcription of theintegrated viral genome takes place only when TAR is bound by the viral regulatoryprotein Tat (trans-activator of transcription). FormatiIt has recently been shown that cellular protein HMGA1 can be recruited in theregulation of transcription from HIV-1 promoter during the latent phase  , 5. DNA-Human immunodeficiency virus type I is a member of the genus Lentivirus, partof the family Retroviridae. It affects the human immune system and causes theacquired immune deficiency syndrome (AIDS). Like the genome of otherlentiviruses, the HIV-1 genome is an RNA molecule, which serves as a templatefor the synthesis of a DNA copy by viral enzyme reverse transcriptase. The DNAcopy is then integrated into the cellular genome forming proviral DNA. However,most of the viral DNA remains non-integrated . This DNBeing integrated into the chromosome of an infected cell, viral DNA can eitherstay silent or be actively transcribed. In other words, the transcription levelcan be low thus resulting in a small number of transcripts without rapidprogression of the infection and is generally referred to as basal (notactivated) transcription. Alternatively, transcription can be active and yielda large amount of RNA and new viral particles. Regulation of the HIV-1 genometranscription, which precludes the fate of the provirus, depends on a largenumber of factors: cis-acting elements of viral DNA, cellular transcriptionfactors, viral trans-activator Tat, and the degree of chromatin condensation.Fig. 1).Transcription starts at the border between the U3and R regions in the 5’-LTR, since the viral promoter recognized by RNApolymerase II (RNAP II) and some other regulatory elements are located in theU3 region. 5’-LTR contains four functional regions partaking in theregulation of the HIV-1 genome transcription: the modulatory region, theenhancer, the promoter, and the leader regions[Fig. 1).These factors are involved in the initiation of transcription[Viral DNA integrated into the cellular genome carries long terminal repeats(LTRs) at its ends. Each of them consists of U3, R, and U5 regions. They co. They ccription.D-ribofuranosylbenzimidazolesensitivity-inducing factor) [Transition of the provirus from a silent to an active state starts with thetranscription initiation. Short abortive transcripts ~60–80 nucleotideslong are synthesized ; they fo factor) . Tocont factor) .Fig. 2)[The viral protein Tat is the key regulator at the elongation stage: it enhancesefficiency of RNA synthesis by several orders of magnitude. BindingFig. 2).Fig. 2A,B). Alternatively,a cellular protein different from P-TEFb but capable of the phosphorylation of CTDRNAP II (as well as the NELF and DSIF repressive factors) may bind to the HIV-1 promoter.However, Tat is not detected in cells at the latent stage of infection. Neitheris it found when the provirus starts exiting from dormancy. In some cases, theTAR– Tat–P-TEFb complex cannot be formed due to mutationsdisrupting the interplay between its components .NeverthAlthough a significant amount of data on the regulation of HIV-1 transcriptionand involvement of various cellular factors in it has been accumulated, manyaspects have not been completely elucidated yet. In particular, the role of twocellular proteins involved in HIV-1 transcription (Ku and HMGA1) remainsunclear. Some available data attest to the positive role of these proteins inthe regulation of transcription, while other studies demonstrate that theirrole is negative. Nevertheless, the accurate mechanisms of involvement of theseproteins in HIV-1 transcription are still to be determined.xrcc6 (Ku70) and xrcc5(Ku80) genes. Ku protein mainly functions in the cell in the form of avery stable heterodimer [Human Ku protein is a heterodimer consisting of two subunits with masses of ~70and 80 kDa, which are known as Ku70 (p70) and Ku80 . These proteinsare encoded by the erodimer . Howevererodimer .Kd value varies within a range of1.5–4.0 × 10-10 M [Fig. 3).The channel predominantly consists ofpositively charged amino acid residues that interact with the negativelycharged sugar-phosphate backbone of the DNA molecule, which explains why Ku canbind DNA in a sequence-independent manner. After binding to the DNA end, Ku canmigrate (slide) along DNA and pause at certain sequences[The Ku70/Ku80 heterodimer is a DNA-binding protein that