Intragenic Transcription Research Articles

Spt5 orchestrates cryptic transcript suppression and transcriptional directionality

Spt5 is a well-conserved factor that manipulates multiple stages of transcription from promoter-proximal pausing (PPP) to termination. Recent studies have revealed an unexpected increase of antisense transcripts near promoters in cells expressing mutant Spt5. Here, we identify Spt5p-restricted intragenic antisense transcripts and their close relationship with sense transcription in yeast. We confirm that Spt5 CTR phosphorylation is also important to retain Spt5’s facility to regulate antisense transcription. The genes whose antisense transcription is strongly suppressed by Spt5p share strong endogenous sense transcription and weak antisense transcription, and this pattern is conserved in humans. Mechanistically, we found that Spt5p depletion increased histone acetylation to initiate intragenic antisense transcription by altering chromatin structure. We additionally identified termination factors that appear to be involved in the ability of Spt5p to restrict antisense transcription. By unveiling a new role of Spt5 in finely balancing the bidirectionality of transcription, we demonstrate that Spt5-mediated suppression of DSIF complex regulated-unstable transcripts (DUTs) is essential to sustain the accurate transcription by RNA polymerase II.

Repression of pervasive antisense transcription is the primary role of fission yeast RNA polymerase II CTD serine 2 phosphorylation.

The RNA polymerase II carboxy-terminal domain (CTD) consists of conserved heptapeptide repeats that can be phosphorylated to influence distinct stages of the transcription cycle, including RNA processing. Although CTD-associated proteins have been identified, phospho-dependent CTD interactions have remained elusive. Proximity-dependent biotinylation (PDB) has recently emerged as an alternative approach to identify protein-protein associations in the native cellular environment. In this study, we present a PDB-based map of the fission yeast RNAPII CTD interactome in living cells and identify phospho-dependent CTD interactions by using a mutant in which Ser2 was replaced by alanine in every repeat of the fission yeast CTD. This approach revealed that CTD Ser2 phosphorylation is critical for the association between RNAPII and the histone methyltransferase Set2 during transcription elongation, but is not required for 3' end processing and transcription termination. Accordingly, loss of CTD Ser2 phosphorylation causes a global increase in antisense transcription, correlating with elevated histone acetylation in gene bodies. Our findings reveal that the fundamental role of CTD Ser2 phosphorylation is to establish a chromatin-based repressive state that prevents cryptic intragenic transcription initiation.

Structures and dynamics of Rpd3S complex bound to nucleosome.

The Rpd3S complex plays a pivotal role in facilitating local histone deacetylation in the transcribed regions to suppress intragenic transcription initiation. Here, we present the cryo-electron microscopy structures of the budding yeast Rpd3S complex in both its apo and three nucleosome-bound states at atomic resolutions, revealing the exquisite architecture of Rpd3S to well accommodate a mononucleosome without linker DNA. The Rpd3S core, containing a Sin3 Lobe and two NB modules, is a rigid complex and provides three positive-charged anchors (Sin3_HCR and two Rco1_NIDs) to connect nucleosomal DNA. In three nucleosome-bound states, the Rpd3S core exhibits three distinct orientations relative to the nucleosome, assisting the sector-shaped deacetylase Rpd3 to locate above the SHL5-6, SHL0-1, or SHL2-3, respectively. Our work provides a structural framework that reveals a dynamic working model for the Rpd3S complex to engage diverse deacetylation sites.

Gene recoding by synonymous mutations creates promiscuous intragenic transcription initiation in mycobacteria.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the deadliest infectious diseases worldwide. Previous studies have established that synonymous recoding to introduce rare codon pairings can attenuate viral pathogens. We hypothesized that non-optimal codon pairing could be an effective strategy for attenuating gene expression to create a live vaccine for Mtb. We instead discovered that these synonymous changes enabled the transcription of functional mRNA that initiated in the middle of the open reading frame and from which many smaller protein products were expressed. To our knowledge, this is one of the first reports that synonymous recoding of a gene in any organism can create or induce intragenic transcription start sites.

Spurious intragenic transcription is a feature of mammalian cellular senescence and tissue aging.

