Drosophila melanogaster, but it is now considered as a general feature of this peculiar portion of the genomes. The phenomenon of TE enrichment in constitutive heterochromatin has been proposed to be the consequence of a progressive accumulation of transposable elements caused by both reduced recombination and lack of functional genes in constitutive heterochromatin. However, this view does not take into account classical genetics studies and most recent evidence derived by genomic analyses of heterochromatin in Drosophila and other species. In particular, the lack of functional genes does not seem to be any more a general feature of heterochromatin. Sequencing and annotation of Drosophila melanogaster constitutive heterochromatin have shown that this peculiar genomic compartment contains hundreds of transcriptionally active genes, generally larger in size than that of euchromatic ones. Together, these genes occupy a significant fraction of the genomic territory of heterochromatin. Moreover, transposable elements have been suggested to drive the formation of heterochromatin by recruiting HP1 and repressive chromatin marks. In addition, there are several pieces of evidence that transposable elements accumulation in the heterochromatin might be important for centromere and telomere structure. Thus, there may be more complexity to the relationship between transposable elements and constitutive heterochromatin, in that different forces could drive the dynamic of this phenomenon. Among those forces, preferential transposition may be an important factor. In this article, we present an overview of experimental findings showing cases of transposon enrichment into the heterochromatin and their positive evolutionary interactions with an impact to host genomes.Transposable elements (TEs) are abundant components of constitutive heterochromatin of the most diverse evolutionarily distant organisms. TEs enrichment in constitutive heterochromatin was originally described in the model organism  Transposable elements (TEs) represent a significant fraction of the eukaryotic genomes, and this fraction is often referred to as the “mobilome” . The mobEukaryotic TEs are classified on the basis of their structure and transposition mechanism into two main classes . Class IIn this review, we resume the current literature on the role of TE insertions connected to the structure and function of the heterochromatin compartment of the eukaryotic genomes.Drosophila [Constitutive heterochromatin is an almost ubiquitous component of eukaryotic genomes  aosophila , and C-bosophila  genomic osophila  are alsoDrosophila suggested evidence of a skewed distribution of TE insertions in the heterochromatin compared to euchromatin [The pervasive presence and accumulation of TEs is a hallmark of heterochromatin. A build-up of TEs in this genomic region has been documented in a large number of evolutionarily distant organisms  are known to contain B chromosomes that are usually riddled with TEs and display a heterochromatic state [An extreme and intriguing example of genomic entities accumulating TEs are B chromosomes ,30. B chic state .Drosophila melanogaster has been previously discussed in detail [The accumulation of TEs in constitutive heterochromatin of n detail ,72,73,74D. melanogaster [Drosophila and other species. Indeed, sequencing and annotation have shown that this peculiar genomic compartment contains hundreds of transcriptionally active genes [D. melanogaster constitutive heterochromatin [The dispersed nature of TEs in the euchromatin can be the cause of chromosomal aberrations generated through ectopic recombination between homologous TE copies  or by trnogaster ,74, as wnogaster ,81. Howeve genes ,83, whicAn alternative hypothesis explaining TE accumulation in constitutive heterochromatin relies on the presence of hotspots for TE insertions. In the I-R hybrid dysgenesis, where the LINE-like I factor undergoes retrotransposition ,85,86, hAt least some TEs belonging to the Chromoviridae, a lineage of the Metaviridae family , expressTy5 retrotransposon of S. cerevisiae integrates at the telomeres and the mating type loci, two transcriptionally silent genomic regions [Ty5 integrase with the Sir4 protein led to a biased integration of the retrotransposon [While the integration bias of many transposable elements has been documented, the preference for heterochromatin is less understood. Studies in plants revealed that while members of the Pseudoviridae family insert randomly, elements of the Metaviridae family preferentially target heterochromatin , confirm regions . The intansposon ,97.Skipper-1, a LTR-retrotransposon of D. discoideum, co-localizes with the centromeric histone variant cenH3 [Skipper-1 is an active CHD-mediated process or an indirect effect resulting from the loss of cells that integrate Skipper-1 in other gene-rich genomic regions.nt cenH3 , althougTy3/gypsy retrotransposons, the CR family, preferentially inserts into the centromeric satellite DNA and associates with the centromere-specific protein CenH3, suggesting an active role in the centromere function and kinetochore formation [In plants, a specific family of ormation ,40.However, many other transposon types display a biased insertion pattern toward heterochromatin.Tetrahymena thermophila [The domesticated transposase Tpb2pn evolved from the PB transposase  associatrmophila .1360 element in D. melanogaster [In some cases, the introduction of TE-related sequences in the integration cassette changes the integration pattern of the transgenes, as demonstrated for the nogaster , suggestEven the SB transposase, resurrected from a fish Tc1-like transposon , prefereTetraodon nigroviridis [Preferential accumulation in the heterochromatin of several TE families has been also observed in oviridis ,101. It oviridis  in the poviridis .D. melanogaster [As discussed in the previous section, the picture of constitutive heterochromatin as the silent part of the genomes should be profoundly reconsidered, at least in nogaster ,103. Indnogaster . D. melanogaster, TE copies are intimately associated with the heterochromatic genes’ body, both in the flanking and intronic regions [D. melanogaster heterochromatic genes [In  regions ,105,106.ic genes ,108, espic genes ,110.Thus, it may be possible that during evolution, TE sequences became functionally integrated within the genes in heterochromatin, acting as regulatory elements that drive gene expression by recruiting specific epigenetic factors, such as HP1 protein. D. melanogaster constitutes a relevant case study of epigenomic conflict. While heterochromatin contains genes that are expressed throughout the development and across tissues, it is tagged with repressive epigenetic marks, such as methylation of H3K9 and H4K20 [Indeed, constitutive heterochromatin of nd H4K20 , in addind H4K20 . Drosophila species. Studies performed in the last decades are consolidating a new perspective of constitutive heterochromatin on the basis of its transcriptional plasticity. Heterochromatin transcription indeed plays a critical role in establishing heterochromatin de novo in the daughter cell after mitosis completion [However, transcription of heterochromatic sequences is not limited to mpletion , as wellmpletion . AlthougExaptation  is an evD. melanogaster, which has been extensively discussed [Het-A, TART, and TAHRE [Very special examples of TE domestication associated with neofunctionalization are the telomere maintenance in iscussed ,60,61. Tnd TAHRE , that avDrosophila genus, this recalls a theory on the telomere origin that suggests that group II introns  could have originated the ancestral eukaryotic telomeres, allowing the formation of primitive t-loops [While this example seems to be limited to the species of the  t-loops , suggestDrosophila telomeric TEs, there is no reported function for the presence of TEs in the telomeres of other species, suggesting an accumulation resulting from the absence of selective pressure at these loci.TE enrichment in telomeric and sub-telomeric regions has been described in diverse species of fungi , vertebrNotably, members of the Athena clade of the Penelope-like retrotransposons identified in Rotifer (Bdelloidea) lack the endonuclease domain, contain short stretches of telomeric repeats at their 3′ end, and are preferentially oriented toward the telomere with their 5′ truncated end .The centromere is the major locus buried in the constitutive heterochromatin. The hallmark of the centromeric DNA, virtually in all eukaryotic chromosomes, is the enrichment in satellite DNA, but very often centromeres are associated with a high frequency of TE insertions that built up the architecture of such complex chromosomal structures  Table 1Table 1.pogo-like transposase [One of the well-known cases of TE exaptation connected to the centromere function is the CENP-B protein . CENP-B nsposase ,126 and nsposase ,127. Thensposase .Whether TEs play a pivotal role in either establishing a functional centromere, evolving new centromeres from scratch, or generating new satellite DNAs are still unsolved questions.D. melanogaster was first highlighted using combined sequencing and chromosomal deletions analyses [Drosophila centromeres range between approximately 200 and 500 kb in size [G2/Jockey-3 retroelement is the most highly enriched sequence in CENP-A chromatin, and it is shared among all centromeres. Since this feature is somehow conserved in related species with divergent centromeric satellites, these results strongly suggest a conserved role of retroelements in centromere specification and function in Drosophila.The massive presence of TEs in the centromeric DNA of analyses . This st in size  and are  in size ,130. Rec in size , by mappDictyostelium dyscoideum centromeric DNA is composed of LTR retrotransposons [The massive occupancy of TEs in the centromeric and pericentromeric DNA regions of the chromosome is also a shared feature in other species. An 86% fraction of the 170–360 Kbp long nsposons . Nearly nsposons .Human centromeric and pericentromeric regions also appear to constitute “soft-landing” platforms for TEs insertions. Indeed, a recent investigation of TE insertions in 5675 genomes has revealed that the preferred insertion sites of LINE elements lay within centromeric DNA . HoweverCryptococcus genus, the presence of full-length TEs is observed only in species with long centromeres and in which the RNAi process is active . By contrast, C. deutereogatti, which lacks the RNAi pathway, has short centromeres void of active TEs [A possible explanation of the target preference and accumulation of TEs in the centromeric DNA comes from studies in fungi. In three species of the pathogenic basidiomycete tive TEs . Since ative TEs  it has bThe amplification of pre-existing TE copies in the constitutive heterochromatin may also have contributed to the birth of pericentromeric repeats.Aegilops speltoides, the 250 bp centromeric satellite shares high similarity to portions of a Ty3/gypsy-like retrotransposons [A. thaliana [A. lyrata [D. virilis [In the plant nsposons . TEs hom. lyrata , as well virilis , and in  virilis .The compact organization of the centromere 8 in rice variety Nipponbare, which contains 65 Kbp of repeats , and theDrosophila, with at least two well-studied examples involving Class II TEs. The first example concerns tandem repeats of the P-element, which are frequently generated during genetic screens in D. melanogaster [D. guanche [P-element tandem repeats are formed by double insertion at the same site [Bari1 element, which is arranged as a regular tandem repeat in the heterochromatin [Bari1 clusters in the deep heterochromatin of D. melanogaster, mapping on the second and the X chromosomes correspondingly [The ability of TEs to form tandem repeats is well documented in nogaster  and natu guanche . It has ame site . The sechromatin . Rollinghromatin . This meondingly ,57.Drosophila [In this context, it has been suggested that satellite DNA can be generated from transposons following different routes. One is the accumulation of TEs that can be detected as long stretches of DNA sequences similar to TEs. The observed sequence similarity between some TEs