mostly interacts withthe free ends of doublestranded DNA, and its biological function is mainlyrelated to this feature. The interaction between the Ku heterodimer and DNA israther strong: the  10-10 M .Accordi 10-10 M , Ku70 anequences, 27.The most well-known and the best studied biological function of Ku is itsinvolvement in double-strand DNA break repair by nonhomologous end joining(NHEJ). Ku also participates in such cellular processes as V(D)J-recombination, mobile element-induced genomic rearrangement, telomere lengthmaintenance, apoptosis, and transcription , 29. OneThe possible mechanisms of Kumediated regulation of transcriptionc-Myc, Hsp70[Participation in transcriptional regulation is one of the numerous functions ofKu. Several mechanisms of transcriptional activation or suppression by Ku havebeen described. The first mechanism is a direct sequence- specific interactionbetween Ku and the promoter region of genes. It has been hypothesized thattranscription of the cellular genes c, Hsp70, U1 snRNc, Hsp70, as wellc, Hsp70 and MMTVc, Hsp70, is reguc, Hsp70.Fig. 4)[in vitro phosphorylated by DNAPK. Specific recruitment ofDNA-PK to the promoter and subsequent phosphorylation of transcription factorsis probably one of the transcriptional regulatory mechanisms.A sequence whose binding to Ku is considered to be truly sequence-specific andmore preferable compared to Ku binding to DNA ends has been identified in theNRE-1 region (negative regulatory element 1) in the LTR of MMTV retrovirus. TheintFig. 4), 35.It Fig. 4) and Oct-Fig. 4), the twoFig. 4). Onlythese sequences were shown to be capable of direct andspecific interaction with the Ku heterodimer in the absence of free DNA ends.All the Ku-binding sites in promoters homologous to the NRE-1 sequence in thepromoter of the GR strain of MMTV and known up to the publication date arereported in second mechanism via which Ku affects the transcription isits direct interaction with transcription factors, including Oct-1, Oct-2[ in vitro. The ability of DNA-PK to interact bothwith transcription factors and a number of nuclear receptors  probablc-jun gene, the AP-1 transcription factor component [Ku can indirectly regulate gene transcription by influencing the expression ofother transcription factors. Thus, in the AGS cell line, Ku positivelyregulates the expression of the gene of the NF-κB p50 subunit . Ku80 alomponent . It shouIL-2 gene promoter, while the amount of the Ku70 subunit boundto this region decreases [Hsp70 gene was attributed tothe Ku70 subunit rather than to Ku80.The Ku70 and Ku80 subunits may have a different effect on transcription. It hasbeen demonstrated that the subunits of the Ku heterodimer dynamically bind tothe promoter of the interleukin 2 (IL-2) gene and interact with the NF45/NF90factor in response to T-cell activation . This acecreases . Inanotecreases , the repthird mechanism of transcriptional regulation is thedirect interaction between Ku heterodimer or its Ku80 subunit and RNAP IIholoenzyme. Ku80 was found to be colocalized with the elongational form of RNAPII and transcription factors specific for the elongation stage  in the nucleus. The C-terminal domain of Ku80 was also found to play akey role in the interplay with these proteins [in vitro [The proteins . Let us in vitro ; howeverfourth mechanism of Ku involvement in transcriptionalregulation is related to its role in the repair of double-strand DNA breaks[The A breaks. Double-Hence, the isolated heterodimer subunits, the heterodimer as a whole, or itscomplex with the DNAPKcs catalytic subunit can be involved in the regulation oftranscription. No common mechanism of action of Ku has been revealed. It ismost likely that there is a specific mechanism of Ku-dependent regulation foreach particular gene. It should be mentioned once again that Ku may act both asan activator and as a suppressor; its effect usually depends on subunits, whichare involved in the regulation. It seems that the catalytic subunit of DNA-PKis not necessarily involved in the Ku-dependent regulation of transcription;however, in certain cases its capability of DNA-dependent phosphorylation oftranscription factors and RNAP II can be the key element of regulation.Role of Ku in HIV-1 transcriptionThe significance of Ku for maintaining the HIV-1 life cycle has beendemonstrated in numerous