Saccharomyces cerevisiae strain harbouring the H3K56A mutation, which is known to perturb chromatin structure and nascent transcription. We identified Puf5  as essential in an H3K56A background. Depletion of Puf5 in this background leads to downregulation of Puf5 targets. We suggest that Puf5 plays a role in post-transcriptional buffering of mRNAs, and support this by transcriptional shutoff experiments in which Puf5 mRNA targets are degraded slower in H3K56A cells compared to wild-type cells. Finally, we show that post-transcriptional buffering of Puf5 targets is widespread and does not occur only in an H3K56A mutant, but also in an H3K4R background, which leads to a global increase in nascent transcription. Our data suggest that Puf5 determines the fate of its mRNA targets in a context-dependent manner acting as an mRNA surveillance hub balancing deregulated nascent transcription to maintain physiological mRNA levels.Gene expression involves regulation of chromatin structure and transcription, as well as processing of the transcribed mRNA. While there are feedback mechanisms, it is not clear whether these include crosstalk between chromatin architecture and mRNA decay. To address this, we performed a genome-wide genetic screen using a  Summary: Identification of a pathway involving the yeast RNA-binding protein Puf5 that ensures maintenance of correct mRNA levels when chromatin alterations lead to changes in nascent transcription. The life of an individual mRNA starts with its synthesis from the DNA template. This process is catalysed by RNA polymerase II and is strongly impacted on by the underlying architecture and dynamics of chromatin. Indeed, the packaging of DNA into nucleosomes affects all stages of transcription, from transcription factor binding to initiation and elongation . NucleosDegradation of mRNA is initiated by the removal of the poly(A) tail, which in yeast is mainly catalysed by the cytoplasmic Ccr4–Not complex . SubsequSaccharomyces cerevisiae, we identify puf5Δ as synthetically lethal with H3K56A, indicating that Puf5 might work as a central player in this buffering system through its targeting of downregulated nascent transcripts in an H3K56A background. Depletion of Puf5 in this background leads to the downregulation of ribosomal protein genes, suggesting that the reason for synthetic lethality is a decrease in translation. We further identify mutations in H3K4 that are also genetically linked to Puf5, indicating that the phenomenon of buffering mRNAs upon chromatin-mediated transcriptional change is widespread. Strikingly, in the case of H3K4R, nascent transcription is upregulated, suggesting a context-specific activity of Puf5. Such a post-transcriptional buffering system would constitute another regulatory layer in gene expression to ensure cellular homeostasis.In this study, we set out to address whether there is a connection between chromatin dynamics and the complex post-transcriptional network of RNA-binding proteins. Could global changes in the underlying histone modification pattern, such as during cancer development or ageing, influence the post-transcriptional life of an mRNA? The rationale for this study comes from observations in mutants affecting acetylation of histone H3K56. Acetylation at this site is important to allow access to chromatin after DNA damage, enhance histone turnover at transcriptionally active chromatin, and to promote nucleosome assembly during S phase . DespiteTable S1). These included several metabolic complexes, genes required for genome stability and members of the nucleosome assembly machinery, as well as components of the Mediator complex . Using this system, the synthetic genetic interaction of H3K56A with pop2Δ could not be reproduced  screen was performed. The H3K56A mutant was used as a query strain, and screened against the entire yeast knockout (YKO) and Decreased Abundancy by mRNA Perturbation (DAmP) collections  Fig. 1AAFig. 1A. complex , its com shuffle  . To determine whether a puf5Δ mutant has aberrant chromatin structure, chromatin immunoprecipitation (ChIP)-seq was used to assess genome-wide deposition of H3, acetylated H3  and H3K56ac. No differences in the genome-wide levels of H3, pan-acH3 and H3K56ac were observed in a puf5Δ mutant . Puf5 is localized in the cytoplasm and nucleus. To test whether cellular localization plays an important role for the potential buffering of mRNAs, we tagged Puf5 with either a nuclear localization signal (NLS) . Interestingly, whereas Puf5 localized to the cytoplasm complemented a Puf5 deletion, nuclear Puf5 only partially complemented a Puf5 deletion    or a nucdeletion D, indicaFig. S2A). This system combines transcription shut-off through a Tet-off promoter with auxin-induced degradation that is mediated by an AID tag. However, this system led to a slight overexpression of Puf5. Therefore, we pre-treated cells with low doses of Dox overnight, which decreased Puf5 transcripts, returning them to wild-type levels . More importantly, in the presence of Dox/Aux, this system recapitulated the synthetic defect between H3K56A and puf5Δ in liquid culture and on agar plates , while in their absence, the strain showed a similar expression programme to an H3K56A strain .To identify genes and pathways that rely on the potential buffering effect of Puf5 in the H3K56A background, we used an auxin-induced degradation (AID) system  to quickpuf5Δ strains. In line with previously published data, we did not observe strong differential gene expression between a wild-type and H3K56A strain , while a puf5Δ deletion showed a significant change in the gene expression programme . Comparing the individual strains after addition of Dox/Aux, it is obvious that only degradation of Puf5 led to significant changes in the transcriptome, indicating that the combined Dox/Aux treatment only affected the degron strain . Acute depletion of Puf5 in the H3K56A background led to upregulation of 238 genes, while 410 genes were significantly downregulated . Upregulated genes were enriched for functions in chromatin organization-, cell wall- and cell cycle-related terms  direct Puf5 targets as identified by eCLIP . These data indicated that the depletion leads to an arrest during the cell cycle. However, cell cycle analysis by fluorescence-activated cell sorting (FACS) indicated that cells did not arrest in any specific cell cycle stage .As additional controls, we performed RNA-seq on wild-type, H3K56A and ed terms A,B, whiced terms C. To ideby eCLIP  , suggesting a partial overlap of substrates with Puf5. Importantly, Puf3 and Puf4 only share a fraction of these transcripts with Puf5  deregulated on the level of nascent transcription and (2) are indeed buffered, that is degraded more slowly in the H3K56A mutant in order to maintain a comparable level of these transcripts to that seen in wild type. To address the first point, we made use of a previously published large dataset comparing nascent transcription in a variety of mutants unable to acetylate H3K56  and inten strain A.Fig. 4Fig. S4A). Additionally, we observed only a mild genetic interaction between H3K56A and xrn1Δ . An overexpression of XRN1 in H3K56A/puf5Δ background only led to a minor rescue of the lethality  suggesting that Xrn1 plays some role in the observed H3K56A/puf5Δ phenotype. However, as in the case for Ccr4, the puf5 lethality in the H3K56A background is, at least to a substantial part, independent of Xrn1. Finally, to directly measure the difference in degradation rates between a wild-type and H3K56A strain, we performed transcriptional shut-off experiments . Using this approach, we identified mRNAs that showed a significantly altered degradation pattern, in particular mRNAs that were stabilized in an H3K56A background . In line with the idea that Puf5 might be able to buffer expression of its target genes in an H3K56A background, differentially degraded mRNAs are strongly enriched for transcripts encoding ribosomal proteins . Of these mutations, H3K4R showed a strong synthetic phenotype in combination with puf5Δ . To test this hypothesis more directly, we performed transcriptional shutoff experiments in an H3K4R background. As was the case for the H3K56A background, the genome-wide impact of thiolutin was comparable between WT and H3K4R . In contrast to what was seen with H3K56A, we identified mRNAs that were degraded faster in the H3K4R background compared to in wild type . Again, gene ontology (GO) enrichment revealed that the transcripts with a significantly different degradation rate were enriched for proteins involved in translation  or transcription    . An addinscripts . Howeverpuf5Δ are of H3K4 and H3K56. Interestingly, both residues and their modification have recently been implicated in playing a major role in buffering gene expression during DNA replication, in which the transcript number of a given gene remains the same although the underlying DNA sequence is duplicated  was from US Biological Life Sciences (D9543-01); 5-FOA was bought from Cayman Chemical (17318); Ampure XP beads were from Beckman Coulter (A63881); Protein-G coupled dynabeads from Thermo Fisher Scientific (10009D) and Thiolutin from Abcam (ab143556). Secondary antibodies against rabbit (7074S) and mouse (7076S) IgG coupled to horseradish peroxidase (HRP) were purchased from Cell Signaling. Antibodies against H3K56ac were from Active Motif (39281), G6PDH (A9521) and Myc (MABE282) from Sigma, iAID-tag (M214-3) from MBL International and β-actin (GTX109639) from GeneTex. Antibody dilutions were as suggested by the manufacturer (Table S12).Genotypes of strains and yeast plasmids used in this work are listed in 595)=1 and 5-fold serially diluted.If not stated otherwise, all strains used were derivatives of W303. Integrations and deletions were performed using one-step PCR-based methods . Yeast w595 of ∼0.15 and grown at 30°C until an OD595 of 0.6–0.8. From each cell culture, 10 ml were collected for RNA extraction and 1 ml of cell culture was diluted in 4 ml of YPD medium containing 0.25 μg/ml of Dox and incubated overnight at 30°C. The following day, the overnight cultures were diluted in 40 ml of YPD medium containing 0.25 μg/ml of Dox to an OD595 of 0.2 and incubated at 30°C to an OD595 of 0.6–0.8. From each cell culture, 10 ml were then collected for RNA extraction and the remaining cell culture was diluted in 40 ml of YPD medium containing 40 μg/ml of Dox and 1 mM Aux to an OD595 of ∼0.2. The cell cultures were then further incubated at 30°C and additional samples were taken at 90 and 240 min.Single yeast colonies were picked and inoculated overnight in 5 ml of YPD medium at 30°C. The next day, cultures were diluted in 20 ml of YPD medium to an OD600nm 0.5–0.6). Cultures were then split and either DMSO or 3 μg/ml of thiolutin  were added. Cells were sampled at time-points of 0, 15, 30 and 60 min. Total RNA was extracted using the hot phenol method (t method was used to measure relative expression. 18S rRNA and time-point 0 were used as reference. RNA-seq libraries were prepared as described below.Cells were grown in 100 ml of YPD (30°C and 200 rpm rotation) to mid-log phase (ODl method . Reverse30 oligonucleotide containing a 8 bp barcode for later identification and a unique molecular identifier (UMI). Reverse transcription used the template switching. After the reverse transcription step, all reverse transcribed samples were pooled into a single 2 ml Eppendorf tube and DNA was purified with Ampure XP  beads at 1:1 (v/v). Purified DNA was eluted in 17 μl H2O and treated with exonuclease . Subsequently, cDNA was amplified and the final PCR product was purified with Ampure XP beads at a volume to volume ratio of 0.8. Finally, 0.8 ng cDNA was tagmented in five replicates. The quality of library was assessed on an Agilent Tapestation and sequenced on an Illumina HiSeq4000 in a 2×75 bp mode to ∼1 million fragments per sample. The sequenced reads were processed using zUMIs (version 2.0.6)  enrichment analysis. Differential gene expression analysis for all conditions can be found in Tables S5–S11. GO term enrichment was performed using Metascape (Bulk 3′ RNA-seq libraries were prepared following the SCRB-seq protocol  using fin 2.0.6)  preprintn 2.0.6) , samtooln 2.0.6)  and featn 2.0.6)  from Rsun 2.0.6)  with then 2.0.6)  approachetascape  and inteetascape .puf5D was carried out using edgeR (Chromatin immunoprecipitation and library production was performed as previously described . The fasng edgeR . The norng edgeR  with the7 cells was spun down (1000 g for 5 min), resuspended in 0.5 ml water, sonicated for 30 s in an ultrasonic bath and ethanol was added to a 70% final volume. The samples were agitated at an Eppendorf Thermomixer Compact at top speed for 10 min, then left overnight at 4°C. Cells were flash spun, resuspended in 0.5 ml water, flash spun again (1000 g for 5 min), and resuspended in 0.5 ml RNase A . The samples were shaken at 37°C for 2 h at an Eppendorf Thermomixer Compact at top speed and then pelleted (1000 g for 5 min). Pellets were washed with 0.5 ml water and resuspended in freshly prepared pepsin solution (5 mg/ml in 50 mM HCl). The samples were shaken at 37°C for 20 min at an Eppendorf Thermomixer Compact at top speed, pelleted (1000 g for 5 min), washed with 0.5 ml water, resuspended in 0.2 ml 50 mM Tris-HCl, pH 7.5 and sonicated in an ultrasonic cleaning bath for 30 s. The suspensions were passed through a 35-μm nylon cell-strainer , propidium iodide (PI) was added to 50 μg/ml final from a 500 μg/ml stock solution in water. The samples were vortexed and placed in the dark. Aliquots (25 µl) of the samples with PI were diluted with 950 µl 50 mM Tris-HCl, pH 7.5 and analysed immediately at a BD FACS CantoII Analyzer at the low speed flow setting. PI detection was carried out with the 488 nm laser and the 670 long pass filter. A total of 10,000 or 50,000 events were analysed.Cell culture containing 2×10