and satellite DNA in several species is indeed one of the main observations that has led to the intriguing hypothesis that satellite DNA may have derived from TEs. This suggestion is supported by observations in osophila  and humaosophila . Alternaosophila , which cParamecium species, the domesticated PiggyBac transposase PiggyMac (Pgm) protein [In the ciliate  protein ,65 and t protein  are invoArabidopsis, genetic loss of two genes MAIL1 and MAIN result in impaired condensation of pericentromeric heterochromatin and upregulation of TE transcription, suggesting a transcriptional repression role. The proteins encoded by MAIL1 and MAIN appear to be derived from a subset of Ty3/gypsy retrotransposons found in angiosperms [In iosperms .An additional role of TEs in the heterochromatin appears to be related to the embryo development. In a recent study, the upstream region of retrotransposons belonging to few active families was identified as the nucleation sites of heterochromatin formation during early stages of embryo development in the fly ,143, andBeing habitual residents of heterochromatin, TEs are obviously linked to human diseases related to heterochromatin defects, since loosening in heterochromatin compaction can cause the de-repression of TEs, thus leading to deleterious physiological changes such as aging, cancer, and neurological disorders. Although it is difficult to establish the direct link between heterochromatic TE copies and the disease itself, some recent papers suggest that this could be the case.It is well established that the conformation of the chromatin in the interphase nucleus has a physiological relevance to cell life. In this view, any perturbation of the chromatin organization in the 3D nucleus can contribute to cell response to environmental stress or to the onset of diseases. Laminopathies are heterogeneous diseases caused by dysfunction of the LAMIN proteins that ensure the correct anchoring of heterochromatin to the nuclear periphery .Heterochromatin relaxation and TE de-repression have been related to the onset of ALS  and tauoDrosophila tauopathy model, Tau dysregulates TEs resident in the heterochromatin, reinforcing the above-described observation in mammals [In a  mammals .Additionally, chromatin undergoes global chromatin compaction upon activation of cell motility in several cell types of cancer metastasis .Similarly, an aging-dependent loss of heterochromatin induces a dysregulation of TEs that can in turn produce genome instability and activation of inflammatory responses .Although more experimental evidence is needed, there is a clear connection between the role of heterochromatic copies of TEs and the onset of pathological phenotypes.About 80 years have passed since Barbara McClintock published her results on the controlling elements in maize, which were considered very controversial at that time by the scientific community. Today, in the post-genomic era, due to the huge amount of genome sequences, transposable elements are still arousing a large amount of interest in the field of genome evolution. In particular, transposable elements are no longer considered only a mere example of genomic parasites, but it is widely recognized that they have colonized all eukaryotic genomes and represent a major force driving the evolution of organisms. The development of new genome sequencing methods and annotation protocols could enforce this vision, opening the possibility of comparing constitutive heterochromatin in thousands of eukaryotic organisms. Such studies are needed to shed light on the dynamics of TE accumulation in the heterochromatin and to resolve the role of TEs in the determination of one of the most elusive chromosome structures, i.e., the centromere. Large-scale comparative analysis will also help determine the impact of TEs on heterochromatic gene expression and to disentangle the complex evolutionary trajectories that lead to TE exaptation and domestication. Since many of these issues are (or they will be) related to human health/diseases, the role of TEs in the constitutive heterochromatin is likely to be even more substantial than we may now imagine, and a multiplicity of their roles and impact on cellular functions and genome evolution will come to light.


As in the prenatal stages, the postnatal period of neurodevelopment is also sensitive to environmental inputs. Often this sensitivity is reflected in children facing adverse conditions, such as maternal separation. This exposure to early life stress (ELS) is implicated in the manifestation of various behavioral abnormalities. Most FASD research has focused exclusively on the effect of prenatal ethanol exposure in isolation. Here, we review the research into the effect of prenatal ethanol exposure and ELS, with a focus on the continuum of epigenomic and transcriptomic alterations. Interestingly, a select few experiments have assessed the cumulative effect of prenatal alcohol and postnatal maternal separation stress. Regulatory regions of different sets of genes are affected by both treatments independently, and a unique set of genes are affected by the combination of treatments. Notably, epigenetic and gene expression changes converge at the clustered protocadherin locus and oxidative stress pathway. Functional studies using epigenetic editing may elucidate individual contributions of regulatory regions for hub genes and further profiling efforts may lead to the development of non-invasive methods to identify children at risk. Taken together, the results favor the potential to improve neurodevelopmental outcomes by epigenetic management of children born with FASD using favorable postnatal conditions with or without therapeutic interventions.Neurodevelopment in humans is a long, elaborate, and highly coordinated process involving three trimesters of prenatal development followed by decades of postnatal development and maturation. Throughout this period, the brain is highly sensitive and responsive to the external environment, which may provide a range of inputs leading to positive or negative outcomes. Fetal alcohol spectrum disorders (FASD) result from prenatal alcohol exposure (PAE). Although the molecular mechanisms of FASD are not fully characterized, they involve alterations to the regulation of gene expression  Also included in the scope of the epigenome are ncRNAs that act at the post-transcriptional level to interfere with the translation of mRNA into proteins, such as microRNAs (miRNAs).TET1 deficiency impairs neurogenesis and leads to the downregulation of neural progenitor proliferation genes , is critical for appropriate synapse formation and transmission through the regulation of genes such as the neurotransmitter receptor Htr5b  and the genome. It enables prenatal and postnatal events to shape neurodevelopment and related behavioral endophenotypes. At each stage of neurodevelopment, epigenetic players are at work. Neurogenesis relies on intergenic de novo DNA methylation by DNMT3A in neural stem cells to maintain active chromatin states for essential genes  are deregulated in the adult mouse brain , indicating that DNA methylation changes occur after PAE  at both the gene , together with adhesion and polarity genes DiGeorge syndrome critical region gene 2 (Dgcr2), and Par-6 family cell polarity regulator alpha , POU class 4 homeobox 3 (Pou4f3), and SRY-box transcription factor 7 (Sox7), are each hypomethylated following ethanol treatment  mRNA following PAE was accompanied by increased promoter methylation  alongside decreased promoter methylation  polycomb group ring finger 6 (Pcgf6), and ring finger protein 1 (Ring1), as well as transcription factor II D complex members general transcription factor II A, 2 (Gtf2a2), and TATA-box binding protein associated factors 8, 10, and 11 , nuclear transcription factor-Y gamma (Nfyc), peroxisome proliferator activator receptor delta (Ppard), and nuclear receptor subfamily 1, group H, member 4 and group D, member 2 , unc-51-like kinase 4 (Ulk4), disabled 2 interacting protein (Dab2ip), and rho/rac guanine nucleotide exchange factor (GEF) 2 (Arhgef2). Conversely, the combination of PAE and ELS led to increased promoter methylation of genes important for immune response, including 21 members of the cytokine-cytokine receptor interaction KEGG pathway  at the Pcdhb2 promoter and a corresponding decrease in gene expression (fold change = −1.23, p < 0.05; Laufer et al., Protocadherins are a group of cell-cell adhesion molecules that are highly expressed in the brain and are involved in establishing single-cell neuronal identity. Many of these protocadherins are encoded in a large genomic locus that consists of three multi-gene clusters (Chen and Maniatis, The clustered protocadherins have also been implicated in ELS, where a comparison of the hippocampal tissue from individuals that have experienced abuse as children and rats that received low levels of maternal care revealed DNA promoter hypermethylation and gene body DNA hypomethylation at the locus, corresponding with a repressive effect (Suderman et al., In an FASD model that includes PAE and ELS in adult mouse hippocampus both hyper- and hypo-methylation in the protocadherin region as well as decreased gene expression resulted from PAE (Alberry and Singh, Oxidative stress is a well-characterized part of FASD etiology. At the cellular level, ethanol acts directly on mitochondria to produce superoxide, hydroxide, and nitric oxide radicals (Wu and Cederbaum, via epigenetic manipulation.FASD is a common neurodevelopmental disorder that represents a societal burden with no effective treatments. Yet, it is entirely preventable. It requires the avoidance of alcohol before and during pregnancy. This is a challenging proposition given that the use of alcohol in reproductive-age women is increasing and many pregnancies are unintended, which is expected to lead to a corresponding increase in FASD (Alberry and Singh, Hoxa1 and its miRNA, miR-10a (Wang et al., Dnmt1 and Mecp2 in the hypothalamus (Bekdash et al., Set7/9, G9a, and Setdb1, as well as the detected changes in histone PTMs, H3K4me2, 3 and H3K9me2 (Bekdash et al., POMC promoter methylation and corresponding decreased gene expression in the hypothalamus (Bekdash et al., To this end, research regarding the prevention of FASD has centered on increasing the availability of methyl donors during development through supplementation of known methyl donors such as folate and choline. In an embryonic cell culture model, co-administration of folic acid prevents the morphological delays and deficits, together with the downregulation of Further consideration should also be paid to the types of experiments used in FASD research. To date, most studies have used bulk tissue or whole embryo inputs for molecular experiments. These have provided many valuable insights to this point, however, given the heterogeneity of the effects of PAE and preferential apoptosis of specific cell types, more focused studies are needed. Specifically, single-cell epigenetic and transcriptomic techniques are now viable for most FASD models. Single-cell or single-cell-type resolution may address many outstanding questions about the outcomes of ethanol exposure at the molecular level. Furthermore, single-cell studies and improved bioinformatic cellular deconvolution methods will aid future translational studies examining the DNA methylation signature of FASD in saliva (Turinsky et al., Neurodevelopment in mammals is a long-lasting, highly orchestrated, and coordinated continuum. It is highly sensitive and responsive to environmental exposures that may determine the outcome and resulting brain function, including neurodevelopmental disorders such as FASD. FASD is highly heterogeneous and a direct result of maternal alcohol consumption during pregnancy. Further, a stressful postnatal environment may worsen the outcomes. This review emphasizes the role of dynamic epigenetic changes during the neurodevelopmental continuum, from prenatal to postnatal stages in the development of FASD, including ncRNAs, DNA methylation, and histone PTMs. We argue that FASD is an epigenetic disorder. Additionally, the early postnatal environment may determine the severity of the effect of PAE. It may be possible to moderate this effect by postnatal epigenetic modifications, which may or may not be driven by pharmaceutical interventions. This perspective offers new hope for the development of effective treatment for FASD. Finally, the strategy of epigenetic intervention discussed here may apply to a variety of neurodevelopmental disorders involving epigenetic marks caused by environmental exposures.All authors contributed to the design and organization of this review. All authors performed a literature review, as well as interpretation and synthesis of findings. BA created the first draft, with all authors substantially contributing to revisions. All authors have approved the final revisions prior to submission. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hi-C and its high nucleosome resolution variant Micro-C provide a window into the spatial packing of a genome in 3D within the cell. Even though both techniques do not directly depend on the binding of specific antibodies, previous work has revealed enriched interactions and domain structures around multiple chromatin marks; epigenetic modifications and transcription factor binding sites. However, the joint impact of chromatin marks in Hi-C and Micro-C interactions have not been globally characterized, which limits our understanding of 3D genome characteristics. An emerging question is whether it is possible to deduce 3D genome characteristics and interactions by integrative analysis of multiple chromatin marks and associate interactions to functionality of the interacting loci.robC to decompose Hi-C and Micro-C interactions by known chromatin marks. ProbC is based on convex likelihood optimization, which can directly take into account both interaction existence and nonexistence. Through ProbC, we discover histone modifications  and CTCF as particularly predictive of Hi-C and Micro-C contacts across cell types and species. Moreover, histone modifications are more effective than transcription factor binding sites in explaining the genome’s 3D shape through these interactions. ProbC can successfully predict Hi-C and Micro-C interactions in given species, while it is trained on different cell types or species. For instance, it can predict missing nucleosome resolution Micro-C interactions in human ES cells trained on mouse ES cells only from these 5 chromatin marks with above 0.75 AUC. Additionally, ProbC outperforms the existing methods in predicting interactions across almost all chromosomes.We come up with a probabilistic method PVia our proposed method, we optimally decompose Hi-C interactions in terms of these chromatin marks at genome and chromosome levels. We find a subset of histone modifications and transcription factor binding sites to be predictive of both Hi-C and Micro-C interactions and TADs across human, mouse, and different cell types. Through learned models, we can predict interactions on species just from chromatin marks for which Hi-C data may be limited. The accumulating evidence suggests that 3D nuclear architecture is important for the gene expression regulation, key element in transcriptional regulation , and it Multiple chromosome conformation capture experimental techniques, for instance Hi-C, have remarkably improved our comprehension of 3D chromatin structure . Among tSimilarly, Micro-C is a Hi-C based method where micrococcal nuclease is used instead of restriction enzymes to fragment chromatin, enabling nucleosome resolution chromosome folding maps. Chromatin is fragmented to mononucleosomes, thereby increasing both fragment density as well as uniformity of spacing. Crucially, Micro-C overcomes the current resolution gap of Hi-C at the fine scale, which now allows us to investigate more detailed chromatin structures. Hi-C and Micro-C technologies have led to the intuitive observation of the genome structure of human and mouse , 8, and Epigenetics is quite important in understanding the basic cellular pathways and processes occurring in chromatin . Even though Hi-C does not directly depend on the binding of specific antibodies, previous analysis has revealed enriched interactions and domain structures around multiple chromatin marks; histone modifications and transcription factor binding sites (TFBSs) , 10. IntDespite such analyses, the ability of histone modifications affect 3D genome shape through the binding sites remains poorly understood across species, cell types, and cell cycles. This can be partially due to the fact that the previous research on the relationships between histone modifications and genomic structures have frequently considered each modification independently, ignoring their joint quantitative effects. However, it has been previously shown that multiple modifications are jointly important in genome shape. For instance, both H3K4me3 and H3K27me3 are known to be effective in TAD formation , 15 whicROBC to decompose genome-wide Hi-C and Micro-C interactions by chromatin marks. PROBC estimates interaction probabilities between chromatin marks based on convex likelihood optimization, which can directly take into account both the interaction existence and nonexistence. Through PROBC, we systematically find 4 histone modifications  and CTCF to be highly predictive of most Hi-C and Micro-C interactions across cell types and mammals when considered together. Similarly, we also identify subset of transcription factor binding sites to be highly predictive of Hi-C and Micro-C interactions. We complete the missing Hi-C and Micro-C interactions and predict intra-chromosomal interactions at a very high resolution. Identified sparse set of chromatin marks account for a large proportion of the accuracy of Hi-C prediction, matching with their known roles, which fail to predict Hi-C and Micro-C interaction when considered independently. These marks and interaction probabilities between them inferred by PROBC can also be interpreted as latent biases in Hi-C and Micro-C experiments. We also discover that chromatin marks are conserved across cell types and species in a robust way: PROBC trained on embryonic stem cells work on IMR90 cells quite accurately, and PROBC trained on mouse keep working well on human. However, we also interpret our cross-species and cross-cell types results carefully as suggested in , v∈R} ∈M2} be the symmetric matrix of interaction probabilities between mark pairs. We assume these interaction probabilities between mark pairs X to be same across all restriction sites which is a global characteristic of genome embedding rather than being a local feature. This assumption is discussed in previous studies N[v] is large and xmncmcn is small. When replaced P(Euv|X) in Eq. where mentclass2pt{minimWe utilize Hi-C data from human IMR90 cells  and humaROBC in Python, and solve several parts of optimization by L-BFGS method. Datasets and code are available at http://www.github.com/seferlab/probc. PROBC is reasonably fast: We can estimate interaction probabilities between marks in less than 15 minutes on human IMR90 cells even without binning. We follow fivefold nested cross-validation to prevent overfitting and optimize for regularization hyperparameters. When we train and test on different chromosomes, the outer step trains PROBC on all chromosomes except the chromosome to be predicted in fivefold nested cross-validation. Within each loop of outer step, we estimate the regularization hyperparameters by utilizing fourfold inner cross-validation. When we train and test on the same chromosome, we apply similar fivefold nested cross-validation on the single chromosome.We implement PIf we forbid the interactions between chromatin marks, we find that only 4 histone modifications  and CTCF out of total 32 chromatin marks  are sufficient to explain the major proportion of Hi-C interactions in human ES cells. This is correct for both unbinned and 5 kb binned cases. These essential set of chromatin marks are significantly conserved when this procedure is repeated across mouse ES and human IMR90 cells with varying number of chromatin marks. Table Among the most important 5 chromatin marks, H3K4me1, H3K27ac are enhancer-specific marks. H3K9me3 is part of heterochromatin and is known to have repressive roles, whereas H3K4me3 is an activating mark. The same subset of histone modifications are important for human IMR90 cells as well, where activating mark H3K9ac and H3K36me3 that is associated with active gene bodies and elongation are also part of the solution. CTCF regulates the 3D structure of chromatin, and it is known to have roles in forming chromatin loops, TADs. It also defines the boundaries between active and heterochromatic DNA . Among tROBC, where λ1=2.5 and λ2=1.0 are found by fivefold nested cross-validation for genome-wide human ES cells. These parameters are important as otherwise PROBC assigns non-zero probabilities to all mark pairs. Previously identified 4 histone modifications have the highest interaction probabilities within themselves in explaining the given Hi-C interactions for human ES cells. Additionally, the interaction probabilities between H3K9ac-H3K4me2, H3K27me3-H3K27me3, H3K4me1-H3K4me1, and H3K9me3-H3K9ac modifications are also high. Both H3K9ac, H3K4me2 are known to exhibit activator features whereas H3K27me3 is known for its repressive characteristics. Results are similar for human IMR90 cells.Figure According to the dendrogram in Fig. ROBC reveals CTCF, RAD21, DNase I, TBP, POLRA2, Suz12, EZH2 in explaining the given Hi-C graph which genome-wide probabilities are shown in Fig. We also examine the independent effect of transcription factor binding sites in human ES cells. PAmong interactions between transcription factor binding sites, RAD21 and SMC3 interaction is highly probable. Both proteins are part of Cohesin complex, and SMC3 is present in all cohesin complex whereas there are multiple paralogs for RAD21. RAD21 is an evolutionarily conserved in all eukaryotes from budding yeast to human. This interaction is also highly probable in human IMR90 cells. Similarly, high probability interaction between TBP binding sites also suggests the importance of binding to TATA box in genome shape formation. Interestingly, CTCF-JUND and CTCF-MAFK interactions are also highly probable which do not belong to the same protein complex. Roles of both MAFK and JUND are different than CTCF: MAFK is basic region and leucine zipper (bZIP)-type transcription factor, whereas JUND protein is a member of the JUN family, and a functional component of the AP1 transcription factor complex. These interactions need to be carefully examined to further develop novel insights about genome shape formation.ROBC can detect false positive interactions and predict novel Hi-C interactions. In such experiments, fivefold nested cross validation is applied independently to each chromosome. We first predict Hi-C interactions from previously identified H3K27ac, H3K9me3, H3K4me3, H3K4me1, and CTCF. Then, we obtain ROC curves by decreasing the Hi-C interaction probability threshold from 1 to 0. According to the pairwise comparison matrix in Fig. ROBC performance declines but it is still reasonable when we train it on one chromosome and test on a different chromosome. As an example, we obtain ROC AUC score of 0.86 when we train with Hi-C interactions on the 6’th chromosome and predict interactions on the 4’th chromosome. The similarity of the chromatin marks found as important across chromosomes suggests the similarity of main properties controlling chromosomal contact formation. Although, there can still be close-grained differences which have not been caught by PROBC.On human ES cells, PROBC outperforms random-forest based approach HiC-Reg, and convolutional neural network-based approach Rambutan for most of the intra-chromosomal interaction prediction in terms of ROC AUC score on human ES cells as in Fig. ROBC with EpiTensor as EpiTensor constructs interactions jointly across multiple species from chromatin datasets, whereas the other methods are based on individual species. While comparing with HiC-Reg in terms of ROC AUC, we divide each count value by the sum of the counts to normalize the HiC-Reg output. The performance difference is