Mammalian aging is characterized by the progressive loss of tissue function and increased risk for disease. Accumulation of senescent cells in aging tissues partly contributes to this decline, and targeted depletion of senescent cells in vivo ameliorates many age-related phenotypes. The fundamental molecular mechanisms responsible for the decline of cellular health and fitness during senescence and aging are largely unknown. In this study, we investigated whether chromatin-mediated loss of transcriptional fidelity, known to contribute to fitness and survival in yeast and worms, also occurs during human cellular senescence and mouse aging. Our findings reveal aberrant transcription initiation inside genes during senescence and aging that co-occurs with changes in the chromatin landscape. Interventions that alter these spurious transcripts have profound consequences on cellular health, primarily affecting intracellular signal transduction pathways. We propose that age-related spurious transcription promotes a noisy transcriptome and degradation of coherent transcriptional networks.

Sense and antisense RNA products of the uxuR gene can affect motility and chemotaxis acting independent of the UxuR protein.

Small non-coding and antisense RNAs are widespread in all kingdoms of life, however, the diversity of their functions in bacteria is largely unknown. Here, we study RNAs synthesised from divergent promoters located in the 3'-end of the uxuR gene, encoding transcription factor regulating hexuronate metabolism in Escherichia coli. These overlapping promoters were predicted in silico with rather high scores, effectively bound RNA polymerase in vitro and in vivo and were capable of initiating transcription in sense and antisense directions. The genome-wide correlation between in silico promoter scores and RNA polymerase binding in vitro and in vivo was higher for promoters located on the antisense strands of the genes, however, sense promoters within the uxuR gene were more active. Both regulatory RNAs synthesised from the divergent promoters inhibited expression of genes associated with the E. coli motility and chemotaxis independent of a carbon source on which bacteria had been grown. Direct effects of these RNAs were confirmed for the fliA gene encoding σ28 subunit of RNA polymerase. In addition to intracellular sRNAs, promoters located within the uxuR gene could initiate synthesis of transcripts found in the fraction of RNAs secreted in the extracellular medium. Their profile was also carbon-independent suggesting that intragenic uxuR transcripts have a specific regulatory role not directly related to the function of the protein in which gene they are encoded.

Transcript elongation by RNA polymerase II in plants: factors, regulation and impact on gene expression.

Transcriptional elongation by RNA polymerase II (RNAPII) through chromatin is a dynamic and highly regulated step of eukaryotic gene expression. A combination of transcript elongation factors (TEFs) including modulators of RNAPII activity and histone chaperones facilitate efficient transcription on nucleosomal templates. Biochemical and genetic analyses, primarily performed in Arabidopsis, provided insight into the contribution of TEFs to establish gene expression patterns during plant growth and development. In addition to summarising the role of TEFs in plant gene expression, we emphasise in our review recent advances in the field. Thus, mechanisms are presented how aberrant intragenic transcript initiation is suppressed by repressing transcriptional start sites within coding sequences. We also discuss how transcriptional interference of ongoing transcription with neighbouring genes is prevented. Moreover, it appears that plants make no use of promoter-proximal RNAPII pausing in the way mammals do, but there are nucleosome-defined mechanism(s) that determine the efficiency of mRNA synthesis by RNAPII. Accordingly, a still growing number of processes related to plant growth, development and responses to changing environmental conditions prove to be regulated at the level of transcriptional elongation.

Abstract P2013: Transcription Fidelity Dysregulation In Heart Failure Pathogenesis

Using deep sequencing combined with computational biology, we found that the failing human hearts (n=3) contain a significant amount of aberrant transcripts arising from widespread spurious intragenic transcription initiations. The same phenomenon was detected in failing mouse hearts (n=3) induced by pressure overload and ischemia injury. Therefore, we hypothesize that spurious transcripts accumulation is a causative factor promoting heart failure (HF). The SET domain containing 2 (Setd2), an H3K36 tri-methylase, is a key regulator for transcription fidelity. The Setd2 modified H3K36me3 prevents RNA polymerase II (Pol II) entry from the gene body and the subsequent cryptic transcription initiation. In the same cohort of failing human and mouse hearts, we found that the SETD2 and H3K36me3 levels were concurrently reduced. To further test our hypothesis, we generated cardiac-specific Setd2-Knockout mice. The mice (n≥8) developed a typical HF progression phenotype: initial compensatory hypertrophy followed by a gradually deteriorating HF with chamber dilation. Transcriptome analysis revealed an accumulation of spurious transcripts in pre-hypertrophic Setd2 -/- cardiomyocytes (n=3). New intragenic transcription initiation sites were identified by DECAP-seq in the knockout (n=3), confirming that the increased aberrant transcripts resulted from spurious Pol II entry. Using ribosome footprinting assay (n=3) to map the translation initiation sites, we found that the aberrant transcripts were partially translated into micro peptides in the Setd2 -/- heart, suggesting that the accumulated aberrant transcripts and micro peptides together promote cardiac hypertrophy. Finally, we developed transgenic mice overexpressing human SETD2 (Tg-MHC-SETD2) in the heart. SETD2 overexpression repressed TAC-induced accumulation of spurious transcripts in cardiomyocytes (n=3). The transgenic mice (n≥6) were resistant to pressure overload stress with less cardiac hypertrophy and improved cardiac contractility. Together, we report that dysregulated transcription fidelity causes adverse aberrant transcripts accumulation leading to cardiac hypertrophy and heart failure. This new mechanism provides potential new therapeutic targets for HF.