In eukaryotes, ribosome biogenesis is driven by the synthesis of the ribosomal RNA (rRNA) by RNA polymerase I (Pol-I) and is tightly linked to cell growth and proliferation. The 3D-structure of the rDNA promoter plays an important, yet not fully understood role in regulating rRNA synthesis. We hypothesized that DNA intercalators/groove binders could affect this structure and disrupt rRNA transcription. To test this hypothesis, we investigated the effect of a number of compounds on Pol-I transcription in vitro and in cells. We find that intercalators/groove binders are potent inhibitors of Pol-I specific transcription both in vitro and in cells, regardless of their specificity and the strength of its interaction with DNA. Importantly, the synthetic ability of Pol-I is unaffected, suggesting that these compounds are not targeting post-initiating events. Notably, the tested compounds have limited effect on transcription by Pol-II and III, demonstrating the hypersensitivity of Pol-I transcription. We propose that stability of pre-initiation complex and initiation are affected as result of altered 3D architecture of the rDNA promoter, which is well in line with the recently reported importance of biophysical rDNA promoter properties on initiation complex formation in the yeast system. In eukaryotes, ribosome biogenesis (RiBi) is a fundamental cellular process in which all three nuclear polymerases are involved. Pol-I transcribes rRNA as a 47S-rRNA precursor which is processed into mature 28S, 18S and 5.8S rRNAs. Pol-II transcribes all mRNAs including those that encode ribosomal proteins, and Pol-III transcribes the 5S rRNA, tRNAs and other small RNAs. Aberrations of RiBi that allow an increased level of ribosome production and consequently an increased rate of protein synthesis which supports cell growth are commonly found in aggressive cancers .Many DNA binding proteins and drugs bind to DNA through interactions with the minor and major grooves, and compounds that can bind into the grooves or compete out pre-bound proteins are capable of inhibiting a variety of cellular processes . AlthougIn recent years it has become evident that selective inhibition of Pol-I transcription has a potential to become a novel and efficient therapeutic approach in the fight against cancer. Pioneering work by the Hannan group has convincingly demonstrated a high sensitivity of cancer cells to inhibition of Pol-I transcription . HoweverThe Pol-I promoter is predominantly composed of guanine and cytosine residues and we can expect that GC-intercalators may efficiently inhibit transcription (similar to ellipticines). However, the promoter also contains four stretches of five AT nucleotides  DNA binding affinity can inhibit Pol-I transcription both in vitro and in cells without having a significant effect on general Pol-II/III transcription. Notably, effect on Pol-I transcription can be detected relatively earlier (30 min after treatment), whereas longer incubation leads to downregulation of Pol-II/III transcription. Importantly, we found that these compounds act predominantly by impairing recruitment of Pol-I to the rRNA promoter without affecting the synthetic ability of Pol-I. We also found that intercalating agent, and topoisomerase I (Top 1) inhibitor camptothecin (CPT) inhibits Pol-I transcription in vitro and its inhibitory activity is not Top 1 mediated.20 and stored at −20 °C. CX-5461 was a kind gift from Ross Hannan (ANU).Camptothecin (CPT), methyl green (MG), crystal violet (CV), propidium iodide (PI), ethidium bromide (EtBr or EB), proflavine (Pro), 4′,6-diamidino-2-phenylindole (DAPI), thionine (Thio), acridine orange (AO) and bisBenzamide (Hoechst 33258) were of analytical grade, purchased from Sigma Aldrich  and reconstituted as 10 mg/mL stock solutions in ddH+/+ and −/−cells were maintained in recommended McCoy’s 5A media  in a 37 °C, 5% CO2 incubator. The culture media was supplemented with 10% FBS (PAA) and 10,000 U/mL penicillin and 10,000 µg/mL streptomycin . Drug treatments were administered as indicated in figure legends at the concentrations stated.HCT116 p53All antibodies are described in All primers and probes are described in Non-specific Pol I driven transcription assays were performed as described  and samp32P-5′-end labelled oligonucleotide containing a sequence identical to the region between −20 and +40 of the template strand in the human rRNA gene promoter. The amount of RNA produced by in vitro transcription reactions was quantified with the aid of a phosphorImager, FLA-7000 (Fuji) and Aida software . The standard deviations were calculated from three independent experiments. Specific Pol I driven transcription reactions were performed as described ,15 usingAnalysis of the specific Pol-II driven transcription was performed using the approach described in  with modThe pcfTAF110 plasmid , a derivAmplified DNA fragment was purified using the PureLink™ PCR Purification Kit (Invitrogen) and immobilized onto Dynabeads M280 magnetic beads (Invitrogen). Bead immobilization was performed as recommended by the manufacturer (10 µg of dsDNA per 100 µL beads) and beads stored at 4 °C in the binding buffer. The binding efficacy of about 90% resulted in a DNA concentration of 90 ng/µL. Prior to transcription reaction setup, beads were washed three times in the transcription buffer and then transferred into new tubes. Typically, 5 µL beads (450 ng DNA) was used per transcription reaction.2, 0.2 mM EDTA, 0.5 mM DTT, 20% glycerol) and 40U RNasin (Fermentas). Reactions were supplemented with various compounds or vehicle, mixed and incubated at 30 °C for 30 min. The transcription reaction was then initiated by adding 10 mM NTP mix  and maintained at 30 °C for 1 h. The transcription was terminated by addition 150 μL stop solution  and immobilized template was removed using a magnetic rack. A total 700 μL of the RLT buffer from the Qiagen RNeasy Micro kit was added to each sample. RNA was purified as recommended by the manufacturer, including on-column DNase treatment but without the addition of carrier RNA. The RNA was eluted using 15 μL water and stored at −80 °C. A total 50 ng of free DNA template was subjected to the same purification procedure and used as a control for DNA removal efficiency, ‘DNA control’.The transcription reaction contains 5 μL HeLa nuclear extract, 450 ng of immobilized DNA template in the transcription buffer  and then were treated with compounds or vehicle (as indicated) for 30 min, followed by the addition of 5EU  and further incubation for 1 h. The following concentrations were used: Actinomycin D (ActD): 3 µM; ethidium bromide (EB): 40 µM; proflavine (Pro): 2 µM; CX5461: 5 µM.Cells were grown until 50–60% confluent on sterilized glass slides  supplemented with Cy5 Azide (Sigma Aldrich) according to manufacturer’s instruction. Nuclei were stained with Hoechst 33342 and images were acquired with a Leica TCS SP5 confocal microscope equipped with an x63 oil-filled objective. Leica Application Suite X (LAS X) software was used for quantification of 5EU incorporation. Box-and-whisker plots of quantification of 5EU incorporation were obtained with Excel. They show the median, the 25% and 75% quantiles, as well as outliers.Cells were fixed with 3.7% paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. Nascent RNA was fluorescently labelled using Click-ITCells were grown until 70% confluent and then treated with compounds for 30 min. The following concentrations were used: ActD: AD1–3 nM; AD2–3 µM; Camptothecin (CPT): 8 µM; ethidium bromide (EB): 40 µM; crystal violet (CV): 20 µM; Pro: 2 µM; CX5461: 5 µM. RNA was purified using the RNeasy purification kit (Qiagen) following the manufacturer’s instructions and the RNA concentration was determined spectroscopically. A total 1 µg of RNA was converted to cDNA using the High Capacity RNA-to-cDNA reverse transcriptase kit . Then 47/45S pre-rRNA levels were analyzed on the LighCycler 480 thermocycler (Roche) using primers and conditions described previously . Pol-II and Pol-III transcripts were analyzed as described previously . All sig6 cells, antibodies listed in ChIP assays were carried out as described previously . In brie50 for each drug using an in vitro Pol-I specific transcription assay. This assay primarily measures the efficiency of the initial stages of transcription which include preinitiation complex (PIC) formation, first phosphodiester bond synthesis and promoter clearance . We found no apparent correlation between the specificity of the DNA binding of the compound and its inhibitory activity  but obse50 concentrations for EtBr, Pro, CPT, DAPI and CV in cells by measuring the level of Pol-I transcription at various compound concentrations , even though it has been reported that EtBr is actively excluded from viable cells [Next, we determined ICtrations . For thi compare ; Table 2le cells .50 concentrations  exhibit similar catalytic properties and share the same overall structure and mechanism of transcription ,32,33,34× 5 IC50  were use× 5 IC50 . To ensu50. As expected, 400 μM α-amanitin completely repressed transcription by Pol-II  at concentrations sufficient to inhibit about 90% of Pol-I transcription as shown in Figure 5A. As a control we used high concentration of ActD (3µM) that inhibits activity of all nucleolar polymerases [Our results suggest that in vitro Pol-I promoter driven, but not non-specific transcription is hypersensitive to the intercalators and grove-binders. We also found that these compounds inhibit Pol-I transcription in cells . Althougymerases . Cells wymerases . As expected, a high concentration of ActD (3 μM) almost completely abolished 5EU incorporation and hence RNA synthesis A,B. If tImportantly, treatment with proflavine and ethidium bromide not only affected the level of nascent RNA synthesis, but also affected the distribution of nascent RNAs. As it is well known, and as shown in It should be noted that we used relatively short treatment times to pin-point earlier effects, and if treatment time is increased to several hours the inhibitory effect of the intercalators is greatly increased .