the most apparent in chromosome 4. The outperformance of PROBC still exists for human IMR90 cells. Additionally, both Spearman and Stratum-adjusted correlation coefficient [ROBC and HiC-Reg, EpiTensor, Rambutan performances is moderate, showing the impact of the difference between the nonlinear approaches these methods are based on. Variables identified as important by HiC-Reg and PROBC overlap. For instance, H3K27ac, H3K9me3, and H3K4me3 also appear to be important according to out of bag (OOB) variable importance measure, that measures the change in OOB error by permuting the feature values. Rambutan performs slightly better than HiC-Reg as it additionally uses nucleotide sequence data. However, in terms of other datasets, it is limited to Dnase I signal.Pfficient  between ROBC predictions with multiple distance decay-based prediction baselines as seen in Fig. u and v is inversely related to their genomic distance. In more detail, P(u and v interact with each other)=αdβ where d is the genomic distance between segments u and v, and optimal α is learned over the training set. in our case, we test the performance for various β exponent values for variety of thresholds as in the figure. According to the figure, PROBC clearly outperforms all three distance decay baselines with β=1,1.5,2. Among the baselines, distance decay baseline with β=1.5 performs the best, but its AUC score is still remarkably lower than PROBC.We also compare PROBC’s performance for TAD and compartment domains prediction on human ES cells respectively. The performance is evaluated for only histone modifications, only transcription factor binding sites, and for combination of histone modifications and transcription factor binding sites. We compare the TAD and compartment domains prediction across different chromatin mark combinations by Normalized Variation of Information (NVI) [ROBC trained only with histone modifications outperforms the case it was trained only with transcription factor binding sites for both TAD and compartment domain prediction. However, the best TAD prediction performance is observed when we combine both mark datasets.In addition to Hi-C prediction, Fig. on (NVI) , which qon (NVI)  is used on (NVI)  has beenROBC followed by TAD prediction method Armatus can correctly predict 12.31% of TAD boundaries correctly. If we look into the false positive boundaries, we found most of them to be near the true TAD boundaries, which can also be inferred from lower NVI scores. The accurracy of TAD prediction increases if we make the matrices smaller by decreasing the resolution. For instance, we can predict 69.83% of TAD boundaries correctly at 100 kb resolution.We have also analyzed the breakdown of TAD prediction in terms of TAD boundaries as shown in Table ROBC indirectly models the chromatin marks contribution to three-dimensional shape via chromatin interactions, we have investigated whether PROBC can forecast modifications to 3D structure explained via perturbations over chromatin marks. We have especially focused on samples where significant chromatin marks are removed due to structural variations. Despang et al. [As Pg et al.  has inveROBC once we have perturbed the input CTCF dataset. We trained PROBC by using human ES cells dataset, and we made predictions across species in mouse ES cells. PROBC has normally predicted a robust TAD boundary between Sox9 and Kcnj2, putting these into separate TADs. Once we mask the four CTCF binding sites at the boundary and additionally mask the whole set of CFCF binding sites, results predicted by PROBC are consistent with the true experiments in which 2 separate TADs are slowly combined into a single TAD. Furthermore, we have investigated the association between CTCF binding sites and formation of TADs via feature attribution approaches. We have estimated SHAP values [We have analyzed whether those modifications in the TAD structure can be predicted by PP values , and aveROBC. Yang et al. [In another experiment, we have investigated whether changes in the structure due to genomic deletions can be predicted by Pg et al.  have foug et al.  has notiROBC. In this case, all chromatin marks corresponding to the deleted region were removed, and the remaining two partitions were added together. TADs inferred by PROBC are compatible with the true discoveries. Without 13q12.2 deletion, a smaller TAD segregation between PAN3 and FLT3 genes are predicted by PROBC, in line with the ground truth. However, once 13q12.2 is deleted, PROBC finds that 2 TADs are fused together and number of interactions between PAN3 and FLT3 increases.In our evaluatuon, simulation of this deletion is achieved by cutting out DS2 region from all chromatin marks input datasets and predicting the interaction matrix via PROBC trained with chromosome-wide human ES and mouse ES cells respectively. We apply fivefold cross validation to Hi-C, histone modifications and transcription factor binding sites datasets. Figure ROBC can predict inter-chromosomal interactions less accurately than the intra-chromosomal interactions. Similar prediction analysis on mouse ES cells while training PROBC over human ES chromosomes is shown in Fig. We predict Hi-C/Micro-C interactions on mouse ES and human ES chromosomes over PPrediction performance between species is lower than the performance between cell types on the same species, showing effective histone modifications may slightly differ across species. Results show the importance of species-specific interactions between histone modifications in genome shape, since the performance decreases even when modifications are transferred from a closer species.ROBC is robust to changes in sparsity parameter λ1 and low-rank parameter λ2 as seen in Fig. λ1 does not significantly decrease the quality of the predictions, while it leads Hi-C data to be explained by fewer chromatin marks. A subset of previously identified histone modifications  and CTCF appear in the solution when λ1≥5. On the other hand, increasing the low-rank parameter λ2 ensures block diagonal/communities structure of the mark interactions matrix, but decreases genome-wide performance especially when λ2≥10. Robustness of PROBC across parameters shows that the reported performance and generated insights in previous sections are quite reliable.PROBC regarding the similarity of the interaction probabilities inferred from two replicate Hi-C experiments that differ only in the choice of restriction enzyme. Specially, we apply PROBC to two enzyme replicates, Mbol and HindIII, carried out in human IMR90 cells. We evaluate the stability by Spearman correlation between the two pairwise interaction probabilities matrices of the pairs of histone modifications on each chromosome as in Fig. Hi-C experiments utilizes a restriction enzyme such as Mbol and HindIII to cleave the DNA after cross-linking, and different restriction enzymes can be used to analyze the sequence dataset multiple times for this purpose. Even though the resulting restriction fragments differ, the complete genome architecture and thus epigenetic and transcription marks should be the same from such replicate experiments. Therefore, we assess the stability of PSimilarly, our methods are stable to changes in resolution as seen in Fig. ROBC to optimally decompose Hi-C interactions in terms of these chromatin marks at genome and chromosome levels. Via our method, we find a subset of histone modifications and transcription factor binding sites to be predictive of Hi-C interactions and TADs across human, mouse, and different cell types. Given Hi-C data is still limited to certain species, accurate prediction of Hi-C interactions at a high resolution without integrating Hi-C data is mainly useful to analyze the 3D genome shape on such species. The similarity of the chromatin marks found as important across chromosomes suggests the similarity of main properties controlling chromosomal contacts. In summary, the analysis performed and all predictions made by PROBC in this work give good insights in exploring the 3D chromatin organization. We find that identified chromatin marks alone carry almost enough information to predict chromosomal structures.We explore the 3D genome organization by investigating how individual chromatin marks such as histone modifications and transcription factor binding sites as well as interactions between these chromatin marks explain Hi-C interactions. We come up with a novel probabilistic generative model-based method PROBC between marks might be relaxed as it is still open whether chromatin marks work differently depending on genome region. Lastly, we can extend PROBC to directly carry out a differential analysis on multiple tissues to predict differential interactions.In the future, we can extend the proposed method to recent multilocus chromatin interaction datasets by using a hypergraph instead of a graph. The assumption of same interaction probability in P


Pseudomonas aeruginosa (Pae) is an opportunistic pathogen showing a high intrinsic resistance to a wide variety of antibiotics. It causes nosocomial infections that are particularly detrimental to immunocompromised individuals and to patients suffering from cystic fibrosis. We provide a snapshot on regulatory RNAs of Pae that impact on metabolism, pathogenicity and antibiotic susceptibility. Different experimental approaches such as in silico predictions, co-purification with the RNA chaperone Hfq as well as high-throughput RNA sequencing identified several hundreds of regulatory RNA candidates in Pae. Notwithstanding, using in vitro and in vivo assays, the function of only a few has been revealed. Here, we focus on well-characterized small base-pairing RNAs, regulating specific target genes as well as on larger protein-binding RNAs that sequester and thereby modulate the activity of translational repressors. As the latter impact large gene networks governing metabolism, acute or chronic infections, these protein-binding RNAs in conjunction with their cognate proteins are regarded as global post-transcriptional regulators. RNA sequencing (RNAseq) has revolutionized the discovery of regulatory RNAs in bacteria . Sr. SrPae spagL gene, encoding the lipid A 3-O deacylase (cystic fibrosis (CF) patients [pagL mRNA in an Hfq-independent manner [The sRNA Sr006 was originally annotated during a single nucleotide resolution transcriptome analysis . A GRILseacylase  52]. Th. ThpagL patients ,77,78. St manner . As a cot manner ,80 and pt manner .E. coli sRNA RyhB . A. Aregula of ReaL .Pae . F. FrgsA iy in Pae , and wasy in Pae .Pseudomonas anaerobically induced RNA I) was identified in the intergenic region between the genes PA14_13970 and PA14_13990 in strain PA14 . P. PPae [vs of PQS . The mecFernandez et al.  providedA recent study proposed a novel function for PhrS as a positive regulator of CRISPR-Cas immunity  141]. H. H141]. Apart from its role as a regulatory RNA, PhrS encodes a highly conserved small 37 aa peptide ,137, andPae features two comprehensive post-transcriptional networks, which are governed by Hfq/Crc [ Hfq/Crc ,142 and  Hfq/Crc , respectPseudomonads, can act in concert with the catabolite repression control protein Crc [Hfq belongs to the Sm/Lsm family of RNA-binding proteins, ubiquitously present in Eukaryotes, as well as in many Eubacteria and Archaea ,145. It tein Crc ,147,148.tein Crc  and virutein Crc ,150,151,tein Crc ,75,152. tein Crc , but alstein Crc ,151.5G-rich sequence [via polyU-tails of their rho-independent terminators [via AA(R)N triplets, wherein A is an adenine, R is a purine and N is any nucleotide. Although the A-rich stretches are predominantly found in mRNAs, several studies have shown that some regulatory RNAs also contain A-rich sequences which enable interaction with the distal surface of Hfq [Many sRNAs bind to the proximal side of Hfq through either an AUsequence ,156,157 minators ,159,160.minators ,163,164.minators ,166,167 e of Hfq ,169. In Pae [The CsrA/RsmA family of RNA-binding proteins governs virulence gene expression in pathogenic bacteria ,177,178,Pae ,179. MemPae ,183,184.Pae ,179,185.Pae ,187. AnaPseudomonas, the hierarchical assimilation of carbon sources is governed by the RNA chaperone Hfq [catabolite