Targeted protein degradation reveals a direct role of SPT6 in RNAPII elongation and termination

SummarySPT6 is a histone chaperone that tightly binds RNA polymerase II (RNAPII) during transcription elongation. However, its primary role in transcription is uncertain. We used targeted protein degradation to rapidly deplete SPT6 in human cells and analyzed defects in RNAPII behavior by a multi-omics approach and mathematical modeling. Our data indicate that SPT6 is a crucial factor for RNAPII processivity and is therefore required for the productive transcription of protein-coding genes. Unexpectedly, SPT6 also has a vital role in RNAPII termination, as acute depletion induced readthrough transcription for thousands of genes. Long-term depletion of SPT6 induced cryptic intragenic transcription, as observed earlier in yeast. However, this phenotype was not observed upon acute SPT6 depletion and therefore can be attributed to accumulated epigenetic perturbations in the prolonged absence of SPT6. In conclusion, targeted degradation of SPT6 allowed the temporal discrimination of its function as an epigenetic safeguard and RNAPII elongation factor.

C-Terminal BRE Overexpression in 11q23-Rearranged and t(8;16) Acute Myeloid Leukemia Is Caused By Intragenic Transcription Initiation

Abstract TextOverexpression of the BRE (Brain and Reproductive organ-Expressed ) gene defines a distinct pediatric and adult acute myeloid leukemia (AML) subgroup (Balgobind et al, Leukemia 2010, and Noordermeer et al, Blood 2011 and 2012). BRE overexpression specifically associates with 11q23/MLL/KMT2A-rearranged AML, occurring in 70% and 50% of 11q23 childhood and adult AML cases, respectively. In adult 11q23-rearranged AML, BRE overexpression significantly associates with younger age. BRE functions in BRCA1-A (Breast Cancer 1) complex mediated DNA damage repair, BRISC (BRCC3 isopeptidase complex) complex mediated mitotic spindle assembly and in protection from apoptosis and senescence. Another constituent of the BRCA1-A/BRISC complexes is BRCC3 which is recurrently mutated in AML, especially in cases with a TET2 mutation (Huang et al., Haematologica, 2015). BRE is relatively highly expressed in blood cells in general but its exact function in hematopoiesis is unknown.By performing genome-wide ChIP-sequencing analyses on primary KMT2A-MLLT3 patient material (n=9) we identified a promoter enriched for active chromatin marks (H3K4me3/H3K27Ac) in BRE intron 4, exclusively in AML cases with BRE overexpression. Both 5' RACE end RNA-sequencing identified strong biallelic expression of a previously unknown C-terminal BRE transcript. This transcript starts near the intragenic promoter, with BRE intron 4 sequences spliced to exon 5 and downstream sequences. RNA-seq and qRT-PCR showed that the downstream transcript is expressed 5-8-fold higher compared to the full-length transcript, explaining BRE overexpression. Of note, two start codons in exon 5 of the downstream BRE transcript may cause the formation of N-terminally truncated BRE isoforms.By performing a qRT-PCR that specifically recognizes the new BRE transcript on samples from a large cohort of pediatric and adult AML (n=195) we observed that it was highly expressed in 60% of 11q23/ KMT2A -rearranged (n=58) and 70% of t(8;16)/ KAT6A-CREBBP cases (n=20), while it was virtually absent from other AML subsets (n=101) and normal hematopoietic tissues (n=14). In gene reporter assays, the AML-specific fusion protein KMT2A-MLLT3 transactivated the intragenic BRE promoter. Further epigenome analyses revealed 97 additional intragenic promoter marks frequently bound by KMT2A in AML with C-terminal BRE overexpression. The bound genes may be part of a context-dependent KMT2A-MLLT3-driven oncogenic program, because they were higher expressed in this AML subtype compared to other cytogenetically-defined AML groups (p<0.01). C-terminal BRE might be a contributor to this oncogenic program because in a case with relapsed AML, we observed an ins(11;2) fusing CHORD1 in frame to BRE exon 5. The CHORDC1 fusion to BRE exon 5 is remarkable because the alternate BRE transcript starts with exon 1b also fused to exon 5. This means that if the CHORDC1-BRE and alternate BRE transcripts would be translated, they would contain a similar C-terminal part of the BRE protein. The CHORDC1 disruption may also contribute to AML pathogenesis because it was recently shown that heterozygous Chord1 (a.k.a. Morgana) inactivation in mice results in the spontaneous development of atypical chronic myeloid leukemia (Di Savino, Blood, 2015). We conclude that abnormal expression of C-terminal alternate BRE sequences specific for AML is caused by distinct, non-random events; by a chromosomal abnormality and by intragenic transcription initiation in 11q23/ KMT2A -rearranged and t(8;16)/ KAT6A-CREBBP AML. These findings warrant further studies to determine whether leukemia-specific alternate BRE contributes to disease pathogenesis and how abnormal intragenic transcription activation in AML is brought about. DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