studies. The Ku70 subunit is a part of thepre-integration complex and interacts with HIV-1 integrase , 52. TheInvolvement of Ku in the transcriptional regulation of HIV-1 was first reportedin the early 2000s. However, the role of Ku in this regulation is still to beclarified. Data attesting both to the positive and negative effects of Ku ontranscription of the HIV-1 genome have been obtained.Ku80 gene expression. These cells supported enhancedexpression from the plasmid carrying the CAT (chloroamphenicolacetyltransferase) gene under the control of the viral promoter from5’-LTR [Ku80 genereduced CAT expression. Hence, Ku80 has a negative effect on transcription fromthe HIV-1 5’-LTR promoter. The negative role of Ku80 has been confirmedusing a human U1 cell line whose genome contained the integrated provirus; thiscell line is used as a model of the latent state of HIV-1. It turned out that adecreased amount of endogenous Ku80 in the cells increases the level oftranscription of the HIV-1 genes, both for basal and TNFα-inducedtranscription.The role of Ku in transcription from viral 5’-LTR was first studied usingthe xrs-6 cell line, a variant of CHO-K1 (Chinese hamster ovary) cells lackingFig. 5)[Fig. 5A)facilitates recruitment of these factors to the HIV-1 promoter[Taking into account that Ku has a negative effect on transcription from otherretroviral promoters  ,33, L. Fig. 5). SeveralFig. 5), which rpromoter. It isapromoter overlappIn their next study , this grAs opposed to the aforementioned findings, Ku plays a positive role in theregulation of transcription from the HIV-1 promoter in human MAGI and CEM-T4cell lines .Further Ku80+/-variant, which were transduced with HIV-1- based lentiviral vectors, were used.It has been preliminarily shown that in the HCT 116Ku80+/- cells with a twofold reduced level of Ku80the level of Ku70 is equally lowered. It turned out that a twofold decrease inthe amount of endogenous Ku in cells reduces the efficiency of viraltranscription. It should be mentioned that, as opposed to data [The positive influence of the Ku heterodimer on transcription of the HIV-1genome was also mentioned in , wherewIt is known that Ku80 is incorporated into the virion during its assembly. Hence, It should be mentioned that the involvement of the previously predictedKu-binding site in HIV-1 LTR in Ku-mediated regulation of the provirustranscription was also refuted in .ReplaceAnother important aspect of this study is that Ku does not affect transcriptionfrom the circular forms of viral DNA. Taking into account that, according to, Ku had et al. who investigated thepossible involvement of both the Ku70/Ku80 and the entire DNA-dependent proteinkinase DNA-PK in the transcriptional regulation of HIV-1. Experiments werecarried out using Jurkat-E4 cells whose DNA carried the integrated HIV-1genome. Thus, this cell line was used as a model of T cells in the latencyperiod of infection. DNA-PK was found on the HIV-1 promoter, and its locationcorrelates with that of RNAP II. It was also determined that transcriptionactivation significantly increases the DNA-PK and RNAP II levels not only onthe promoter, but also on the transcribed region of the genome.We would like to draw special attention to study  by S. Tyin vitro experiments showed that DNA-PK predominantly phosphorylatesSer2 rather than Ser5 or Ser7 in the heptapeptide repeats YSPTSPS. Consideringthe fact that phosphorylation of Ser2 is required to activate elongation, ithas been hypothesized that involvement of DNA-PK can be important, mostly atthe elongation phase of transcription. DNA-PK presumably directly interactswith RNAP II at the HIV-1 promoter, and DNA-PK can act as a factorphosphorylating polymerase and eliminating elongation block. At any rate,parallel distribution of DNA-PK and RNAP II along the provirus and theirsimultaneous recruitment in response to transcription activation allow one tosuggest that DNA-PK (and Ku as its component) is an element of a largetranscriptional complex that is involved in HIV-1 gene expression.It has also been demonstrated  thatDNAFurthermore, it has been demonstrated that DNAPK has a positive effect ontranscription from HIV-1 5’-LTR and lentiviral vectors . KnockdoSummarizing the role of the Ku protein in the regulation of HIV-1transcription, it should be mentioned that the currently available