repression control protein Crc [carbon catabolite repression (CCR), Hfq and Crc form a repressive complex on target mRNAs, the encoded proteins of which are involved in uptake and/or utilization of carbon and nitrogen sources other than the preferred one [In rone Hfq  and the tein Crc ,195,196 rred one ,147,197.rred one  by bindirred one , therebyrred one . A recenrred one  and 244 rred one , respectrred one .proton motive force (PMF) [hfq deletion strain showed an increased pyocyanin production [crc deletion strain [Several transcriptome, proteome and metabolome analyses have implicated Hfq and Crc in the regulation of iron metabolism and transport ,171, as ce (PMF) ,199  ,149,150.oduction ,149,150 oduction  and biofoduction    b [193,20. putida .carbon storage regulator CsrA, which primarily serves as a translational repressor and pleiotropic regulator of various processes in E. coli [rsmA and rsmN/F causes additional phenotypes when compared with a single rsmA deletion [The RNA-binding proteins RsmA  and RsmN E. coli ,216,217. E. coli ,182,218.deletion ,218,219.deletion ,184. Mortype VI secretion system (T6SS) [wzx, wzy and wzz genes, the products of which are involved in regulation and assembly of the O-antigen, and the wbp operon, which is important for the biosynthesis and assembly of the nucleotide sugars of the O-unit [Pae isolates during chronic CF lung infections [wzz2 expression can be transcriptionally repressed by AmrZ [wbpA and wbpH genes appears to be directly governed by the T3SS transcription regulator ExsA [The Rsm proteins govern the synthesis of many proteins involved in the transition between acute and chronic virulence phenotypes ,185,220 m (T6SS) ,227, algm (T6SS) ,230,231 m (T6SS) . Vice vem (T6SS)  and exopm (T6SS)  , which contribute to biofilm formation [Another direct target of Rsm proteins is the response regulator PprB , which iormation   and diguanylate cyclases (DGCs), which impact the c-di-GMP levels, which in turn widely affects gene expression and function [Last but not least, Rsm proteins appear to modulate the expression of several transcripts encoding function ,250.Pseudomonads. The Rsm RNAs contain multiple GGA motifs, which serve as Rsm binding sites [The function of the Rsm proteins is controlled by Rsm-binding RNAs, which act by titration ,219,253.ng sites ,256,257.ng sites . The seqng sites ,260,261.rsmW transcription was observed in early stationary phase [rsmW, rsmY and rsmZ expression is elevated in biofilms [RsmY and RsmZ are the main RsmA/N/F binding RNAs ,185,218,ry phase . On the ry phase . Moreovebiofilms ,262. Howbiofilms , highligrsmY and rsmZ is directly controlled by the TCS GacA/S  and traription; ), which,rsmY transcription [rsmZ transcription by binding to AT-rich region upstream of the rsmZ promoter  and HptB/HsbA/D synthesis, respectively [The stability and turnover of RsmZ and RsmY RNAs in strain PAO1 is modulated by several regulators, which can affect the stability of both RNAs  [RNase-sensitive gradient profiling by mass spectrometry (GradR) [Many candidate regulatory RNAs have been revealed in bacteria owing to the development of novel high-throughput technologies. Recent examples include Gradseq) ,283 or R (GradR)  for the  (GradR) ,287,288.Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) [peptide nucleic acids (PNAs), which can act as precision antimicrobials selectively targeting species-specific mRNAs under certain conditions [Unravelling the RNA-based regulation of processes controlling metabolism, cell growth, biofilm formation, antibiotic susceptibility and other virulence traits of (multi)drug-resistant pathogens such as the ESKAPE class (species) ,290 willspecies) ,292. Sucnditions ,298,299.nditions ,302,303.nditions ,305,306 nditions  during anditions .Pae.Moreover, the discovery of new RNA chaperones such as ProQ ,313,314,


YAP and TAZ are transcriptional cofactors that integrate several upstream signals to generate context-dependent transcriptional responses. This requires extensive integration with epigenetic regulators and other transcription factors. The molecular and genomic characterization of YAP and TAZ nuclear function has broad implications both in physiological and pathological settings.Yes-associated protein (YAP) and TAZ are transcriptional cofactors that sit at the crossroad of several signaling pathways involved in cell growth and differentiation. As such, they play essential functions during embryonic development, regeneration, and, once deregulated, in cancer progression. In this review, we will revise the current literature and provide an overview of how YAP/TAZ control transcription. We will focus on data concerning the modulation of the basal transcriptional machinery, their ability to epigenetically remodel the enhancer–promoter landscape, and the mechanisms used to integrate transcriptional cues from multiple pathways. This reveals how YAP/TAZ activation in cancer cells leads to extensive transcriptional control that spans several hallmarks of cancer. The definition of the molecular mechanism of transcriptional control and the identification of the pathways regulated by YAP/TAZ may provide therapeutic opportunities for the effective treatment of YAP/TAZ-driven tumors. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif  are transcriptional coactivators, which were initially identified as downstream effectors of the Hippo signaling pathway . The HipIn this review, we will concentrate on the “nuclear” function of YAP/TAZ, with a particular focus on cancer. We will provide a detailed overview of the role of YAP/TAZ as transcriptional coactivators and their interaction with other transcriptional components. Then, we will summarize the main networks of transcription factors (TFs) that are controlled by and that interact with YAP/TAZ. Finally, we will present a panoramic view of the transcriptional programs regulated by YAP/TAZ in cancer. It should be pointed out that many of the cited studies focused on either YAP or TAZ and only some performed a comparative analysis on both cofactors. Thus, despite the broad functional redundancy of YAP and TAZ, specific findings may not necessarily concern both factors.YAP/TAZ are defined as transcriptional cofactors, since they are endowed with transactivation activity but cannot bind DNA. Consequently, they need to form complexes with DNA-binding proteins in order to regulate gene expression. While several TFs have been reported to interact with YAP/YAZ (see paragraph 3), collective evidence indicates that the transcriptional enhanced associate domain (TEAD) TFs are the main partners which mediate both transactivation and growth-promoting activities ,30,31,32Less is known concerning YAP/YAZ-dependent gene repression. Most of the current knowledge comes from the study of Estarás and colleagues, which examined the transcriptional repression of mesendoderm genes during the early differentiation of human embryonic stem cells. By chromatin immunoprecipitation coupled to next generation sequencing (ChIP-seq) and GRO-seq, they observed that YAP–TEAD selectively counteract the Activin/SMAD2,3/Wnt-dependent induction of mesendoderm genes by inhibiting p-TEFb-dependent elongation and by recruiting the negative elongation factors NELF .(i)TFs may modulate the YAP/TAZ-dependent transcriptional landscape by boosting the activation of a subset of potential YAP/TAZ targets. This selection may be dictated in cis by the presence of cognate TF-binding motifs which favor the colocalization of YAP/TAZ with other TFs on proximal and distal regulatory regions, as is the case for AP-1 or MRTF/SRF ,44.(ii)TFs may enhance the binding of YAP/TAZ to TEAD loci that would otherwise be low-affinity sites for YAP/TAZ binding. This is the case of MYC target genes, whereby MYC promotes the recruitment of YAP/TAZ on constitutive TEAD-bound loci, which, in the absence of MYC expression, are not bound or regulated by YAP .(iii)Co-operating TFs might directly interact with YAP/TAZ-TEAD and can be recruited to YAP/TAZ-bound loci in a manner that is independent of the presence of their cognate DNA motif, as is the case of ZEB1 . In thes(iv)There are a number of examples where YAP/TAZ have been proposed to regulate transcription in a TEAD-independent manner, as is the case of p73, mutant p53, and the regulation of osteogenic programs by RUNX2 and SNAIL/SLUG ,48,49,50(v)The chromatin-independent association of YAP/TAZ can also contribute to the integration/coordination of transcriptional responses. This is the case of β-Catenin and YAP/TAZ, which are both components of the WNT destruction complex or the cytoplasmic association of YAP/TAZ with SMADs ,51.Biochemical data and recent genome-wide chromatin studies indicate that YAP/TAZ interact with chromatin and regulate transcription primarily by associating with TEADs. Nonetheless, several reports document the association of YAP/TAZ with other TFs, suggesting the existence of intense transcriptional integration with other pathways for context-dependent transcriptional regulation. A review of the literature reveals emerging common themes in the way YAP/TAZ may integrate transcription :(i)TFs mThus, the interaction with other TFs can lead to the remodeling and potentiation of YAP/TAZ-dependent transcription, the co-regulation of common target genes shared with the other TFs, the concerted activation of distinct transcriptional programs, or the enhancement of the activity of other TFs on their target genes. The following is a summary of the most relevant interaction of YAP/TAZ with TFs and cofactors.AP-1. Activator protein 1 (AP-1) is a group of dimeric TFs composed of JUN, FOS, and ATF family proteins. AP-1 proteins belong to the basic leucine zipper (bZIP) family and bind DNA as either homo- or heterodimers. Both gain- and loss-of-function studies have revealed specific roles for individual AP-1 components in cell proliferation, differentiation, apoptosis, and other biological processes. ChIP-seq analyses show that a high fraction of YAP/TAZ–TEAD peaks (70–80%) colocalizes with AP-1 and contains both a TEAD and an AP-1 motif [-1 motif ,44. Co-i-1 motif . YAP/TAZ-1 motif ,52,53. C-1 motif .E2F. E2F TFs are the final effectors of the cyclin-dependent kinase (CDK)–RB–E2F axis, which drives cell cycle progression. Mutations in this pathway are frequent in cancer and lead to uncontrolled DNA replication and cell division. In Drosophila, the YAP/TAZ orthologue Yorkie (Yki) and dE2F1 share the binding of promoters of a large fraction of genes that are cooperatively activated during development. This concerted control of cell growth genes explains the pleiotropic effect of loss of function mutations of dE2F1 over Yki activation [tivation . Co-regutivation .MYC. c-MYC is a basic Helix-Loop-Helix leucine-zipper TF that dimerizes with MAX to regulate gene expression. In physiological conditions, it is activated by growth factor stimulation, thereby controlling the expression of a large number of genes involved in cell growth and metabolism [tabolism ,58. In ctabolism ,58. MYC-tabolism . MYC protabolism .Myb–MuvB complex. The Myb–MuvB complex is a master regulator of genes expressed at the G2 and M phase of the cell cycle, which complements the activity of E2F factors in the control of cell cycle progression. During the S-phase, the MuvB complex  dissociates from the repressive DREAM complex  to bind to b-Myb. This Myb–MuvB complex then activates genes-regulating mitosis and cytokinesis. The expression of these genes is controlled by YAP/TEAD, which engage distal enhancers by binding b-Myb. Both complexes promote enhancer–promoter contacts required for transcriptional activation [tivation . b-Myb iMRTF/SRF. Myocardin-related factors (MRTFs) are G-actin-binding coactivators that localize mainly in the cytosol in resting cells. As a result of mechanical  or biochemical stimulation , MRTFs shuttle to the nucleus where they bind to their cognate TF, the serum response factor (SRF). The MRTF/SRF complex drives the expression of genes relevant for fibrosis, including ECM proteins, integrins, cytokines, many components of the actomyosin cytoskeleton, through the binding of CC(A/T-rich)6GG cis-elements, called the CArG