1192. H-NS-like Proteins in Pseudomonas aeruginosa Coordinately Silence Intragenic and Antisense Transcription

BackgroundThe H-NS-like proteins MvaT and MvaU act coordinately as global repressors in Pseudomonas aeruginosa by binding to AT-rich regions of the chromosome, which include horizontally acquired genes and numerous virulence factors. Although cells can tolerate the loss of either protein, identifying their combined regulatory effects has been challenging because the loss of both proteins is lethal due to induction of the prophage Pf4 and subsequent superinfection of the cell. Although in other bacteria, H-NS promotes cellular fitness by inhibiting intragenic transcription from AT-rich target regions, preventing them from sequestering RNA polymerase, a role for MvaT and MvaU in repressing transcription from intragenic promoters has not been demonstrated.MethodsHere we utilize a parental strain that cannot be infected by Pf4 phage to identify the combined MvaT and MvaU regulon. RNA-seq was utilized to identify genes differentially expressed in cells lacking MvaU or both MvaU and MvaT. ChIP-seq was utilized to identify genes directly regulated by MvaT and MvaU in wild-type cells. Further, ChIP-seq was performed in cells of the parental strain and cells lacking both MvaT and MvaU to map genome-wide σ 70-dependent promoters that were active in the presence or absence of both H-NS-like proteins.ResultsWe demonstrate that the loss of both MvaT and MvaU, but not MvaU alone, leads to increased sense, antisense, and intragenic transcription from loci directly controlled by these proteins. We further show that the loss of MvaT and MvaU leads to a striking redistribution of RNA polymerase containing σ 70 to those genomic regions vacated by these proteins.Loss of MvaT and MvaU causes global changes in gene expression Loss of MvaT and MvaU results in increased sense and antisense transcription Loss of both MvaT and MvaU results in genome-wide redistribution of RNA polymerase ConclusionOur findings suggest that the ability of H-NS-like proteins to repress intragenic transcription is a universal function of these proteins and describe a second mechanism by which MvaT and MvaU may contribute to the growth of P. aeruginosa.Disclosures All Authors: No reported disclosures

H-NS-like proteins in Pseudomonas aeruginosa coordinately silence intragenic transcription.

The H-NS-like proteins MvaT and MvaU act coordinately as global repressors in Pseudomonas aeruginosa by binding to AT-rich regions of the chromosome. Although cells can tolerate loss of either protein, identifying their combined regulatory effects has been challenging because the loss of both proteins is lethal due to induction of prophage Pf4 and subsequent superinfection of the cell. In other bacteria, H-NS promotes the cellular fitness by inhibiting intragenic transcription from AT-rich target regions, preventing them from sequestering RNA polymerase; however, it is not known whether MvaT and MvaU function similarly. Here, we utilize a parental strain that cannot be infected by Pf4 phage to define the collective MvaT and MvaU regulon and demonstrate that the combined loss of both MvaT and MvaU leads to increased intragenic transcription from loci directly controlled by these proteins. We further show that the loss of MvaT and MvaU leads to a striking redistribution of RNA polymerase containing σ70 to genomic regions vacated by these proteins. Our findings suggest that the ability of H-NS-like proteins to repress intragenic transcription is a universal function of these proteins and point to a second mechanism by which MvaT and MvaU may contribute to the growth of P. aeruginosa.