−/− and p53+/+ cells as described previously [As a complimentary approach we also measured the effect of intercalators and grove binders on transcription by Pol-II and Pol-III by determining relative expression levels of 18 house-keeping genes  and of three Pol-III dependent genes in intercalator treated and untreated HCT116 p53eviously .Compound concentrations in this experiment were sufficient to inhibit about 90% of Pol-I transcription A. We fouIn cells expressing wt p53  all compTogether, these results indicate that intercalators and groove binders affect Pol-I transcription in cells but have significantly less effect on the transcription by Pol-II and Pol-III, thereby confirming the high sensitivity of Pol-I to compounds that may affect the 3D-structure of DNA.50 concentration. To exclude any potential effects of p53 activation by these drugs (activation of p53 leads to dissociation of SL1 from the rRNA promoter [−/− cell line)  assays using a selection of intercalators from each of the main groups: , crystal violet (CV) and ethidium bromide (EtBr) respectively) and analyzed the occupancy of basal Pol-I transcription factors  at the rDNA before and after treatment at ICpromoter ) we usedll line) . We found that UBF occupancy was affected by proflavine but not by other drugs. Surprisingly, none of the drugs significantly affected SL1 occupancy at the rRNA promoter in contrast to two other intercalating agents known to affect initiation of transcription (9HE ). InsteaCamptothecin (CPT) is a compound that inhibits rRNA synthesis in humans by inhibiting Topoisomerase 1 (Top 1) which plays an important role in Pol-I elongation ,42. As wIntriguingly, CPT represses Pol-I specific transcription under all conditions B, strongThe known selective inhibitors of Pol-I transcription are DNA intercalators and we hypothesize that changes in the structure of DNA induced by intercalators (or groove binders) are important for Pol-I inhibiting activity. Our hypothesis is based on work by Denissov and colleagues who investigated the topology of active rDNA repeats , our worIn the first study a new core-helix model has been suggested which implies a very important role of 3D-structure of the rDNA transcription unit for the productive synthesis of rRNA. DNA intercalators and groove binders are known to affect the 3D-structure of DNA and we hypothesize that Pol-I transcription would exhibit high sensitivity to these changes induced by such agents, compared to Pol-II and Pol-III (the results that we have observed for ellipticines). By performing in vitro and in cell-based transcription assays, we demonstrate that DNA intercalators and groove binders inhibit Pol-I transcription regardless of their sequence specificity. The tested compounds do not influence the catalytic properties of the enzyme  and haveSupported by in vivo occupancy studies , our resThe promoter consists of three key elements: upstream binding site (UCE), core region and linker region ,57. DuriFurthermore, structural analysis of the SL1-homologous ‘Core Factor’ (CF) and an initially transcribing Pol I complex in the yeast system ,48,49) uThus, we propose that the three-dimensional architecture of the rDNA promoter within the pre-initiation complex plays an important and previously not fully recognized role in the sensitivity of rDNA transcription to various chemical agents and, in particular, to DNA intercalators and groove binders. This can have certain implications for our under-standing of the sensitivity of cells to environmental contamination.One of the unexpected results of this study is the finding of Top 1 independent inhib-itory effect of Camptothecin (CPT). Our data strongly suggests that CPT repress Pol-I tran-scription by not only inhibiting Top 1 and affecting Pol-I elongation, but also by interfering with the initial stages of transcription via a Top 1 independent mechanism, and this was not previously recognized.


Gene expression is an essential process for cellular growth, proliferation, and differentiation. The transcription of protein-coding genes and non-coding loci depends on RNA polymerases. Interestingly, numerous loci encode long non-coding (lnc)RNA transcripts that are transcribed by RNA polymerase II (RNAPII) and fine-tune the RNA metabolism. The nucleolus is a prime example of how different lncRNA species concomitantly regulate gene expression by facilitating the production and processing of ribosomal (r)RNA for ribosome biogenesis. Here, we summarise the current findings on how RNAPII influences nucleolar structure and function. We describe how RNAPII-dependent lncRNA can both promote nucleolar integrity and inhibit ribosomal (r)RNA synthesis by modulating the availability of rRNA synthesis factors in trans. Surprisingly, some lncRNA transcripts can directly originate from nucleolar loci and function in cis. The nucleolar intergenic spacer (IGS), for example, encodes nucleolar transcripts that counteract spurious rRNA synthesis in unperturbed cells. In response to DNA damage, RNAPII-dependent lncRNA originates directly at broken ribosomal (r)DNA loci and is processed into small ncRNA, possibly to modulate DNA repair. Thus, lncRNA-mediated regulation of nucleolar biology occurs by several modes of action and is more direct than anticipated, pointing to an intimate crosstalk of RNA metabolic events. C. elegans and D. melanogaster, however, also contain approximately 19,000 and 140,000 protein-coding genes, respectively [C. elegans [Cellular growth and development rely on the orderly expression of thousands of genes in a spatiotemporal manner. The human genome contains approximately 19,000 protein-coding genes that are differentially expressed in individual cells and developing tissues [ectively ,3. Thus, elegans ,6. Thus,Here, we review the surprisingly intimate links between RNAPII and ribosome biogenesis in the nucleolus. Ribosome biogenesis is a prime example of cellular homeostasis and requires RNA transcripts from all three RNA polymerases, including RNAPII-dependent nucleolar lncRNA. Mounting evidence suggests that RNAPII-dependent lncRNA are intertwined with the nucleolus and critical for the fine-tuning of rRNA synthesis. The expression of such transcripts is often induced by cellular stress and deregulated in cancer. We discuss how lncRNA modulate the structure and function of the nucleolus both in trans and in cis, thereby mediating a surprisingly direct crosstalk of RNAPII with the nucleolus that may become a future therapeutic target.Indeed, the discovery of regulatory non-coding RNA transcripts was a major advance in molecular biology . The hum3–104 47S pre-rRNA transcripts per minute and fuels a steady-state level of 106–107 ribosomes [The nucleolus is a membrane-less subcellular compartment and is best known for producing ribosomes ,15. Riboibosomes ,31.1-phase [The linearity in the steps of ribosome biogenesis is reflected by the nucleolar tripartite structure. Human interphase nucleoli are multi-layered condensates that contain three sub-compartments: the fibrillar center (FC), the dense fibrillar component (DFC), and the granular component (GC) ,33. The 1-phase ,45,46,47Intriguingly, however, in 1974, the Roeder lab noticed that the inhibition of RNAPII with the fungal poison α-Amanitin disrupts nucleoli, although the activity of RNAPI is not affected by the drug . This suThere is increasing evidence that trans-acting lncRNA facilitate a regulatory crosstalk of RNAPII transcription with the nucleolus. The protein-coding gene TBRG4 encodes the intronic, snoRNA-containing lncRNA transcript SLERT, which functions as a critical regulator of ribosome biogenesis . SLERT cSimilarly, the lncRNA LETN is essential for nucleolar integrity and sustained proliferation of cancer cells  Figure c. LETN iVarious other trans-acting RNAPII-dependent lncRNA function as structural transcripts to support nucleolar integrity. The McStay lab determined a 207 kb stretch of the DNA sequence immediately proximal and 379 kb distal to the rDNA array, and assembled consensus proximal junction (PJ) and distal junction (DJ) contigs from sequenced bacterial artificial chromosomes . Both PJAlu-repeat transcripts are another class of lncRNA that modulate the nucleolus. Alu elements are frequent repetitive elements in the genome of mammals and can be transcribed by both RNAPII and RNAPIII ,59. SomeHowever, deregulated activity of RNAPII and an aberrant RNA metabolism can be pathogenic . SeveralC9orf72 gene [C9orf72 locus also forms G4 structures, which causes the stabilisation of the lncRNA in nucleoplasmic foci. Intriguingly, the authors could show that one of the RNA-binding proteins (RBPs) that associates with the abortive transcript is NCL. The mislocalisation of NCL causes nucleolar stress, impairs ribosome biogenesis, and triggers the toxic accumulation of untranslated mRNA in patients with neurodegenerative diseases.Some neurogenerative diseases like dementia and amyotrophic lateral sclerosis are further characterised by an abnormal expansion of a repeated GGGGCC hexamer sequence in the non-coding region of the f72 gene ,66. The f72 gene . As a ref72 gene . MoreoveCircular RNA are RNAPII-dependent lncRNA species that form upon back-splicing of pre-mRNA and regulate various pathophysiological processes in mammals, for instance, by sponging of transcripts or scaffolding of proteins ,70. One In summary, RNAPII-dependent lncRNA are a major determinant of the nucleolar structure and function. Such transcripts can either support the formation of hyperactive nucleoli in fast proliferating cells like cancer, or be pathologic by diminishing nucleolar integrity, as observed in many neurodegenerative diseases. The fine-tuning of nucleolar functions often occurs via scaffolding and sponging function, but it is currently unclear how the interactions with nucleolar proteins are regulated and how widespread the impact on gene expression is.The nucleolus is multi-functional . BesidesIndeed, nucleolar sequestration of RBPs has been linked to the accumulation of nucleolar lncRNA. Such transcripts are produced by RNAPI upon various kinds of stress, including heat shock. Surprisingly, however, they are not encoded in the rDNA locus, but arise from specific regions in the IGS that are transcribed in a stress-specific manner. IGS-derived lncRNA sequesters a subset of nucleoplasmic proteins, such as the E3-ligase HDM2, in the nucleolus, which triggers a cellular stress response that involves the stabilisation of downstream effectors like p53 ,78. FurtS. cerevisiae) and DNA at CG-rich regions at the rDNA promoter  [The stress-induced production of IGS transcripts is an intriguing addition to the well-described rDNA silencing function of promoter-associated RNA (pRNA) in unperturbed cells, where only about 50% of rDNA genes comprise open chromatin and are transcribed by RNAPI . pRNA ar humans) ,86,87,88S. cerevisiae [Interestingly, the silencing of yeast 35S rDNA loci prevents both spurious RNAPII activity and unwanted recombination events at nucleolar loci ,90. RNAPrevisiae ,92 and trevisiae ,94. Likerevisiae ,96.Could the human nucleolus also give raise to lncRNA that are produced by RNAPII directly from nucleolar loci? And if so, what are their functions? Indeed, the loss with DNA methylation at the rDNA locus enhances RNAPII transcription in human cells . The co-Moreover, the mammalian IGS also encodes RNAPII-dependent lncRNA antisense transcripts. The promoter and pre-rRNA antisense transcripts (PAPAS) are well characterised. PAPAS are a heterogenous population of 12–16 kb lncRNA that originate from random antisense promoters in the 5′ region of the IGS  Figure a. PAPAS More recently, a direct function for nucleolar RNAPII transcription in promoting rRNA synthesis and nucleolar integrity has been reported . The autN. crassa [N. crassa hypersensitive to DNA damage. These findings point towards an RNA-dependent crosstalk of the nucleolus with the DNA damage response. However, future studies are required to further characterise the relevance of nucleolus-derived transcripts for gene expression.The loss of CpG methylation upon co-deletion of DNMT1 and DNMT3a strongly increases the recombination rate of human rDNA loci, but does not induce DNA damage , suggest. crassa . The treIn 1896, Pianese identified enlarged nucleoli as a feature of cancer cells . RibosomNevertheless, we have just begun to understand the relevance of RNAPII for modulating the expression of rDNA genes and the integrity of the nucleolus. What are the regulatory principles that control the activity of nucleolar RNAPII and the fate of nucleolar RNAPII-dependent lncRNA? What enzymes regulate RNAPII activity in the nucleolus? How does RNAPII find a nucleolar promoter? Is the regulatory function of such transcripts due to a quantitative change in level or are there also qualitative differences? Recent findings in the DNA repair field suggest that RNAPII can recognise DSBs irrespective of the genomic context by the assembly of a de novo RNAPII promoter that contains the TATA-binding protein TBP as well as the transcriptional kinase CDK9 ,121. Int