data arerather controversial. The possible reason is that different cell lines anddifferent viral systems were used. Thus, most data attesting to negativeregulation have been obtained using rodent cells, which obviously cannot be anadequate model for processes occurring in HIV-1-infected human cells.Nevertheless, the data obtained using human cells provide ground for drawingsome reliable conclusions.The first general conclusion is that Ku-mediated regulation of transcriptiondepends on viral LTR. The regulation mechanism remains unclear, but one shouldnot rule out the possibility of direct binding of the heterodimer to LTR,although the putative Ku-binding site within LTR probably is not the keyelement in Kudependent regulation of HIV-1 transcription, as opposed to MMTVand HTLV-1.Second, the integrated provirus is crucial for Kumediated transcriptionalregulation of HIV-1 and HIV-1-based lentiviral vectors. Although HIV-1transcription can occur from circular DNA, it is clear that Ku is not involvedin its regulation. This can be explained by the fact that the heterodimer isrecruited to the provirus either during integration or immediately after it.Let us mention once again that there remain many questions concerning themechanism of involvement of Ku in the transcriptional regulation of HIV-1. Evenif the incorporation of the Ku70/Ku80 heterodimer or the entire DNA-PK into thetranscription complex is considered to be an established fact, the mechanismthrough which they influence HIV-1 gene expression has not been elucidated yetand it is important to clarify their role in HIV-1 transcription.Role of HMGA1 in HIV-1 transcriptionHMGA1 , theDNA-binding non-histone chromatin protein, is another cellular protein whoserole in the HIV-1 life cycle has not been studied sufficiently. HMGA1 carriesthree DNA-binding motifs that preferentially bind to the DNA minor groove inATrich regions (A/T hook) . HoweverFig. 6). HMGA1plays a crucial role in the regulation of enhanceosome assembly or disassembly, thusaffecting transcription. It has been repeatedly demonstrated that HMGA1directly interacts with other chromatin-re-modeling proteins and transcriptionfactors [Actually, all the high-mobility group proteins are capable of binding both DNAand proteins, which allows them to get involved in a large number of processes. Alteratd c-Rel), 63.TheInvolvement of HMGA1 in the life cycle of HIV-1 has been demonstrated in manystudies. This protein was detected within the pre-integration complex. HMGA1 wSome rather ambiguous evidence in support of the involvement of HMGA1 in HIV-1transcription has been obtained to date.Fig. 7)[Fig. 7).This site resides at the borderof the repressive nucleosome nuc-1, which exists in the provirus near thetranscription initiation site in the latent phase and degrades aftertranscription of the viral genome is activated. HMGA1was found to facilitate binding of AP-1, an important inducible HIV- 1transcription activator, to viral DNA in response to external stimuliactivating viral expression. HMGA1 is possibly involved in the reorganizationof nuc-1 by competing for this site and making it free for AP-1. Hence, it hasbeen suggested that HMGA1 can positively regulate HIV-1 transcription[Putative HMGA1 binding sites have been detected by DNase I footprinting in the-187/+230 region within HIV-1 LTR . The intcription.Fig. 7).This makes it possible to recruit the ATP-dependent chromatin-remodeling complexSWI/SNF to the repressive nucleosome: a process required for efficient activationof viral transcription.The role of HMGA1 as an architectural transcription factor involved in thereorganization of nucleosome nuc-1 has been confirmed. HMGA1 wFig. 8)[Fig. 8A). Secondly,binding of 7SK to HMGA1 regulates the amount of free HMGA1 that can interactwith DNA and functions in various processes.The mechanism is believed to depend on the nature of the particular gene.Another possible mechanism via which HMGA1 can be involved in transcriptionregulation has recently been proposed .It turnFig. 8). First, Fig. 8A[In the case of HIV-1 transcription regulation, Sp1 can be a factor thatinteracts with HMGA1 and, thus, is recruited to the elongation complex. On onehand, this factor is known to be involved in HIV-1 transcription,while oFig. 8A and, theFig. 