The PWWP2A Histone Deacetylase Complex Represses Intragenic Spurious Transcription Initiation in mESCs.

SummaryTranscriptional fidelity depends on accurate promoter selection and initiation from the correct sites. In yeast, H3K36me3-mediated recruitment of the Rpd3S HDAC complex to gene bodies suppresses spurious transcription initiation. Here we describe an equivalent pathway in metazoans. PWWP2A/B is an H3K36me3 reader that forms a stable complex with HDAC1/2. We used CAGE-seq to profile all transcription initiation sites in wild-type mESCs and cells lacking PWWP2A/B. Loss of PWWP2A/B enhances spurious initiation from intragenic sites present in wild-type mESCs, and this effect is associated with increased levels of initiating Pol-II and histone acetylation. Spurious initiation events in Pwwp2a/b DKO mESCs do not overlap in genomic location or chromatin features with spurious sites that arise in Dnmt3b KO mESCs, previously reported to function in the suppression of intragenic transcriptional initiation, suggesting these pathways function cooperatively in maintaining the fidelity of transcription initiation in metazoans.

The Transcriptomic Landscape of Cupriavidus metallidurans CH34 Acutely Exposed to Copper.

Bacteria are increasingly used for biotechnological applications such as bioremediation, biorecovery, bioproduction, and biosensing. The development of strains suited for such applications requires a thorough understanding of their behavior, with a key role for their transcriptomic landscape. We present a thorough analysis of the transcriptome of Cupriavidus metallidurans CH34 cells acutely exposed to copper by tagRNA-sequencing. C. metallidurans CH34 is a model organism for metal resistance, and its potential as a biosensor and candidate for metal bioremediation has been demonstrated in multiple studies. Several metabolic pathways were impacted by Cu exposure, and a broad spectrum of metal resistance mechanisms, not limited to copper-specific clusters, was overexpressed. In addition, several gene clusters involved in the oxidative stress response and the cysteine-sulfur metabolism were induced. In total, 7500 transcription start sites (TSSs) were annotated and classified with respect to their location relative to coding sequences (CDSs). Predicted TSSs were used to re-annotate 182 CDSs. The TSSs of 2422 CDSs were detected, and consensus promotor logos were derived. Interestingly, many leaderless messenger RNAs (mRNAs) were found. In addition, many mRNAs were transcribed from multiple alternative TSSs. We observed pervasive intragenic TSSs both in sense and antisense to CDSs. Antisense transcripts were enriched near the 5′ end of mRNAs, indicating a functional role in post-transcriptional regulation. In total, 578 TSSs were detected in intergenic regions, of which 35 were identified as putative small regulatory RNAs. Finally, we provide a detailed analysis of the main copper resistance clusters in CH34, which include many intragenic and antisense transcripts. These results clearly highlight the ubiquity of noncoding transcripts in the CH34 transcriptome, many of which are putatively involved in the regulation of metal resistance.

Cdk9 and H2Bub1 signal to Clr6-CII/Rpd3S to suppress aberrant antisense transcription.

Mono-ubiquitylation of histone H2B (H2Bub1) and phosphorylation of elongation factor Spt5 by cyclin-dependent kinase 9 (Cdk9) occur during transcription by RNA polymerase II (RNAPII), and are mutually dependent in fission yeast. It remained unclear whether Cdk9 and H2Bub1 cooperate to regulate the expression of individual genes. Here, we show that Cdk9 inhibition or H2Bub1 loss induces intragenic antisense transcription of ∼10% of fission yeast genes, with each perturbation affecting largely distinct subsets; ablation of both pathways de-represses antisense transcription of over half the genome. H2Bub1 and phospho-Spt5 have similar genome-wide distributions; both modifications are enriched, and directly proportional to each other, in coding regions, and decrease abruptly around the cleavage and polyadenylation signal (CPS). Cdk9-dependence of antisense suppression at specific genes correlates with high H2Bub1 occupancy, and with promoter-proximal RNAPII pausing. Genetic interactions link Cdk9, H2Bub1 and the histone deacetylase Clr6-CII, while combined Cdk9 inhibition and H2Bub1 loss impair Clr6-CII recruitment to chromatin and lead to decreased occupancy and increased acetylation of histones within gene coding regions. These results uncover novel interactions between co-transcriptional histone modification pathways, which link regulation of RNAPII transcription elongation to suppression of aberrant initiation.