Urinary bladder cancer is one of the ten major cancers worldwide, with higher incidences in males, in smokers, and in highly industrialized countries. New therapies beyond cytotoxic chemotherapy are urgently needed to improve treatment of these tumors. A better understanding of the mechanisms underlying their development may help in this regard. Recently, it was discovered that a group of proteins regulating the state of chromatin and thus gene expression is exceptionally and frequently affected by gene mutations in bladder cancers. Altered function of these mutated chromatin regulators must therefore be fundamental in their development, but how and why is poorly understood. Here we review the current knowledge on changes in chromatin regulators and discuss their possible consequences for bladder cancer development and options for new therapies.Urothelial carcinoma (UC) is the most frequent histological type of cancer in the urinary bladder. Genomic changes in UC activate MAPK and PI3K/AKT signal transduction pathways, which increase cell proliferation and survival, interfere with cell cycle and checkpoint control, and prevent senescence. A more recently discovered additional category of genetic changes in UC affects chromatin regulators, including histone-modifying enzymes , transcription cofactors , and components of the chromatin remodeling complex SWI/SNF . It is not yet well understood how these changes contribute to the development and progression of UC. Therefore, we review here the emerging knowledge on genomic and gene expression alterations of chromatin regulators and their consequences for cell differentiation, cellular plasticity, and clonal expansion during UC pathogenesis. Our analysis identifies additional relevant chromatin regulators and suggests a model for urothelial carcinogenesis as a basis for further mechanistic studies and targeted therapy development. Urinary bladder cancer is one of the ten major cancers worldwide with a higher incidence in males, in smokers, and in highly industrialized countries . The preFGFR3, by oncogenic RAS mutations, by alternate PIK3CA oncogenic mutations or PTEN loss, or by other mutations in MAPK and PI3K/AKT pathway components. (2) Cell cycle and checkpoint control are disturbed by TP53 inactivation as well as RB1 mutations and deletions, which are both much more frequent in invasive than in papillary UC, or by homozygous deletion, promoter hypermethylation, and mutations in CDKN2A, encoding regulators of both TP53 and RB1, which are observed across all stages of the disease. Additional relevant changes in cell cycle control include inactivating mutations in CDKN1A (encoding p21CIP1) and ATM as well as amplification of CCND1 encoding Cyclin D1. (3) Loss of function of TP53 and RB1 is expected to impede the establishment of senescence in response to replication stress and genomic instability. In addition, telomerase is activated in almost all UC, independent of grade and stage, most frequently by activating mutations in the hTERT promoter.Genomic changes in UC alter three regulatory systems in a rather consistent manner . . The baIntermediate cells, in keeping with their designation, have an intermediate degree of characteristics and express KRT7, little if any KRT5, and no KRT14. At least a subset of intermediate cells can proliferate during acute injury and contribute to regeneration of the urothelial layer, whereas the response to chronic injury depends more on basal cells ,18,19.The luminal superficial umbrella cells are larger, multinucleated, and terminally differentiated. They express uroplakins, specialized proteins required for the barrier function of the urothelium, and specifically KRT20. To accommodate varying filling volumes in the bladder, the thickness of the epithelium can adapt by intermediate cells shifting against each other, and umbrella cells can adjust their apical surface area [Urothelial regeneration following injury is stimulated by several growth factors from urothelial cells and mesenchymal cells in the underlying submucosal connective tissue. Injured urothelium secretes EGF-like growth factors  to stimulate proliferation. Signaling through FGF receptors activated by FGF2 contributes to this process. Reciprocal epithelial-mesenchymal signaling, with basal cells secreting SHH to stimulate WNT secretion in mesenchymal cells, is likewise activated during rapid regeneration ,22.Differentiation in the urothelium is regulated by a network of transcription factors, which is still incompletely understood ,24. ThesTP63 gene encodes various isoforms of a p53 family transcription factor. The most prominent isoforms are the full length variant TAp63 and the N-terminal truncated ΔNp63 isoform with opposing functions in the regulation of differentiation and proliferation that carry over into tumors with a basal cell phenotype. The ΔNp63 variant is the most strongly expressed isoform in the basal layer of stratified epithelia. It maintains the proliferation potential of progenitor cells and controls epithelial morphogenesis through recruitment of epigenetic regulators, including repressive complexes containing histone deacetylases (HDAC) [The s (HDAC) ,25. ΔNp6KDM6B) and BRG1/BRM (SMARCA4/A2) and the histone methyltransferase MLL2 (KMT2D) [The transcription factor ZNF750 controls epithelial tissue homeostasis by repression of progenitor genes and upregulation of differentiation genes through interaction with the transcription factor KLF4 and chromatin regulators like KDM1A and HDAC1 . ZNF750  (KMT2D) . These f (KMT2D) .Pparg mutations prevented maturation of superficial umbrella cells, and Pparg inactivation in basal cells resulted in squamous rather than urothelial differentiation. Further, Pparg mutant mice presented with persistent inflammation in the urinary tract and activated NFκB signaling. Accordingly, Pparg re-expression prevents squamous differentiation [The nuclear receptor PPARγ has been shown to promote specifically urothelial over squamous differentiation. In mice, ntiation . Additiontiation ,31,32,33To investigate the transcription factors involved in human urothelial differentiation, a model system using cultures of normal human urothelial (NHU) cells has been employed . These cInduction of urothelial differentiation in this model system is achieved by blocking EGFR signaling and activating PPARγ. This leads to the expression of intermediate transcription factors specifying the differentiated urothelial cell phenotype, including forkhead box A1 (FOXA1), interferon regulatory factor 1 (IRF1), GATA-binding protein 3 (GATA3), and E74-like ETS transcription factor 3 (ELF3) . ConcomiReflecting their increased plasticity, a different protocol applying high dose calcium only results in the stratification and squamous differentiation of primary urothelial cells. This state is characterized by expression of KRT5/6, KRT14, and S100A9 and further epidermal differentiation markers like Loricrin, Filaggrin, and Involucrin . Of noteCharacteristics of basal and luminal differentiation states are reflected in the recently defined molecular subtypes of urothelial carcinoma. The recent consensus classifies invasive urothelial carcinomas into six subclasses, mainly on the basis of gene expression patterns . The subCDKN2A deletions. In contrast, mutations in TP53 (and RB1) are relatively rare in this subtype. The frequency of mutations in the KDM6A histone demethylase gene is likewise highest in this subtype. Importantly, this subtype resembles the predominant molecular subtype of non-invasive papillary urothelial tumors [Luminal subtypes are characterized by the expression of markers of urothelial differentiation, including uroplakins and KRT20, which reflect the activity of the transcription factors FOXA1, GATA3, and PPARγ. This applies in particular to the luminal papillary subtype characterized by oncogenic mutations activating the growth factor receptor FGFR3 and l tumors . Luminall tumors ,42).TP53 mutations and ERBB2 amplification are prevalent in this subtype, but also mutations and gene fusions activating the lineage-specific oncogenes PPARG and RXRA (encoding the standard PPARγ heterodimerization partner) [The second luminal subtype  contains the largest number of genomic changes and displays the highest cell cycle activity of all subtypes. partner) .ELF3 gene, which encodes a transcription factor acting downstream of PPARγ in urothelial differentiation [A third, rarer luminal subtype  contains more stromal cells, especially fibroblasts. Mutations in the ntiation , are enrntiation ,39.Stromal-rich tumors contain an even higher fraction of various stromal cell types, but additionally of immune cells. Urothelial differentiation markers are less prominently expressed.TP53 and RB1 mutations are frequent. BASQ tumors often contain many immune cells, including cytotoxic T-cells.The basal-squamous subtype (BASQ) is characterized by the expression of differentiation markers of basal urothelial cells (see above) and a variable expression of markers of squamous differentiation. Regions with histological squamous differentiation are detectable in up to 40% of these tumors. Gene expression patterns indicate active EGFR and STAT3 signaling. TP53 and RB1 are concomitantly inactivated. Neuroendocrine-like cancers have the worst prognosis of all UC subtypes, followed by BASQ tumors [Neuroendocrine-like cancers constitute the rarest subtype. Most are morphologically identifiable as ‘small cell carcinomas’, while others can only be assigned to this subtype by their expression patterns. Typical of neuroendocrine-like cancers, Q tumors .Squamous cell carcinoma (SCC) of the bladder is regarded as a distinct histologic entity from urothelial carcinoma. These rarer cancers usually arise following chronic inflammation of the bladder, caused, e.g., by Schistosoma infection or catherization. Genomic alterations in SCC are however similar to those in BASQ UC, suggesting that they arise by a similar mechanism. Presumably, aberrant urothelial regeneration during chronic inflammation results in squamous metaplasia that can progress to SCC . The plaRegulation of the chromatin structure is required for transcription regulation, genome replication, mitosis, DNA repair, formation of constitutive heterochromatin, and other nuclear processes. Cellular plasticity, lineage choices, and cell differentiation are controlled by dynamic epigenetic mechanisms. At the core of epigenetic regulation, establishment, maintenance, and changes of cell differentiation states are associated with chromatin states, delineating the basic transcription patterns of each cell type. These long-term stable chromatin configurations are then dynamically adapted to various functional requirements, including cell replication, specific cell function, metabolic demands and various stresses  and removed by DNA demethylases (the ‘erasers’) and additional mechanisms, such as ‘passive’ dilution by replication without remethylation (reviewed in ). It is Histone modifications mediate both stable and dynamic chromatin states. For instance, heterochromatin, active and inactive enhancers, active and repressed genes, promoters, and gene bodies are each distinguished by patterns of specific histone modifications, including lysine and arginine methylation and lysine acetylation. Phosphorylation of histones is particularly relevant for DNA repair and mitosis. Overall, more than 200 different histone modifications are known (listed in ), and thEP300) and CBP (gene: CREBBP). Notably, like these two proteins, many HATs have paralogs with overlapping but not identical functions, like the pairs GCN5/PCAF (KAT2A/KAT2B) and MYST3/MYST4 . In its function as a co-activator, each HAT interacts with a set of cell-type-specific or ubiquitous transcription factors to establish and maintain open chromatin structures at enhancers  and