9);HMGA1 may compete withviral protein Tat for binding to TAR RNA. This results in a negative effect ofHMGA1 on HIV-1 transcription both in the presence and absence of Tat. Theinfluence of overexpression and knockdown of the HMGA1 geneand 7SK L2 RNA on transcription from the HIV-1 promoter was studied in thepresence and absence of Tat. HMGA1 was found to reduce both the basal andTat-activated transcription from the HIV-1 promoter, which is partiallyrecovered upon 7SK L2 RNA overexpression. Based on this experiment, the modelof HMGA1- mediated repression has been proposed[Another mechanism via which HMGA1 influences HIV-1 transcription was uncoveredwhile studying the expression of a reporter gene under the control of viral5’-LTR from a plasmid . In thisFig. 10). AccordiFig. 9Structures of the 7SK L2and TAR RNA regions interacting with HMGA1. The HMGA1 binding site in both RNAsis shown in green. The TAR region responsible for the interaction with Tat andCycT1 is shown in gray [Fig. 11)[A different model of the repressive effect of HMGA1 on transcription from theHIV-1 promoter has also been proposed . Factors in gray . in the in gray .It has  in gray . Moreove in gray .A modelFig. 11). HMGA1 gene significantlyreduces the levels of CTIP2 and P-TEFb/7SK snRNP recruited to the viralpromoter and consequently recovers the level of transcription from it. Hence,it is assumed that a mechanism of HMGA1-mediated negative transcriptionalregulation similar to that shownin Fig. 11 maybe realized on the viral LTR. Nevertheless, in the case of HIV-1 it remainsunclear which DNA region or the LTR-bound factor is involved in recruitment ofthe HMGA1/CTIP2/7SK snRNP complex. The role of HMGA1 binding to TAR in theHMGA1/CTIP2-mediated repression of basal transcription is also unclear. Neitherhas it been studied whether HMGA1 has a direct effect on the binding of CTIP2to viral DNA, similar to what happens with the transcription activator AP-1[It has been demonstrated that when interacting with the HIV-1 promoter, HMGA1and CTIP2 synergistically repress basal transcription .Knockdotor AP-1.Hence, HMGA1 can both activate and repress HIV- 1 transcription. Its positiveeffect was seen in induced transcription, while a negative influence was shownfor basal transcription. Possibly, some external factors trigger the switch ofprotein partners of HMGA1 and subsequent alteration in the mechanism of HMGA1-mediated transcriptional regulation.Although the features of HIV-1 transcription have been extensively studied,many aspects still have not been fully elucidated. It is well known thatelongation of transcription of the viral genome takes place after the viralregulatory protein Tat binds to TAR RNA, which interacts with the multiproteintranscription elongation factor P-TEFb for its recruitment to the viralpromoter. Cyclin-dependent kinase Cdk9 within P-TEFb performs phosphorylationof RNAP II that is required for elongation. However, a question arises: how isthe transcription of the latent provirus activated in the absence of Tatprotein? It was assumed that the phosphorylation of RNAP II, which is requiredfor eliminating transcription block and transition to the elongation stage, canbe activated by cellular factors. Some cellular factors may recruit P-TEFb tothe promoter.Regulation of HIV-1 transcription is a process that involves many cellularproteins; however, the role of some of them is not fully clear. Two cellularproteins described in this review, Ku and HMGA1, are among these “unclearfactors.” Data attesting to both the stimulative and repressive effectsof these proteins on the expression of HIV-1 genes has been obtained. Theirrole is often particularly prominent upon basal transcription.The majority of studies performed on human cells demonstrate that the Kuheterodimer activates transcription from the HIV-1 promoter. The importance ofthe DNA-PK catalytic subunit for the activation of transcription has beenreported in a number of studies. A hypothesis has been put forward that DNA-PKis involved in the transcription elongation stage . Let us invitro seems to suppress the basal transcription of the HIV-1 genes andis important for maintaining latency [The architectural factor HMGA1 may influence the chromatin composition uponHIV-1 transcription regulation. In this case, HMGA1 has a positive effect. Onthe other hand, the interplay between HMGA1 and TAR demonstrated  latency .Recruit