boxes. Genome-wide chromatin association studies in fibroblast reveal the co-regulation of genes by MRTF/SRF and YAP/TAZ-TEAD, which is mainly driven by the co-presence of their cognate DNA-binding motifs on promoters and enhancers. This creates codependency in the expression of these common target genes, whereby the expression and the activity of one of the two complexes are necessary and sufficient to activate the other TF complex, thus leading to gene transactivation [tivation . This crtivation . In additivation . Given tRUNX. The Runt-domain TFs (RUNXs) are generally involved in lineage differentiation and cancer development, although with functions that depend on the context and the specific RUNX involved. RUNX2, which controls mesenchymal stem cells differentiation along the osteogenic lineage, is upregulated during the epithelial-to-mesenchymal transition (EMT) and its expression correlates with poor prognosis in breast cancer [t cancer . RUNX2 it cancer , cell trt cancer . On the t cancer .TRPS1. TRPS1 is a transcriptional repressor that belongs to the family of GATA TFs. These TFs are able to act as pioneers which regulate enhancer functions by altering their chromatin accessibility. In breast cancer cells, where TRPS1 is frequently overexpressed and amplified, TRPS1 physically interacts with TEAD and colocalizes with TEAD-bound loci. On these loci, TRPS1 promotes the recruitment of corepressor complexes  along with their associated histone deacetylases (HDAC1 and HDAC3). This leads to the loss of H3K27ac at promoters and enhancers, loss of accessibility, and decreased promoter–enhancer contact, all of which result in the repression of YAP-dependent genes [nt genes . The oveZEB1. ZEB1 is a transcriptional repressor known for its regulation of cellular motility, survival stemness, and EMT. In cancer, ZEB1 has been implicated in the regulation of early dissemination, metastasis, and therapy resistance. In triple-negative breast cancer, its association with YAP/TEAD enhances the transcription of a subset of YAP/TAZ targets. The binding of ZEB1 to the WW domain of YAP is required to target ZEB1 to these genes, while the presence of the E-box, which is the ZEB1 cognate DNA-binding motifs, is dispensable [pensable . This ispensable .SNAIL and SLUG. SNAIL and SLUG are TFs known to regulate EMT and the differentiation of the mesenchymal lineage and cancer stem cells. Their function is essential for skeletal stem/stromal cells’ self-renewal, differentiation, and proper bone development. These osteogenic programs are coordinated by the formation of protein complexes that activate the transcriptional activity of YAP/TAZ. Protein–protein interactions are mediated by the WW domain of YAP/TAZ and the repressor domain of SNAIL/SLUG [AIL/SLUG . These iAIL/SLUG . ConsideAIL/SLUG .NICD/RBPJ. Notch signaling is regulated by the interaction of membrane-bound ligands that engage Notch receptors on juxtaposed neighboring cells, leading to the proteolytic release of the Notch intracellular domain (NICD) fragment from the membrane. NICD translocates to the nucleus and binds the TF recombining binding protein suppressor of hairless (RBPJ) and the nuclear effector Mastermind-like, thus forming a complex that activates the transcription of the Notch target genes. YAP/TAZ and Notch crosstalk at different levels due to the YAP/TAZ-mediated transcription of Notch ligands and receptors and the YAP/TAZ–NICD co-regulation of common genes [on genes . This ison genes . Similaron genes . This inERBB-4-ICD. EGFR family member v-Erb-b2 avian erythroblastic leukemia viral oncogene homolog 4 (ERBB-4) is a receptor protein tyrosine kinase, which undergoes proteolytic processing by membrane proteases (γ-secretase) in response to ligands. The resultant soluble intracellular domain (ICD) of ERBB-4 is translocated to the nucleus, functioning as a transcriptional regulator [egulator . The PPxegulator . The ERBegulator .β-catenin. β-catenin (βCAT) is a TF that is regulated by the WNT signaling pathway. Once stabilized, it shuttles to the nucleus, where it associates with LEF/TCF TFs to activate gene expression. It is required for a wide range of developmental processes, and it is frequently activated in cancer cells. The WNT/βCAT pathway and YAP/TAZ are intertwined at many different levels, both in the cytoplasm where their upstream regulation occurs and in the nucleus where they activate gene expression. In the developing heart, YAP and β-catenin co-regulate the expression of SOX2 and SNAI2, two key TFs that support cardiomyocytes proliferation by binding their respective cognate DNA motifs found at the SOX2 and SNAI2 loci [AI2 loci . SimilarAI2 loci .TBX5. TBX5 is a member of the T-box family of TFs (TBXs), characterized by a conserved 180 amino acid DNA-binding domain (T-box). TBXs regulate a variety of developmental processes, including specification of mesoderm, development of the heart, vasculature, and limbs, and tumorigenesis. Evidence suggests the transcriptional cooperation of YAP/TAZ and TBX5 in a β-catenin-independent way. In the developing heart, YAP/TAZ and TBX5 co-regulate the expression of cardiac genes by favoring the recruitment of histone acetyltransferases. Mutations of TBX5, which are identified in the Holt–Oram syndrome, impair TAZ binding and hamper gene activation [tivation . This suSMADs. SMADs are TFs that are regulated by the superfamily of TGF-β and BMPs pathways. These signal transduction pathways regulate the cell cycle and differentiation and are altered in cancer. Activatory SMADs, SMAD2,3 (TGF-β-dependent), and SMAD1,5,8 (BMPs-dependent), once activated, associate with SMAD4 and translocate to the nucleus to activate transcription. Negative feedback regulation depends, in part, on their binding to the inhibitory SMAD6,7. Hippo signaling, YAP/YAZ activation, and TGF-β signaling intersect at different levels, in part by the association of YAP/TAZ with SMADs, which is regulated by the Hippo pathway and restrains SMADs within the cytoplasm [ytoplasm . Given tytoplasm , it willERG. ERG belongs to the family of ETS TFs. Several observations support the oncogenic role of ERG and other ETS factors in prostate carcinogenesis [ogenesis . ETS-famogenesis . This ilFOXO1. Evidence linking YAP activity to the cellular responses to oxidative stress and its cytoprotective role during recovery from oxidative damage leads to the analysis of FOXO1. FOXO1 belongs to a family of TFs possessing a conserved DNA-binding domain termed the “Forkhead box”. ChIP on selected regulatory regions of the MnSOD, a critical gene in the antioxidant response, reveals that both YAP and FOXO1 bind and regulate MnSOD expression. In vivo genetic analysis shows that FOXO1 and YAP prevent cardiac cell death in response to oxidative stress [e stress . FOXO1 we stress . Future PITX2. Paired-like homeodomain transcription factor 2 (Pitx2) is found to be upregulated in the border-zone ventricular cardiomyocyte nuclei after infarction in Hippo-deficient mice and is required for YAP-induced regenerative responses [esponses . Genomicesponses .RELA (p65). RELA associates with either NF-kB1,2 to form the transcriptionally competent NF-kB complex. This complex is the effector of signals transduced following stimulation by a number of factors, including TNFa, TLRs, and TCR/BCR. The p65 subunit (RELA) of the NF-kB complex is shown to bind YAP/TEAD in response to TNFα stimulation. This leads to the assembly of the YAP/TEAD/p65 complex, which activates the expression of chemokines (IL-6 and CCL2) and glucose metabolism genes. This program supports migration in breast cancer cells, suggesting the crosstalk between NF-kB signaling and YAP/TAZ in cancer cells [er cells . Previouer cells .Mutant-TP53 (Gain of function). TP53 is a tetrameric TF that regulates cellular responses to stress conditions such as DNA damage, metabolic, and activation of oncogenes. It is frequently inactivated by loss of function mutations, which impair its tumor-suppressive activity. TP53 can also acquire the gain of function mutations which are positively selected during tumor evolution due to their pro-oncogenic activities. Among these activities, the mutant-TP53 gain of function promotes cell cycle by regulating transcription for a subset of cell cycle genes which are co-activated by NF-Y and YAP. On these genes, the presence of a CAAT motifs consensus sequence for NF-Y binding is required to recruit the mutant-TP53/NF-Y/YAP trimeric complex, which stimulates transcription by enhancing promoters acetylation [tylation .TP73. TP73 belongs to the TP53 family and shares tumor-suppressive activity functions with TP53. Several reports show the association of p73 with YAP to form transcriptionally active complexes. YAP acts as a competitor of the ITCH E3 ubiquitin ligase, which binds and targets p73 for ubiquitin-mediated degradation [radation . The binradation . Their tradation ,84.Initial studies concerning the activity of YAP/TAZ as transcriptional coactivators were focused on the analysis of the promoter-driven transcription of a few available target genes  ,85. ThisSome common themes are emerging concerning the type of enhancers regulated by YAP/YAZ in development and cancer. Consistently with their activity as de-differentiating factors, YAP/TAZ are able to bind and regulate developmental and lineage specification enhancers. For instance, during pancreatic development, YAP/TEAD regulate the activity of a subset of temporarily regulated enhancers. On these enhancers, YAP/TEAD integrate the combinatorial control of gene expression along with other lineage-specific TFs as HNF1B, ONECUT, PDX1, FOXA1, and GATA6, which colocalize with TEAD on co-regulated enhancers. This suggests a central role for YAP as a signal responsive regulator of multipotent pancreatic progenitors . In addiATAC-seq analysis performed on adult cardiomyocytes reveals that, once they are reprogrammed by YAP into fetal-like progenitors, these cells lose nucleosomes and acquire open chromatin regions at a subset of YAP-bound loci. These genomic sites show a significant gain of topological contacts between promoters and enhancers, supporting the hypothesis that YAP overexpression alters local chromatin conformation by enhancing the looping between YAP-bound distal regions and promoters of target genes . The newIt is worth mentioning that there are also enhancers that are bound and repressed by YAP/TAZ. This aspect, which to date has received less attention, suggests context-dependent transcriptional regulation by YAP/YAZ. During the early stages of embryonic development, YAP/TAZ recruit the NuRD repressor on “switch-enhancer elements” bound by TEAD, SMAD2/3, and OCT4, thus preventing the expression of the mesendoderm specification genes . This inIn cancer cells, YAP/TAZ have been mapped to a subset of super-enhancers mainly associated with the expression of cell growth genes ,39. SupeThe evidence collected so far highlights a prominent role of epigenetic processes in the regulation of transcription by YAP/TAZ, both in Drosophila and in mammalian cells ,99. ThisThe SWI/SNF complex. Genome-wide studies investigating chromatin structures in different cellular contexts report alterations in chromatin accessibility upon YAP/TAZ overexpression or depletion. Independent reports suggest that the SWI/SNF complex (switch/sucrose non-fermentable complex) might be a conserved chromatin remodeling partner of YAP/TAZ [ YAP/TAZ . In Dros YAP/TAZ . Depleti YAP/TAZ . Notably YAP/TAZ . In mamm YAP/TAZ . These i YAP/TAZ . Overall YAP/TAZ . Indeed, YAP/TAZ . Thus, Y YAP/TAZ . Furtherp300 and BRD4. The acetylation of chromatin at several histone residues is generally associated with transcriptional activation; importantly, YAP/TAZ are able to recruit selected histone acetyltransferases (HATs), mainly at enhancers, in order to activate gene expression. Enhancers bound by YAP/TAZ are heavily acetylated on H3K27 [on H3K27 ,43, a chon H3K27 . The recon H3K27 . p300 maon H3K27 .YAP/TAZ can enhance chromatin acetylation by recruiting BRD4 ,108. BRDChromatin methylation. Post-translational modifications of histones may affect their charge, impairing the affinity of DNA–nucleosomes binding and, thus, the