DNA Methylation and Histone H1 Jointly Repress Transposable Elements and Aberrant Intragenic Transcripts.

DNA methylation and histone H1 mediate transcriptional silencing of genes and transposable elements, but how they interact is unclear. In plants and animals with mosaic genomic methylation, functionally mysterious methylation is also common within constitutively active housekeeping genes. Here, we show that H1 is enriched in methylated sequences, including genes, of Arabidopsis thaliana, yet this enrichment is independent of DNA methylation. Loss of H1 disperses heterochromatin, globally alters nucleosome organization, and activates H1-bound genes, but only weakly de-represses transposable elements. However, H1 loss strongly activates transposable elements hypomethylated through mutation of DNA methyltransferase MET1. Hypomethylation of genes also activates antisense transcription, which is modestly enhanced by H1 loss. Our results demonstrate that H1 and DNA methylation jointly maintain transcriptional homeostasis by silencing transposable elements and aberrant intragenic transcripts. Such functionality plausibly explains why DNA methylation, a well-known mutagen, has been maintained within coding sequences of crucial plant and animal genes.

CRYPTIC TRANSCRIPTION IS ASSOCIATED WITH AGE IN MAMMALIAN STEM CELLS

Aging is a multifaceted process that challenges organisms with stresses resulting from the dysregulation of cellular processes. Unsurprisingly, given how tightly regulated it is under normal conditions, transcription is one of the key pathways disrupted during aging. Indeed, dysregulation of transcription contributes to the activation of transposable elements, the loss of cellular identity, and decreased stem cell potency with age. Our previous work identified intragenic cryptic transcription (CT) as a novel type of age-associated transcriptional dysregulation that limits the lifespan of yeast and worms. Continuing this work, we show for the first time that CT increases with age in mammalian stem cells. Increased CT is associated with disrupted chromatin structure, particularly with the reduction of H3K36me3, a histone modification known to inhibit CT throughout eukaryotes. We propose that an age-associated reduction in H3K36me3 in actively transcribed gene bodies drives disruption of chromatin structure in these regions, resulting in an open chromatin state. This open chromatin state is permissive for the entry of RNA Pol II, which can then initiate transcription from within the gene body. These aberrant cryptic transcripts may contribute to the pathological load of mammalian aging.

Abstract P5-05-08: Molecular cross-talk between retinoic acid and NOTCH1 signaling pathways: Role in triple negative breast cancer