promoters. Other HATs function mostly in the deposition of newly formed nucleosomes following DNA replication or in DNA repair as components of multiprotein complexes. Histone acetylation is removed by hydrolysis catalyzed by histone deacetylases (HDACs). Among the 18 HDACs in human cells, subclass I enzymes  catalyze the bulk of histone deacetylation. However, HDACs have non-histone protein substrates as well, and some are located predominantly in the cytoplasm or shuttle between cytoplasm and nucleus . Acetylated histones are specifically recognized by a large number of nuclear proteins containing PHD domains or bromodomains  and histone deacetylases (HDACs). Many HATs are transcriptional co-activators, like p300 (gene iewed in ).The function of lysine methylation depends on the site and the number of methyl groups. Histone methylation is in general less dynamic than acetylation. For instance, trimethylation of lysine 9 (H3K9me3) is characteristic of constitutive heterochromatin, whereas H3K4me3 marks active promoters. H3K27me3 is a repressive modification found especially at genes regulated during development, lineage choice, or cell differentiation. Methylation at H3K36 distinguishes actively transcribed gene bodies. In a similar fashion, methylation at various sites of other canonical and of variant histones  is associated with different chromatin states. Accordingly, different histone methylases from several enzyme and gene families attach methyl groups, and conversely, histone demethylases with various specificities remove them. Most histone demethylases, with KDM1A as a notable exception, are dioxygenases (like the TET family of DNA demethylases) and use α-ketoglutarate as a co-substrate. In the context of UC, the KMT2 family and EZH2 have received most attention. The KMT2 H3K4 histone methylases  are components of multiprotein complexes that activate transcription at promoters and enhancers (reviewed in ). EZH2 iSMARCA4) or BRM (gene SMARCA2) in different isoforms of the complex. ARID1A and ARID1B are further essential components present in alternative isoforms. ARID1A is particularly frequently inactivated in cancer [SMARCA6). A number of chromodomain protein (CHD) subunits are named for a domain recognizing methylated histones. Chromatin remodelers form another class of chromatin regulators. They interact with histone-modifying proteins and DNA-binding transcription factors to establish and change overall chromatin structure. In particular, several multiprotein complexes move nucleosomes along DNA, thereby creating denser packaging or more open structures. Chromatin remodelers like the SWI/SNF complex are therefore required for both gene repression and activation, but remodelers are also necessary for DNA repair and replication or for exchange of histone variants (like the SWR1 complex). In the context of cancer, the SWI/SNF complex has received most attention, as several of its components are mutated at significant frequencies in many cancer types, including UC . n cancer ,64. HeliSTAG2 and another frequently mutated chromatin regulator gene, KDM6A, are located on the X-chromosome, which may contribute to the overall male gender bias of UC [At a higher level of organization, chromatin looping defines local segments along the DNA strand, within which enhancers and promoters can interact. It is organized among other factors by the DNA-binding ‘isolator’ protein CTCF and by the cohesin complex . Oneas of UC .Histone variants provide another option for chromatin regulation; they mark and support the function of specific genome regions such as centromeres, telomeres, active genes, and regions involved in nucleolar functions. H2AX is essential for DNA repair. Only a few studies to date have addressed the function of histone variants in UC ,73. As iIn addition, various types of non-coding RNAs are involved in the regulation of chromatin states and gene expression. Long non-coding RNAs act at many steps of gene expression regulation, and several are involved in the establishment and maintenance of chromatin structures (reviewed in ). AltereKDM6A (encoding histone demethylase UTX), KMT2C, KMT2D (encoding histone methyltransferases MLL2 and MLL3), CREBBP, EP300 (histone acetyltransferases), and ARID1A (a SWI/SNF component).Several studies have consistently revealed that chromatin regulators, specifically chromatin remodelers and histone-modifying enzymes, are exceptionally frequently mutated in UC ,7,68,77,https://software.broadinstitute.org/Morpheus, accessed on 31 August 2021). Expression values for the 720 epigenetic regulators were extracted for tumor and normal samples, and fold-change expression across tumor samples as compared to normal samples was calculated. Data on mutations and genetic changes in the TCGA BLCA dataset were obtained from Robertson et al. [To obtain a comprehensive overview of genomic alterations affecting chromatin regulators in UC, we downloaded a list of 720 genes categorized as chromatin regulators in the EpiFactors database, a manually curated database for epigenetic regulators and their complexes . Note thn et al. ,82 and fKMT2C, KMT2D, and KDM6A, and the SWI/SNF subunit ARID1A, along with TP53 (a transcription factor), each in more than 100 tumors. Genes mutated in at least 10 tumors encoded many additional histone writers, erasers, and components of chromatin remodeling and polycomb complexes, as well as a few transcription factors, coactivators, and corepressors, including FOXA1 in 14 cases, PPARGC1A (a PPARγ coactivator) in 19 cases, and RB1 (a corepressor) in 83 cases. Each of the three TET DNA demethylase genes was mutated in at least 10 cancers, most frequently TET1, in 30 cases. DNMT3A and DNMT1 were each affected by mutations in 10 cases. This latter result suggests that DNA methylation changes in UC [Among the 720 chromatin regulator genes, 665 contained nonsynonymous mutations in at least one of the 412 UC samples; 187 were mutated in at least 10 tumors. es in UC ,52,53,54KMT2C, KMT2D, KDM6A, ARID1A, and others.These numbers have to be considered in the light of the high mutation rate in UC (up to 10/Mbp), with 100–200 genes mutated in typical cases. In addition, our present analysis does not distinguish the type of mutation in the respective genes, such as missense, nonsense, or other types of truncating mutations. In evaluating the significance of mutations, the ratio of nonsynonymous to synonymous mutations would also have to be considered for each individual gene , 11q, 1ing EZH2  and BRCAing EZH2 . While iing EZH2 . Anothery others . The ency others . Its funr cancer . The E3 r cancer  but has r cancer . WhetherJAK2, doubling as a histone kinase in addition to its function in STAT signaling), 13q , 16p (including CREBBP), 17p , and 22q13 (encompassing BRD1 and HDAC10). Homozygous deletions at 3p, 4q, 14q, and 18q each contained only a single chromatin regulator gene, namely FOXP1, ING2, RCOR1, and ZNF516, respectively. RB1 was most frequently subject to homozygous deletions in 37 cancers, followed by three genes on 8p.A total of 225 chromatin regulator genes were homozygous deleted in at least one UC, with 39 homozygous deleted in at least 10 cases. NCOR1 was identified as a significantly mutated gene by several studies [KAT2A) to acetylate histones [In addition to those genes discussed in detail in the following subsection, several are good candidates for further studies.  studies . It enco studies ,96, but  studies . ZNF516  studies , especiahistones . No dedihistones ), but FOhistones . The INGhistones .Employing a twofold change cut-off, 58 genes were identified as down-regulated in UC compared to normal tissues, whereas 50 were upregulated. These genes are comprehensively listed in the KAT6B  and KAT2B (PCAF), class IIA HDACs 4 and 9, and the class III (sirtuin) HDAC SIRT1, the KDM6B demethylase, as well as components of SWI/SNF, namely SMARCA2/BRM, SMARCD3/B60c, and DPF3/BAF45c. Notably, other components of the SWI/SNF multiprotein complex are upregulated ; PRDM12ated see B. No dedAURKA/B, TTK, GSG2/HASPIN, PBK and others) and regulators of DNA replication and cell cycle progression  and DNA methylation . Like the upregulation of these factors, upregulation of chromatin assembly factors  may simply reflect the increased proliferation of urothelial cancer cells compared to quiescent normal urothelium. Interestingly, CAF1 is involved particularly in the deposition of variant histones and can influence differentiation and reprogramming in this manner [BRCA1/2, RAD51, UBE2T/FANC2, CHEK1, PCNA, TOP2A) is also upregulated, possibly reflecting an increased requirement in cancer cells subject to replication stress. The histone methyltransferase EZH2, a central component of the PRC2 polycomb complex, is well known to be upregulated in UC [KAT2A (GCN5), with concomitant downregulation of its close paralog KAT2B, is a common finding in cancer [String analysis of upregulated genes yields an entirely different core network, dominated by histone kinases associated with mitosis , was muiewed in . Like CDn reader , is accon reader ). HoweveFOXA1, which directs luminal differentiation. These genes in addition comprise PBK  and HJUnd SIRT1 . A likewnd SIRT1  in accorBRCA1 and BRCA2 were on average upregulated , but a significant number of mutations and—for BRCA2—homozygous deletions was found. This likewise suggests heterogeneity among UCs in their capacity for homologous recombination repair . BaPolycomb complexes are established as key regulators of epidermal differentiation . PRC1 anKDM6A mutations in UC [ASXL3. ASXL proteins in general serve to link chromatin and chromatin-modifying protein complexes. ASXL3 is less well studied than its paralogs but was shown to promote BAP1 binding to its histone substrate like its paralogs [Gene repression by PRC1 is partly mediated via histone H2A mono-ubiquitination, resulting in the H2AK119ub1 modification. This modification is removed by a protein complex containing the deubiquitinase BAP1 as the central unit. Inactivating mutations of BAP1 and deletion of its gene at 3p are prominent in renal cell carcinoma  but havens in UC . Our anaparalogs . Its dowBRCA1 and BRCA2 expression across UC, but also relatively frequent mutations in both genes and even homozygous deletions of BRCA2. Inactivation of homologous recombination repair is thought to occur relatively infrequently in bladder cancer and biallelic inactivation of BRCA1 or BRCA2 is rare. Mutational signatures reflecting homologous recombination repair deficiency (‘BRCAness’) are found in fewer than 10% of all invasive UCs [BRCA1 and BRCA2 is prevalent, potentially in a different subset of cancers. In particular, loss of KMT2C in UC cells was shown to decrease the expression of DNA repair genes. Moreover, Kmt2c knockout in mice induced ureter tumors with a high level of DNA double-strand breaks [KMT2C mutations were moreover more sensitive to PARP inhibitors like olaparib [As suggested by its name, BAP1 (BRCA1-associated protein 1) influences also DNA repair (reviewed in ), especisive UCs . The pred breaks . This geolaparib . Howeverolaparib . As moreAnother interesting factor highlighted by our analysis is the class III histone deacetylase SIRT1. SIRT1 deacetylates H4 but also many non-histone proteins, including TP53, as well as other proteins involved in DNA repair and checkpoints. In some cancer types, SIRT1 is upregulated and therefore considered as a target for specific inhibitors . PharmacThe trithorax-like factors regularly inactivated by mutations in UC are known to be involved in the differentiation of many cell lineages . OnKDM6A loss of function and FGFR3 oncogenic mutations. Intriguingly, active FGFR3 leads to repression of many enhancers of luminal urothelial genes that are normally bound and activated by KDM6A [The luminal-papillary subtype expresses a particularly broad range of urothelial differentiation markers and is characterized especially by frequent by KDM6A . This anThe function of the PRC2 catalytic subunit EZH2, a direct antagonist of KDM6A with respect to H3K27 methylation, is intriguing in this context. As in epidermal differentiation , EZH2 suFOXA1 may contribute. However, despite activation of urothelial differentiation, these tumor cells