degree of compaction of the chromatin structure. The methylation of lysine 4 of histone H3 is associated with gene activity: typically, H3K4me1 is observed at enhancers and H3K4me1 is observed at gene bodies, while H3K4me3 is abundant at promoters [romoters . H3K4 meromoters ,113,114.romoters . Firstlyromoters . EvidencThe NuRD repressor complex. The repression of transcription by YAP/TAZ is mainly associated with the recruitment of the NuRD complex, which possesses both histone deacetylase (HDAC) and ATP-dependent chromatin-remodeling activity [activity . This coactivity ,118. In activity . Similaractivity . Mechaniactivity .YAP/TAZ can phase-separate to control transcription, possibly by forming transcription factories whereby distal enhancers, promoters, and transcriptional coactivators are co-opted by YAP/TAZ in order to activate gene transcription. YAP is shown to phase-separate in response to hyper-osmotic stress. YAP nuclear condensates are also identified in vivo in the kidney medulla as a result of the high osmolarity of this area. YAP condensates, which are driven by its intrinsically disordered transactivation domain, first form away from transcriptional complexes and then subsequently colocalize with nascent RNA once transcription is activated. These condensates are proximal but do not completely overlap with RNA Pol II signals, possibly hinting at their prominent role in shaping the spatial organization of enhancers, promoters, and their associated coactivators . SimilarHyper-transcription is a state of a strong and global increase in RNA synthesis often associated with potent cell proliferation ,122,123.The dysregulation of YAP/TAZ has been reported in a wide variety of human cancers ,135,136.YAP/TAZ control cell proliferation by coordinating the expression of cell cycle genes, which constitute a core gene signature that almost invariably is activated by YAP/TAZ in multiple contexts. As previously discussed, this core cell cycle program is integrated by the activity of other TFs, such as MYC, E2F, AP-1, and MYB, both in normal and transformed cells ,59,139. The AXL receptor tyrosine kinase is described as a key mediator of YAP-dependent oncogenic activities in human hepatocellular carcinoma (HCC). The ectopic expression of YAP increases expression levels of AXL protein and mRNA in the immortalized hepatocytes cell line MIHA. The knockdown of either YAP or AXL in primary HCC cell lines hampers tumor growth and metastasis . SimilarYAP/TAZ activity can also lead to the upregulation of the RAS/MAPK pathway: for example, in thyroid cancer whereby, due to NF2 inactivation, YAP leads to the increased expression levels of the three RAS genes and the activation of the RAS/MAPK pathway . SimilarEarly studies based on the ectopic expression of either YAP or TAZ in human mammary epithelial cells showed the emergence of EMT phenotypes, including loss of cell–cell contact and cell scattering ,149. ThiYAP/TAZ can control EMT at different levels, both by regulating the expression of EMT genes and master regulators and by establishing TFs regulatory networks. This dual control is exemplified by ZEB1, a transcriptional repressor essential for EMT. In skin squamous cell carcinomas, the hybrid-mesenchymal state, which is associated with aggressive metastatic growth and stemness, is due to the high expression of ZEB1. In these tumors, the loss of the protocadherin FAT1 leads to the activation of the CAMK2–CD44–SRC pathway that promotes YAP nuclear translocation, which in turn controls ZEB1 expression . The detYAP/YAZ may also link the metabolic reprogramming of cancer cells to their acquisition of mesenchymal and migratory properties. A study on neural crest cells has recently shown that the Warburg effect could trigger EMT by promoting YAP1-TEAD1 nuclear interaction, which in turn regulates the activation of the expression of SOX9, ETS1, and ZEB2, key TFs driving neural crest delamination and migration. This is due to the YAP-dependent activation of enhancers . This meWhile mechano-transduction is one of the main upstream pathways controlling YAP/TAZ activity, these two cofactors themselves control the expression of genes altering actin and cytoskeletal dynamics or remodeling the ECM ,135,153.The regulation of the cytoskeleton is also relevant within the non-tumor component of the tumor microenvironment: the activation of YAP in cancer-associated fibroblasts (CAFs) is required for promoting matrix stiffening, cancer cell invasion, and angiogenesis. In CAFs, YAP, apart from the well-known target genes AMOTL2, ANKRD1, and CTGF, regulates the expression of several cytoskeletal regulators, including ANLN, SDPR, and DIAPH3 . The regThe initial evidence of the involvement of YAP/TAZ in determining the migratory and invasive phenotype of cancer cells comes from Lamar et al., where multiplexed in-vivo assays (Luminex-based) show the pro-metastatic function of YAP in breast and melanoma tumors. This is dependent on the interaction with TEAD, and the increased metastatic activity on these cells correlates with increased YAP transcriptional activity . It is iBone morphogenic protein 4 (BMP4) is identified as a direct target of TAZ in MCF10A. BMP4 upregulation associated with increased BMP4-SMAD1/5 signaling and BMP4 stimulation is sufficient to induce cell migration. BMP4 silencing partially inhibits TAZ-induced migration, suggesting that BMP4 signaling is one of the pathways engaged by TAZ to confer migratory properties to cancer cells .Whole-genome profiling analysis leads to the identification and validation of target genes repressed by TAZ that may play a role in regulating cell migration . In breaBesides, YAP can affect cell migration and tumor metastatic dissemination by controlling the expression of miRNAs and long noncoding RNAs (lncRNAs). Recently, it was published that the YAP/TEAD/NuRD complex can repress the LncRNA NORAD. Mechanistic studies suggest that NORAD sponges several proteins involved in cellular migration; among these, S100P, a calcium-binding protein, is a prominent target required for the NORAD-mediated suppression of metastasis. NORAD expression is negatively correlated with prognosis and metastatic progression in breast and lung cancer, possibly suggesting a general role of this lncRNA in cancer . In glioIn order to survive in hostile microenvironments characterized by poor nutrient availability, cancer cells must reprogram their cellular metabolism to fulfill their needs for rapid growth. These crucial adaptive responses are at least partially regulated by YAP/TAZ.The investigation of the connection between metabolic reprogramming and metastatic growth in isogenic colon cancer cell lines reveals a positive regulatory loop linking glucose metabolism and metastatic growth. In these cells, YAP upregulates the glucose transporter GLU3, thus leading to increased glucose uptake. This activates the glycolytic enzyme pyruvate kinase 2 (PKM2), which is associated with YAP/TEAD, and increased their transcriptional activity. The YAP/TEAD/PKM2 complex drives the expression of genes linked to glycolysis, nucleotide synthesis, and glutathione-dependent detoxification pathways. The silencing of either YAP or GLUT3 leads to the reduction of metastatic growth in vivo, underscoring how the metastatic phenotype may depend on glucose-driven metabolic reprogramming of cancer cells. In the light of previous evidence showing that glycolysis can activate YAP through AMPK and PFK1 ,167, ple+ intestinal stem cells and in colorectal cancer cells, YAP can regulate cholesterol metabolism by controlling the expression of ZMYND8, a TF of the zinc finger-MYND family. In these cells, ZMYND8 associates to SERBP2 to drive enhancer–promoter interaction and thus upregulates genes of the mevalonate pathway, the main metabolic pathway controlling sterol synthesis. The loss of ZMYND leads to the demise of intestinal stem cells and cancer cells and is associated with loss of intestinal regeneration and delayed tumorigenesis [Bertero and collaborators described a metabolic switch triggered by the stiffening of the tumor microenvironment, which is essential for cancer progression and metastasis. YAP/TAZ regulate the expression of GLS1 (glutaminase) and the aspartate/glutamate transporter SLC1A3, both in tumor cells and in cancer-associated fibroblasts. This boost in glutaminolysis is needed for the TCA cycle-dependent regulation of amino acid biosynthesis and other biosynthetic pathways . Lipid migenesis . This acigenesis . Thus, Yigenesis  or, as iigenesis .A relevant part of the transcriptional programs controlled by YAP/YAZ in cancer cells concerns the establishment of paracrine and autocrine signaling. In particular, expression profiling and loss of function studies in MDA-MB-231 cells have allowed the identification of a set of cytokines  and hematopoietic growth factors (GMCSF and GCSF), of which the expression levels are dependent on YAP/TAZ. This is associated with the activation of STAT3 and NFKB signaling. The supplementation of these cytokines promotes invasiveness in in vitro assays while blocking CXCR2 signaling (CXCR2 is the receptor for CXCL1-3 and IL8), loosens the endothelial monolayer and promotes invasion and extravasation . This paWithin the tumor microenvironment, cancer cells interact with immune cells often to evade or suppress the immune system. There is emerging evidence that YAP/TAZ may trigger immunosuppression by diverse mechanisms. In tumor cells, YAP/TAZ can decrease the expression of programmed death-ligand 1 (PD-L1), a surface protein that activates the immune checkpoint, thus contributing to the repression of T-cell function ,182,183.Ferroptosis is a novel type of regulated cell death, which is iron-dependent and characterized by the accumulation of peroxidated lipids ,187. TheRecent studies have revealed that YAP/TAZ can promote ferroptosis in cancer cells. Yang et al. showed that in renal cell carcinomas, ferroptosis susceptibility is regulated by cell density and confluency. Mechanistically, low cell density increases TAZ activity and leads to the accumulation of the epithelial membrane protein 1 (EMP1), which, in turn, induces the expression of NADPH oxidase 4 (NOX4), a key regulator of lipid peroxidation for ferroptosis . A similOverall, this suggests that ferroptosis may be a selective liability of YAP/TAZ-driven tumors that could be exploited for therapeutic purposes.De-differentiation is a process that confers plasticity to cells, thereby enhancing adaptive responses, in particular during development, tissue regeneration, and oncogenic transformation. This process, crucial for the function of progenitors and stem cells, is often hijacked during tumor evolution to sustain tumor progression towards more aggressive and metastatic states and to promote drug resistance ,197,198.We have certainly gained considerable knowledge of how YAP/TAZ control transcription. Overall, it is generally accepted that YAP/TAZ play a pivotal role in controlling distal enhancers and in linking enhancer activation to their associated genes. This implies a crucial role for mechanisms in controlling three-dimensional (3D) chromatin organization and looping, which must be engaged or at least negotiated by YAP/TAZ in order to shape and integrate transcriptional responses. Understanding these processes is undoubtedly one of the key challenges that lie ahead. It is also emerging that YAP/TAZ are predominantly controlling transcription by epigenetic mechanisms and by establishing an extensive network of functional and physical interaction with several TFs which orchestrate context-dependent transcriptional responses. Further biochemical studies and genome-wide chromatin mapping will be essential to extend and possibly complete the entire catalog of TFs, cofactors, and chromatin modifiers that participate in YAP/TAZ-dependent transcriptional regulation. Considering that YAP/TAZ sit at the crossroads of signaling pathways controlling development, regeneration, and cancer, a full description of their context-dependent activity as transcriptional cofactors will offer a unique opportunity for designing molecular therapies to modulate their function in the relevant clinical settings.