Abstract Triple Negative Breast Cancer (TNBC) represents 10-20% of all breast cancers and it is characterized by poor prognosis and high recurrence rate. The heterogeneity of the disease and the absence of well-defined molecular targets have so far challenged successful therapeutic strategies. NOTCH1 has been found to act as a driver oncogene in a small subset of TNBC characterized by constitutive activation of the protein, acting as a transcription factor. Preclinical studies support an anti-tumor activity of All-Trans Retinoic Acid (ATRA) in specific subsets of breast cancer. By screening a large panel of breast cancer cell lines recapitulating the heterogeneity of TNBC we identify a specific subset sensitive to the anti-proliferative activity of ATRA. These cell lines (N-TNBC cell lines) are characterized by a Notch1 intragenic fusion transcript conferring gain of function activity to the protein. Indeed, sequence analysis reveals that the cell lines harbor an interstitial deletion in the NOTCH1 gene encompassing the negative regulatory region (NRR) domain. These cell lines depend on Notch active signaling for their proliferation since their cell growth is impaired by Notch inhibitors (γ-secretase inhibitors, e.g. DAPT, PF-3084014). Proliferation assays reveal that ATRA and γ-secretase inhibitors act synergistically in inhibiting cancer cell growth in N-TNBC cell lines suggesting the existence of a cross talk between the molecular pathways engaged by retinoic acid and NOTCH1. By using retinoic acid receptors (RARs) agonists and antagonists as well as RAR specific silencing experiments we identify RARα as the retinoic acid receptor responsible of the anti-proliferation activity of ATRA in N-TNBC cell lines. In particular N-TNBC cell lines respond to RARα activation by inducing high amounts of the onco-supressor protein RARβ. This feature is unique in ATRA sensitive TNBC cell lines and does not occur in ATRA sensitive luminal cell lines arguing for the existence of a retinoic acid specific mechanism of action in N-TNBC. Since RARs act as transcription factors inside the cells, to gain insights into the molecular pathway at the basis of the observed ATRA/NOTCH1 cross talk, we performed RNAseq analysis of ATRA and/or DAPT treated N-TNBC cells. Gene set enrichment analysis reveal that ATRA is able to directly affect NOTCH1 transduction pathway by modulating the expression of NOTCH1 target genes. In particular, in two out of three N-TNBC cell lines ATRA directly inhibits the NOTCH1 expression at a transcriptional level and its downregulation is increased by ATRA/DAPT combinations. Pathway analysis has allowed the identification of putative molecular hubs responsible for the synergistic effects observed and therefore likely at the basis of the crosstalk between ATRA/NOTCH pathways. These findings are of clinical interest since both the retinoid and NOTCH signaling display crucial physiologic activities and their pleiotropic effects could impinge on the success of therapeutic options based on their pathway modulation. The newly discovered specificity of ATRA action in the context of NOTCH1 addicted TNBC provides new tools for the identification of patients candidates benefitting from strategies targeting the ATRA/NOTCH axis. Citation Format: Paroni G, Zanetti A, Bolis M, Vallerga A, Troiani M, Fratelli M, Kurosaki M, Terao M, Garattini E. Molecular cross-talk between retinoic acid and NOTCH1 signaling pathways: Role in triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-05-08.

Casein kinase 2 mediated phosphorylation of Spt6 modulates histone dynamics and regulates spurious transcription.

CK2 is an essential protein kinase implicated in various cellular processes. In this study, we address a potential role of this kinase in chromatin modulations associated with transcription. We found that CK2 depletion from yeast cells leads to replication-independent increase of histone H3K56 acetylation and global activation of H3 turnover in coding regions. This suggests a positive role of CK2 in maintenance/recycling of the histone H3/H4 tetramers during transcription. Interestingly, strand-specific RNA-seq analyses show that CK2 inhibits global cryptic promoters driving both sense and antisense transcription. This further indicates a role of CK2 in the modulation of chromatin during transcription. Next, we showed that CK2 interacts with the major histone chaperone Spt6, and phosphorylates it in vivo and in vitro. CK2 phosphorylation of Spt6 is required for its cellular levels, for the suppression of histone H3 turnover and for the inhibition of spurious transcription. Finally, we showed that CK2 and Spt6 phosphorylation sites are important to various transcriptional responses suggesting that cryptic intragenic and antisense transcript production are associated with a defective adaptation to environmental cues. Altogether, our data indicate that CK2 mediated phosphorylation of Spt6 regulates chromatin dynamics associated with transcription, and prevents aberrant transcription.

The evolutionary impact of intragenic FliA promoters in proteobacteria.

In Escherichia coli, one sigma factor recognizes the majority of promoters, and six 'alternative' sigma factors recognize specific subsets of promoters. The alternative sigma factor FliA (σ28 ) recognizes promoters upstream of many flagellar genes. We previously showed that most E. coli FliA binding sites are located inside genes. However, it was unclear whether these intragenic binding sites represent active promoters. Here, we construct and assay transcriptional promoter-lacZ fusions for all 52 putative FliA promoters previously identified by ChIP-seq. These experiments, coupled with integrative analysis of published genome-scale transcriptional datasets, strongly suggest that most intragenic FliA binding sites are active promoters that transcribe highly unstable RNAs. Additionally, we show that widespread intragenic FliA-dependent transcription may be a conserved phenomenon, but that specific promoters are not themselves conserved. We conclude that intragenic FliA-dependent promoters and the resulting RNAs are unlikely to have important regulatory functions. Nonetheless, one intragenic FliA promoter is broadly conserved and constrains evolution of the overlapping protein-coding gene. Thus, our data indicate that intragenic regulatory elements can influence bacterial protein evolution and suggest that the impact of intragenic regulatory sequences on genome evolution should be considered more broadly.