do not exit the cell cycle. This might be explained partly by the loss of TP53 and especially RB1 inactivation, which prevent cell cycle exit and terminal differentiation. Loss of cell cycle control in general and RB1 activity specifically may account for the overexpression of many genes involved in DNA replication and cell cycle progression as revealed by our analysis . These mutant stem cells expand and replace normal stem cells. While the hematopoietic system remains largely functional, clonal hematopoiesis increases the risk for leukemia. The transformation to leukemia requires additional mutations that activate oncogenes (like FLT3 or RAS) or inactivate tumor suppressors (like TP53 or CEBPA) [KMT2C inactivating mutations have been shown to enhance the self-renewal capacity of hematopoietic stem cells [Analogous observations have been made in other tissues (reviewed in ). The phr CEBPA) . Interesem cells .Accordingly, there is no evidence that the mutations in chromatin regulator genes detected in normal urothelium severely affect its functionality or grossly impede its differentiation ,173. MorKDM6A mutations appear to be particularly frequent in the luminal-papillary subtype, both in muscle-invasive UC and non-invasive urothelial tumors, and are often accompanied by FGFR3 mutations. The study by Barrows et al. [Several questions in particular need to be addressed to validate and clarify this hypothesis. First, by which mechanisms does inactivation of chromatin regulators enhance self-renewal of urothelial stem cells? Second, in which respect do KRT14-positive stem cells with mutant chromatin regulators differ from normal ones? While not grossly disturbed, to which extent is their differentiation ability impeded in detail? Third, do all chromatin regulator mutations favor carcinogenesis to the same extent? Fourth, do they favor the development of specific molecular subtypes? For instance, s et al.  providess et al. . Moreoves et al. .The ideas on the function of chromatin regulator mutations in urothelial carcinogenesis outlined above have important implications for targeted therapeutic approaches. Targeted therapies have been relatively unsuccessful in UC, with the recent exception of FGFR inhibitors in the papillary-luminal subtype ,42. On tWhile restoration of its function might come too late to suppress tumor growth, lack of a chromatin regulator may sensitize cancers to the inhibition of different chromatin regulators or other processes that are essential for tumor growth, i.e., it may generate synthetic lethality. For instance, ARID1A-deficient tumors are particularly sensitive to inactivation of its paralog ARID1B, which leads to dissociation of the remaining SWI/SNF complexes with alternative composition ,184. In In this review, we re-analyzed data from the TCGA comprehensive investigation of genetic alterations in urothelial carcinoma with respect to chromatin regulators and summarized the state of the literature on their functions in this cancer type. Our data analysis revealed additional candidate factors and relations that merit further investigation in urothelial carcinoma to improve the understanding of pathogenetic mechanisms in this cancer. The literature analysis led us to propose a hypothesis on the biological function of the most frequently inactivated—trithorax-like—chromatin regulators in urothelial carcinogenesis. Throughout these analyses, we could not help noting that despite much recent progress, bladder cancer remains under-researched. In particular, mechanistic studies addressing the molecular and cellular consequences of chromatin regulator alterations, specifically in urothelial cells, are sparse. Hopefully, the current review will not only alert to that fact, but provide information and ideas that help to ameliorate these deficiencies.


Plasmodium falciparum, is based on an orchestrated and tightly regulated gene expression program. In general, eukaryotic transcription regulation is determined by a combination of sequence-specific transcription factors binding to regulatory DNA elements and the packaging of DNA into chromatin as an additional layer. The accessibility of regulatory DNA elements is controlled by the nucleosome occupancy and changes of their positions by an active process called nucleosome remodeling. These epigenetic mechanisms are poorly explored in P. falciparum. The parasite genome is characterized by an extraordinarily high AT-content and the distinct architecture of functional elements, and chromatin-related proteins also exhibit high sequence divergence compared to other eukaryotes. Together with the distinct biochemical properties of nucleosomes, these features suggest substantial differences in chromatin-dependent regulation. Here, we highlight the peculiarities of epigenetic mechanisms in P. falciparum, addressing chromatin structure and dynamics with respect to their impact on transcriptional control. We focus on the specialized chromatin remodeling enzymes and discuss their essential function in P. falciparum gene regulation.The highly complex life cycle of the human malaria parasite,  Plasmodium falciparum, a unicellular eukaryotic parasite, causes the most severe and deadly form of the human disease malaria. In 2019, 229 million cases of malaria infection with about 409,000 deaths were reported, mainly affecting children under the age of five : F: FPfApiAfe cycle ; AP2-G wfe cycle ; AP2-I ife cycle ; AP2-O afe cycle , while gfe cycle . Still tfe cycle ,40. Altefe cycle ,41, or ufe cycle ,43,44.cis-acting elements and trans-acting factors is strongly influenced by the packaging of the genomic DNA into chromatin. Nucleosome positioning and dynamics control the accessibility of the regulatory DNA elements for the trans-acting factors, as histones would mask the binding sites and inhibit DNA sequence recognition. Therefore, nucleosome-positioning plays an essential role, and well-defined chromatin architectures can be observed at regulatory regions. At gene promoters, positioned nucleosomes (+1) just downstream of the TSS can be observed, and the promoter region directly upstream is generally depleted of nucleosomes . T. TPf nucleosomes . For Pf  regions . The majrich DNA .Pf nucleosome, which can be observed in vitro and is maintained also in vivo [Plasmodium, were shown to inhibit the folding of the nucleosome array into compact higher order structures of chromatin. The absence of higher-order chromatin structure correlates well with the assumption that Pf chromatin exhibits high accessibility [Pf.Besides the differences in nucleosomal properties, the nucleosome repeat length is highly divergent as well when compared to other eukaryotes. With a repeat length of 155 bp, the spacing of the nucleosome cores is maintained by DNA linkers with only 8 bp. Short nucleosome spacing is an intrinsic biochemical property of the  in vivo ,60. The  in vivo . Such shsibility ,62,63,64In addition to canonical histones, eukaryotes express histone variants throughout the cell cycle, which differ in amino acid sequence. Sequence variation occurs predominantly in the (N-)terminal histone tails and leads to novel and different sites of post-translational modifications, potentially impacting their function and interaction with chromatin-modifying enzymes . Among eP. falciparum, a homologue of the universally present H2A.Z was identified, but no H2A.X. Surprisingly, Apicomplexa additionally express an unusual H2B variant histone, called H2B.Z, whose function is still unclear. Genome-wide profiling revealed similar binding sites of PfH2A.Z and PfH2B.Z and coimmunoprecipitation experiments confirmed the existence of nucleosomes containing both H2A.Z and H2B.Z in the same octamer [Toxoplasma gondii, indicating a role in the regulation of gene expression .P. falciparum, although they have not been characterized in detail. Histone H3.3 has eight amino acids substitutions compared to canonical H3 (one amino acid exchanged in human H3.3) and is believed to preferentially bind GC-rich repetitive regions, independent of transcriptional activity, potentially contributing to the regulation of var gene expression and immune evasion [Histones H3.3 and CenH3, the two universal variants replacing histone H3, are present in  evasion . The sec evasion ,71.P. falciparum exhibits striking differences to other eukaryotes with an unusually large proportion of constitutively acetylated histones and a high number (500) of identified PTMs, including several novel modifications specific to Plasmodium or Apicomplexa [P. falciparum, are tightly regulated, and their presence is limited to specific life cycle stages [Histones are the target for post-translational modifications (PTMs), mainly comprising acetylation, methylation and phosphorylation of the histone amino termini, which alter histone properties and their interactions with DNA and chromatin proteins, affecting the functionality of the underlying DNA. The combinatorial nature and functional impact of these epigenetic modifications are defined as the “histones code” amplifying the information content and plasticity of chromatin with respect to the regulation of all DNA-dependent processes ,73. Herecomplexa ,74,75. Ae stages . The pree stages ,76,77.Nucleosomal distribution on DNA is described by the terms “nucleosome occupancy” and “nucleosome positioning”: Occupancy describes the probability with which a certain base pair is covered by a nucleosome, while nucleosome positioning is a measure of the probability of a given base pair to serve as start, dyad or end position of a nucleosome .Pf, large genomic regions lack positioned nucleosomes or even appear to lack histone octamers on DNA at all. Several studies have addressed the nucleosomal landscape in P. falciparum, using Sonication-ChIP, MNase-ChIP, MNase-Seq and other methods, such as ATAC-seq and FAIRE, to detect nucleosome-free regions [Drosophila chromatin have revealed the existence of nucleosomes with different stability [Plasmodium falciparum chromatin structure. The only study so far using limited MNase digestion conditions was performed by Kensche and colleagues [The reduced stability and loss of sequence-dependent positioning of plasmodial nucleosomes in vitro is reflected by the genome-wide analysis of chromatin structure in vivo. In  regions ,79,80,81tability ,82 and, tability ,82. Takilleagues .P. falciparum is framed by positioned nucleosomes upstream and downstream of the coding region  var genes pointing to its putative contribution to epigenetic memory in var gene expression [Studies mapping the genomic localization of nucleosomes containing the histone variants H2A.Z and H2B.Z identified them in the intergenic regions of euchromatin domains, particularly enriched at gene promotors ,67,68,69cription . Interespression .Pf nucleosomes in general do not quite follow known eukaryotic principles. The highly divergent underlying determinants—DNA and histone properties—seem to shape a very different chromatin landscape in Plasmodium falciparum, and potential novel mechanisms may have evolved to allow for the tightly regulated gene expression program in the parasite. Not only the histone variant distribution, but also the occupancy and positioning of P. falciparum and have not been reported to a comparable extent in any other eukaryote. Progressing through the erythrocytic life cycle, major changes in nucleosome occupancy were reported, obtained via high MNase digestion and Hi-C techniques ,79,84: tPlasmodium falciparum using chromosome conformation capture techniques . Oneobserved .As described in the previous sections, gene transcription is impacted by a combination of epigenetic features shaping the chromatin landscape: the main determinants are the variations of nucleosome occupancy and nucleosome positioning at specific DNA elements and histone variants, in addition to the dynamic histone PTMs as well as higher-order chromatin structures. Furthermore, long noncoding RNAs ,89 have P. falciparum: Kensche and colleagues identified 4821 dynamic nucleosomes with 80 percent being located in euchromatic intergenic regions, mainly at promotors. The dynamics of these nucleosomes clearly correlate with temporal transcriptional activity of the downstream gene. Toenhake and colleagues identified 4035 accessible regions, whereof 90 percent are located in intergenic regions. The majority was found to be associated with one or more putative promotors and to correlate in accessibility score with abundance of the downstream gene product.Genome-wide MNase-seq  and ATACvar genes—is maintained by heterochromatin protein HP1, histone deacetylase HDA2 and the histone lysine methyltransferase SET2 and marked by H3K9me3 rendering these genes heterochromatic [var gene-switching mechanism by silencing the gene locus via its sense—activating via its antisense—lncRNA [gdv1 antisense RNA [For some genes with high transcriptional variation, the epigenetic mechanisms were investigated in more detail (reviewed in ): the grhromatic ,95,96,97hromatic ,99, and e—lncRNA ,100. Sexense RNA ,102.These examples illustrate the multilayered nature of epigenetic regulation, but concomitant nucleosome occupancy and positioning were hardly taken into consideration. However, in all of the described processes, nucleosome occupancy determines any interactions with the underlying DNA locus and certainly contributes to the unveiled regulatory mechanism. The importance of nucleosome occupancy was highlighted in a machine-learning model, wherein the relevance of individual epigenetic features in relation to the entirety of transcription regulation was assessed : a collePlasmodium falciparum, as the fundamental differences between chromatin proteins in humans and Plasmodium will provide new insights into the evolution and mechanisms of chromatin dynamics and may reveal new therapeutic options.All DNA-dependent processes require dynamic changes in chromatin organization to exert their DNA specific activities. For this, eukaryotic cells have developed numerous enzymes that change the organization of DNA packaging . ChromatPlasmodium falciparum . In thiRemodeling enzymes and the large multiprotein complexes they form exert a direct ATP-dependent effect on nucleosomes. The enzymes alter histone–DNA interactions, resulting in the eviction, exchange and assembly of individual histones or histone octamers, changing the structure and stability of nucleosomes or the movement of histone octamers on DNA to reposition nucleosomes . InThe essential molecular function of this enzyme family is the movement of nucleosomes in order to provide accessibility to certain DNA regions. However, the exact regulatory mechanisms of this process—with respect to which nucleosomes are recognized to be moved and what is the target position—have not been completely uncovered. There is some evidence of “high affinity” and “low affinity” nucleosomes representing a putative mechanism or at least one aspect of defining reaction educts and products . TherefoThe functionally different subfamilies of SWI/SNF ATPases are grouped by the homology of the helicase region according to Flaus et al.  profile studies in  ATPases  and shar ATPases . During  ATPases . In compP. falciparum, and Apicomplexa in general, remain poorly characterized so far. In a pioneering study, Ji and Arnot identified and classified the first SWI2/SNF2 enzyme (Snf2L) with approximately 60% sequence homology to the ATPase domain of the yeast ISWI remodeler [The individual remodeling enzymes in emodeler . Since temodeler ,128. A gemodeler . The autP. falciparum. This astonishing lack of known interactors in combination with the reduced number and high divergence in sequence indicates that the chromatin-remodeling system in Plasmodium differs from known mechanisms in higher eukaryotes.In general, perhaps with the exception of Chd1, all chromatin-remodeling enzymes are part of large multiprotein complexes with characteristic binding partners that link the enzymes to chromatin modifiers, chromatin and DNA binding motifs and alter the functionality of the complexes . InteresPlasmodium falciparum chromatin is unusual in many respects, from the reduced number of transcription factors to high nucleosome dynamics and the divergent chromatin-remodeling enzymes/complexes. Therefore, we hypothesize that P. falciparum possesses a highly regulated but distinct chromatin system when compared to other eukaryotes. Here we address how these differences may affect the regulatory network and the transcriptional program throughout the parasite life cycle  viagulation ; or (c) P. falciparum. Based on the reduced number of identified Pf enzymes with high sequence divergence and the absence of any known interacting subunits, as there are in higher eukaryotes, we expect functional divergence. We propose that Pf remodeling complexes—closely linked with the nucleosome landscape and transcription-factor binding—build a complex regulatory network exhibiting major differences in comparison to other eukaryotes. The decryption of this system is indispensable to understanding the mechanism of transcription regulation in the parasite and will provide new insights and novel approaches for fighting malaria.How remodeling enzymes pave the way for transcription-factor and polymerase binding and how they are regulated is completely unexplored in


Insulin (InsR) and insulin-like growth factor-1 (IGF1R) receptors mediate the metabolic and growth-promoting actions of insulin and IGF1/IGF2, respectively. Evidence accumulated in recent years indicates that, in addition to their typical cell-surface localization pattern and ligand-activated mechanism of action, InsR and IGF1R are present in the cell nucleus of both normal and transformed cells. Nuclear translocation seems to involve interaction with a small, ubiquitin-like modifier protein (SUMO-1), although this modification is not always a prerequisite. Nuclear InsR and IGF1R exhibit a number of biological activities that classically fit within the definition of transcription factors. These nuclear activities include, among others, sequence-specific DNA binding and transcriptional control. Of particular interest, nuclear IGF1R was capable of binding and stimulating its cognate gene promoter. The physiological relevance of this autoregulatory mechanism needs to be further investigated. In addition to its nuclear localization, studies have identified IGF1R in the Golgi apparatus, and this particular distribution correlated with a migratory phenotype. In summary, the newly described roles of InsR and IGF1R as gene regulators, in concert with their atypical pattern of subcellular distribution, add a further layer of complexity to traditional models of cell signaling. Furthermore, and in view of the emerging role of IGF1R as a potential therapeutic target, a better understanding of the mechanisms responsible for nuclear IGF1R transport and identification of IGF1R interactors might help optimize target directed therapies in oncology. The insulin-like growth factors (IGFs) are a family of mitogenic growth factors, cell-surface receptors and binding proteins that are involved in normal growth, development and differentiation of most organs and tissues as well as in a number of pathological conditions ,2. IGFs IGF2 shares a ~62% amino acid homology with IGF1. Ontogenetic studies in rodents have demonstrated that IGF2 is expressed at very high levels during the prenatal period . On the IGF1R gene was disrupted by homologous recombination [Both IGF1 and IGF2 activate a common receptor, the IGF1 receptor (IGF1R), which signals mitogenic, antiapoptotic and pro-survival activities . The IGFbination .The IGF system includes, in addition, a series of six high-affinity IGF binding proteins (IGFBP1-6) that carry IGF1 and IGF2, but not insulin, in the circulation and extracellular spaces. For the most part, the IGFBPs inhibit IGF’s metabolic and proliferative actions, although some IGFBPs display IGF-potentiating effects ,15. In sThe classical view on hormonal signaling endorses the dogma that this intercellular communication system constitutes an unidirectional cascade that initiates with the binding of an extracellular ligand to a specific, or cognate, receptor or to closely related receptors, followed by receptor activation, usually in the form of a phosphorylation event. This energetic state is then transmitted downstream to a series of cytoplasmic proteins that, in a sequential and highly orderly manner, convey this signal to the nucleus, where genes are turned ‘on’ or ‘off’. In turn, this cascade leads to different, often opposite, biological activities. There is a certain degree of crosstalk between ligands and receptors, but the hormonal signaling is primarily unidirectional, i.e., from cell surface to nucleus.Receptor internalization, or endocytosis, has been commonly regarded as a mechanism required for receptor degradation, usually in endosomes and lysosomes. Most of the trafficking pathways and players involved in this process have been extensively characterized over the years, including caveolins, clathrin-coated pits, etc. ,20. It iEarly experimental evidence of nuclear InsR presence was provided forty-five years ago by Goldfine and Smith . Using pIn terms of attempts to elucidate the mechanistic aspects associated with nuclear InsR presence, electron microscopy allowed Podlecki et al.  in 1987 IGF1R promoter-binding proteins led us to the identification of the IGF1R in cell nucleus. Briefly, human IGF1R promoter fragments were labeled with biotin, followed by incubation with nuclear extracts of breast cancer cells, and purified over streptavidin beads. Bioinformatic analyses of proteins in eluates allowed us to identify approximately 100 nuclear proteins that were either directly linked to the IGF1R gene promoter or, alternatively, part of multimeric protein complexes associated with this DNA region [Proteomic profiling analyses aimed at characterizing the entire collection of A region .IGF1R promoter-binding proteins were typical nuclear proteins, including DNA helicases, DNA repair proteins, elongation factors, RNA splicing proteins, etc. The presence of IGF1R in association with its cognate promoter was unexpected as it contradicted established dogmatic notions [IGF1R promoter region in ER α-depleted, but not ER α-containing, breast cancer cells. These results are consistent with competition between IGF1R and ER α proteins for binding to consensus GC elements in the IGF1R promoter [IGF1R gene. The autoregulation of IGF1R expression by nuclear IGF1R is described below.Most of the identified  notions . While Ipromoter ,34. In oNuclear translocation of InsR and IGF1R is attained via proteosomal, lysosomal and endocytic pathways . Of noteEvidence in support of the hypothesis that IGF1R is transported to the nucleus along microtubules was provided by studies showing that IGF1R co-localizes with α-tubulin . ImportiThe question of whether InsR/IGF1R SUMOylation constitutes a mandatory prerequisite for nuclear internalization is still a matter of controversy . Sehat eThe ability of IGF1R to directly bind DNA was demonstrated by means of electrophoretic mobility shift assays using biotin-labeled oligonucleotides . At a geIGF1R promoter [In order to examine IGF1R subcellular distribution and to gain further insight into its transcriptional role, Jamwal et al.  recentlypromoter . Of intepromoter . In summIGF1R transcription might be regulated by nuclear receptor concentrations. Co-transfection experiments in breast cancer cell lines using an IGF1R expression vector along with an IGF1R promoter-luciferase reporter construct demonstrated that IGF1R stimulated the activity of its cognate promoter. In correlation with the extent of IGF1R binding to promoter DNA, the level of promoter activity stimulation was higher in ER α-depleted than in ER α-containing cells [IGF1R promoter regulation. Thus, whereas IGF1R stimulated its cognate promoter, InsR inhibited IGF1R promoter activity  . Of inteRecent mass spectrometry (MS)-based proteomic analyses, designed to identify nuclear IGF1R interactors, allowed us to detect a series of novel proteins that have not been previously linked to the IGF1 signaling pathway  Table 1Table 1. InsR and IGF1R can dimerize to form hybrid InsR/IGF1R, composed of one α−β InsR hemireceptor linked to an α−β IGF1R hemireceptor ,49,50. TThe role of insulin in mediating the nuclear accumulation of hybrid InsR/IGF1R was recently investigated in corneal epithelial cells . Stress-1250/1251 under basal conditions. The impact of this phosphorylation event was investigated using a phospho-mimetic mutant form of IGF1R. This mutant was shown to be more rapidly internalized than the wild-type IGF1R. Upon IGF1 stimulation, both the wild-type and phospho-mimetic version of IGF1R rapidly accumulated within the Golgi apparatus. On the other hand, a phospho-impaired version of the receptor was retained in the plasma membrane. The localization of IGF1R in the Golgi correlated with a migratory phenotype. Taken together, the data are consistent with the idea that Tyr1250/1251 phosphorylation drives the translocation of IGF1R to the Golgi apparatus, where it might contribute to invasiveness. Of clinical relevance, these results may partially explain the lack of efficacy of current approaches aimed at targeting the cell-surface IGF1R for therapeutic purposes.A recent study from the laboratory of Rosemary O’Connor employed a novel approach to address aspects related to IGF1R activity in malignant cells . Using mUsing a combination of proteomic analyses along with silencing experiments, we discovered a complex, bi-directional interplay between nuclear IGF1R and nucleolar protein NOM1 . The nuccyclin D1 gene promoter, suggesting that it may serve as a transcriptional regulator. Furthermore, a number of studies reported that full-length EGFR is involved in a variety of gene regulatory activities, including transcription, DNA replication and DNA repair [Endocytosis and endosomal sorting are involved in the nuclear import of additional tyrosine kinase receptors . EGFR waA repair ,61.TH gene regulation, with relevance in dopaminergic neuron development. It is reasonable to conclude that nuclear import constitutes a widespread mechanism among cell surface tyrosine kinase receptors. This machinery impinges upon multiple physiological and pathological processes and largely expands the spectrum of classical intracellular communication paths.Likewise, the platelet-derived growth factor receptor (PDGFRα) was shown to be present in nuclear fractions of alveolar rhabdomyosarcoma along with IGF1R . AuthorsThe effect of nuclear InsR/IGF1R on the mitogenic capacity of cells and, more specifically, the impact of this pattern of subcellular distribution on our ability to target the IGF1R for therapeutic purposes, has been the topic of major interest in recent years . The relThe association between nuclear IGF1R and clinical outcome in pediatric gliomas was recently studied by Clement et al. . Immunohjun and fam21 promoters in fresh prostate cancer samples containing abundant nuclear IGF1R. A correlation between IGF1R and jun expression was observed in malignant prostate epithelium, suggesting the existence of a potentially novel mechanism of carcinogenesis. The growth factor amphiregulin participates in the nuclear accumulation of IGF1R in lung adenocarcinoma by allowing the binding of IGF1R to importin-β1 [To gain new insights into the clinical significance of nuclear IGF1R, Aleksic et al.  conducteortin-β1 . This prNuclear IGF1R was shown to phosphorylate proliferating cell nuclear antigen (PCNA), a key player in the DNA damage tolerance pathway . The co-Given the potential role of IGF1R as a therapeutic target in oncology, it was of translational relevance to assess the impact of nuclear IGF1R on the clinical outcome. Using an immunohistochemical approach, Asmane et al.  demonstrThe prognostic value of breast-cancer-specific nuclear InsR was investigated in tissue microarrays from 900 patients with primary invasive breast cancer . NuclearGenome-wide analysis was recently employed to identify genes and signaling pathways affected by nuclear InsR and, more specifically, to evaluate the impact of this nuclear presence on insulin function . ChIP-seEvidence that the nuclear InsR pathway is functionally involved in conditions associated with insulin resistance was provided by animal experiments showing a reduction in the levels of chromatin-bound InsR in an ob/ob mouse, a model of insulin resistance . Of inteIn mechanistic terms, a strong physical interaction between InsR and transcriptional co-regulator HCF-1 was detected. HCF-1 knockout prevented InsR binding to DNA, suggesting that this transcription factor is directly responsible for InsR binding to chromatin. Taken together, nuclear InsR has a key role in the regulation of gene expression, and this molecular mechanism directly impinges upon the physiological and pathological actions of insulin.Grave’s disease (GD) is an autoimmune disorder in which the thyroid gland is enlarged and becomes over-active. IGF1R was shown to be overexpressed by orbital fibroblasts derived from patients . In addiThe identification of InsR and IGF1R in the nucleus of normal and cancer cells has generated a significant level of interest in recent years ,74. CuriThe transcriptional role of InsR/IGF1R is not a trivial one. The ability of these important receptors to regulate gene expression at a genomic level provides a further layer of biological control. Moreover, and given the capacity of these receptors to sense (and respond to) ambient concentrations of insulin and IGF1, it is expected that the novel nuclear roles of InsR/IGF1R might be of major metabolic relevance.Finally, the role of nuclear InsR/IGF1R in cancer biology is of exceptional translational relevance . A bette


Long noncoding RNAs (lncRNAs) are composed of nucleotides located in the nucleus and cytoplasm; these are transcribed by RNA polymerase II and are greater than 200 nt in length. LncRNAs fulfill important functions in a variety of biological processes, including genome imprinting, cell differentiation, apoptosis, stem cell pluripotency, X chromosome inactivation and nuclear transport. As high throughput sequencing technology develops, a substantial number of lncRNAs have been found to be related to a variety of biological processes, such as development of the testes, maintaining the self-renewal and differentiation of spermatogonial stem cells, and regulating spermatocyte meiosis. These indicate that lncRNAs can be used as biomarkers and potential therapeutic targets for male infertility. However, only a few comprehensive reviews have described the role of lncRNAs in male reproduction. In this paper, we summarize recent findings relating to the role of lncRNAs in spermatogenesis, their potential as biomarkers for male infertility and the relationship between reproductive arrest and transgenerational effects. Finally, we suggest specific targets for the treatment of male infertility from the perspective of lncRNAs. Only 4% of the human genome features RNA that is translated into protein . The remLncRNAs are composed of nucleotides located in the nucleus and cytoplasm, which are transcribed by RNA polymerase II and greater than 200 nt in length. The level of transcription for genes transcribing lncRNAs is lower than those of protein-coding genes ,25,26. LWith the development of high throughput sequencing technology, many functional lncRNAs have been discovered, and many of these lncRNAs are thought to be involved in the formation of spermatozoa ,34,35,36LncRNAs do not directly encode for proteins. However, an increasing number of studies have rejected their classification as “junk” and confirmed the important biological functions that they play in a variety of biological processes, including genome imprinting , cell diAccording to their mechanism of action, lncRNAs can be classified into four types : signal,Chromatin has two states: the euchromatin state, which is open and can be transcribed, and the heterochromatin state, which is a compact DNA-protein structure that cannot be transcribed . Pre-traDuring transcription, RNA poll II binds to the core promoter via transcription factors. DNA then undergoes local deconvolution and transcription is suspended due to the low level of phosphorylation at the C terminal domain (CTD) of RNA poll II. At this stage, lncRNAs are able to inhibit the binding of the RNA poll II complex to the promoter to complete transcriptional interference ,75. DuriPost-transcriptional regulation is mainly concerned with the processing, translocation, and stability of the mRNA obtained by transcription ,78. DuriSpermatogenesis is an intricate developmental process in which SSCs differentiate from gonadal cells to form type A spermatogonia, type B spermatogonia, primary spermatogonia, secondary spermatogonia, and finally spermatozoa ,82,83. DThe process of spermatogenesis includes the proliferation and differentiation of SSCs, as well as the maturation of sperm. SSCs are male germline stem cells that support spermatogenesis and maintain male fertility ,85. The Spermatogenesis is essential for male reproduction, and is a complex process which has been well described in many studies on protein-coding genes, mRNA and small RNAs. While during the regulation of this complex physiological process, whether the initiation and maintenance of spermatocyte meiosis, or the maturation of sperm ultimately relies on the lncRNAs participation. Current studies show that more than sixlncRNAs are dynamically expressed during sperm meiosis. For example, in mouse testes, 1700108J01Rik and 1700101O22Rik, two testis-specific lncRNAs, are expressed in round spermatids at prophase and participate in post-transcriptional gene regulation  demonstrLncRNAs can regulate gene expression at the pre-transcriptional, post-transcriptional, and translational levels ,102. ResSpermatogenic arrest is one of the causes of dysfunction in the male reproductive system. During this process, many lncRNAs are involved in the disruption of differentiation of specific spermatogenic cell types, thus inhibiting the formation of spermatozoa. For example, the knockout of testis-specific lncRNA 1 (Tslrn1) in mice testes reduced sperm production, rendering the mice infertile. This demonstrated that the abnormal expression of lncRNAs can affect male reproduction . In miceIn livestock, research on the proliferation of bovine male germ stem cells detected high expression levels of lncRNA H19 in bovine testicular tissue . LncRNA A testis-specific lncRNA, Tesra, was identified during the meiotic phase of mouse spermatogenesis. This lncRNA is widely present in germ cells and the extracellular environment. Nuclear Tesra can prompt Prss42/Tessp-2 promoter activity and its binding to the promoter, and could therefore regulate the process of meiosis from secondary spermatocytes to round spermatids ,127. AnoResearch into human sperm has identified many lncRNAs related to spermatogenesis and male fertility, several of which are tissue-specific  134]. F. F134]. 2 inhalation in pregnancy results in neurological dysfunction in male offspring. It may be associated with the increased expression of lncRNA Malat1, and increased Malat1 perhaps then acts as a brain growth regulator by regulating the expression of ApoE. While ApoE plays an active role in maintaining brain development [Changing the environment and behavior of the parental generation can trigger epigenetic variation thus resulting in different environmentally adapted traits. Some of these traits are retained and passed on from parent to offspring by epigenetic regulation, thus resulting in changes in the expression of heritable genes without altering the gene sequence and intergenerational inheritance of the trait. Several studies have shown that ncRNAs play an essential part in intergenerational inheritance in this manner. For example, a high-fat diet in mice altered the abundance of sperm miRNAs in the sire, causing elevated levels of gene damage and reactive oxygen species levels in sperm. These metabolic disturbances were also observed in the offspring . In addielopment ,147. Furelopment  showed telopment ,149,150.elopment  showed telopment . In miceelopment ,152. In LncRNAs are involved in the regulation of many processes in male reproduction, particularly in the proliferation, differentiation, and meiosis of SSCs as they develop into mature spermatozoa. As research progresses, the functions of an increasing number of lncRNAs have been verified and shown to be advantageous in the regulation of male reproduction, including NLC1-C, Neat1, H19, lncNONO-AS, Mrhl, and lncRNA-Gm2044 . Gaining