Saccharomyces cerevisiae has approximately 200 copies of the 35S rDNA gene, arranged tandemly on chromosome XII. This gene is transcribed by RNA polymerase I (Pol I) and the 35S rRNA transcript is processed to produce three of the four rRNAs required for ribosome biogenesis. An intergenic spacer (IGS) separates each copy of the 35S gene and contains the 5S rDNA gene, the origin of DNA replication, and the promoter for the adjacent 35S gene. Pol I is a 14-subunit enzyme responsible for the majority of rRNA synthesis, thereby sustaining normal cellular function and growth. The A12.2 subunit of Pol I plays a crucial role in cleavage, termination, and nucleotide addition during transcription. Deletion of this subunit causes alteration of nucleotide addition kinetics and read-through of transcription termination sites. To interrogate both of these phenomena, we performed native elongating transcript sequencing (NET-seq) with an rpa12Δ strain of S. cerevisiae and evaluated the resultant change in Pol I occupancy across the 35S gene and the IGS. Compared to wild-type (WT), we observed template sequence-specific changes in Pol I occupancy throughout the 35S gene. We also observed rpa12Δ Pol I occupancy downstream of both termination sites and throughout most of the IGS, including the 5S gene. Relative occupancy of rpa12Δ Pol I increased upstream of the promoter-proximal Reb1 binding site and dropped significantly downstream, implicating this site as a third terminator for Pol I transcription. Collectively, these high-resolution results indicate that the A12.2 subunit of Pol I plays an important role in transcription elongation and termination. S. cerevisiae (yeast) begins with transcription of the 35S gene by RNA polymerase I (Pol I) to synthesize the 35S ribosomal RNA (rRNA). The 35S rRNA is then co- and post-transcriptionally cleaved to produce the 18S, 5.8S, and 25S rRNAs , thereby allowing transcription of those genes ,13. Reb1The current model for Pol I termination at T1 is similar to the “torpedo” model for Pol II termination ,10. Pol rpa12Δ strains grow very slowly compared to wild-type (WT) at 30 °C [Schizosaccharomyces pombe revealed that protein-protein interactions between Reb1 and the A12.2 subunit are specifically required for transcriptional arrest and termination of Pol I ECs [S. pombe Pol I [S. cerevisiae Pol II [One of the known Pol I subunits involved in both intrinsic RNA cleavage  and tranat 30 °C  and are at 30 °C . Structuol I ECs . This obbe Pol I  and S. ce Pol II . In addie Pol II  and is de Pol II ,25,26. RPA12, we observed re-ordering of median polymerase occupancy throughout the 35S gene, favoring the 5′ end of the template. Furthermore, we identified robust changes in Pol I occupancy in response to the rDNA template sequence between the WT and rpa12Δ strains, confirming that A12.2 affects Pol I nucleotide addition in vivo. Our experiments also demonstrated that the rpa12Δ strain is termination deficient in vivo, with significant rpa12Δ Pol I occupancy observed throughout the IGS. Strikingly, rpa12Δ Pol I occupancy increases dramatically directly upstream of the promoter-proximal Reb1 binding site, indicating that the rpa12Δ Pol I EC is paused or halted stably at this site. These findings provide insight into A12.2′s role in growing cells and pose intriguing new questions about Pol I termination and the conserved genomic architecture of the IGS. Many biochemical and structural analyses have implicated A12.2 in a myriad of activities. Here, we use native elongating transcript sequencing (NET-seq) to investigate how deletion of this subunit influences Pol I activity in vivo. This technique allows us to reproducibly determine Pol I occupancy with single-nucleotide resolution, providing detailed insight into Pol I transcription elongation properties in living cells ,28,29. US. cerevisiae strain [RPA12. In brief, early log-phase cultures were collected via filtration, then flash-frozen in liquid nitrogen and cryogenically lysed by a mixer mill. Pol I elongation complexes were isolated via immunoprecipitation with anti-HA magnetic beads. Associated RNA transcripts were isolated using an acidic phenol-chloroform extraction. The RNA transcripts were 3′ appended with a DNA linker and reverse transcribed to produce complementary DNAs (cDNAs). The cDNAs were circularized and amplified by PCR to produce high-throughput sequencing libraries. Finally, these libraries were sequenced as previously described [NET-seq library generation was performed exactly as previously described using the original HA-tagged Rpa135 e strain  and a deescribed .Sequencing trimming, alignment, and data formatting were performed as previously described . Unix scrpa12Δ yeast strains were aggregated. To generate Figures, the counts were normalized to the sum of all signals on the positive strand of the 35S gene (corresponding to negative strand RNA transcripts). Analyses were performed using the following packages: R (version 4.0.2) [Data analyses methods in R (version 4.0.2) were based on previous literature . In brien 4.0.2) , dplyr (n 4.0.2) , plyr (vn 4.0.2) , ggplot2n 4.0.2) , ggseqlon 4.0.2) , ggpubr n 4.0.2) , matrixSn 4.0.2) , hexbin n 4.0.2) . The Difn 4.0.2)  was usedrpa12Δ yeast strains bearing HA-tagged Pol I as previously described [rpa12Δ). The amplitude at each position corresponds to the number of polymerases mapped to that position, representing a snapshot of Pol I occupancy. The counts were normalized to the sum of Pol I occupancy from the 35S transcription start site (position 1) to the first termination site (position 6739). To determine the reproducibility among replicates of the same strain, the Spearman correlation test was deployed (rpa12Δ). This test ranks the peaks displayed in rpa12Δ strains indicated high reproducibility. These results reveal that NET-seq is an ideal tool to determine the effect of the deletion of A12.2 on Pol I occupancy in vivo.NET-seq was first developed to investigate the occupancy of Pol II at single-nucleotide resolution in vivo . We receescribed . The resrpa12Δ strains. The median occupancy for WT and rpa12Δ were plotted in t-test was performed at each position to determine whether there was a significant change between strains. Based on the p-value, we determined whether there was a significant difference in occupancy and indicated this with either a green (increased occupancy) or a black (decreased occupancy) line below the histogram for the rpa12Δ strain as compared to WT. These results reveal that there is an increase in Pol I occupancy at the 5′ end of the rDNA in the rpa12Δ strain as compared to WT and indicates that the deletion of A12.2 results in a significantly different occupancy pattern of Pol I across the template. Furthermore, there is an increase in Pol I occupancy in the ETS 2 region in rpa12Δ yeast. To examine these patterns further, the moving average across 300 nucleotides was plotted for the median occupancy . It is reasonable to expect that there would be an increase in Pol I backtracking in the rpa12Δ strain, though NET-seq cannot be used to detect this effect.We demonstrated that NET-seq performed within yeast strains is highly reproducible, therefore, we can use this technique to draw conclusions about the occupancy of Pol I in the WT vs. ccupancy B. This grpa12Δ strain, suggesting defects in transcription elongation. This finding is consistent with the results of transcription run-on experiments performed by Prescott, et al. [rpa12Δ Pol I by our lab [rpa12Δ strain were sequence-dependent. We generated a DiffLogo (rpa12Δ strain (shown on top) vs. WT (shown below). The patterns shown in RPA12 repositions the polymerases on the rDNA template with respect to WT. In the rpa12Δ strain, Pol I is paused up- and downstream of G/C-rich rDNA regions, and there is a difference in sequence enrichment directly surrounding the LNT in the RNA:DNA hybrid as compared to WT. These results confirm that the RPA12 deletion perturbs nucleotide addition by Pol I and demonstrate that the effects of this deletion are at least partially sequence dependent. This observation further supports the intimate relationship between template sequence and Pol I occupancy in vivo [The results shown in , et al.  and prev our lab . However our lab ,29. As aDiffLogo  to visua in vivo ,29. A12.2 deletion  of the 5S gene. In fact, this is the only location in the IGS where Pol I occupancy is greater in the WT strain than the rpa12Δ strain. These data show that a very small subset of WT Pol I ECs read through both the primary termination site (T1) and the secondary fail-safe site (T2), with the ECs ultimately halting upon encountering the 3′ end of the 5S gene, likely due to collision with Pol III or its associated factors. By contrast, rpa12Δ results in significantly increased Pol I EC read-through of T1 and T2, as well as the 5S gene, suggesting that this deletion renders the polymerases termination deficient. We also observed persistent nd IGS 2 B. To detrpa12Δ Pol I EC is much more stable than WT in vitro [rpa12Δ Pol I ECs to read through the Pol III EC-occupied 5S gene. Compared to WT, we see consistent rpa12Δ Pol I occupancy throughout IGS 2, culminating in a cluster of positions with sharply increased occupancy approximately 215 base pairs upstream of the transcription start site for the adjacent 35S gene  may ongation . The resrpa12Δ Pol I occupancy on the DNA template in the top 2.5% occupied positions. The cumulative effect of this may explain the observed 5′ preference in occupancy in the rpa12Δ strain as well as the significant increase in spacer region occupancy. Overall, these findings show that nucleotide addition by rpa12Δ Pol I is perturbed in vivo, consistent with previous in vitro studies [The template positions demonstrating statistically significant changes in polymerase occupancy displayed unique sequence trends within the RNA:DNA hybrid, and overall sequence-specific repositioning . We iden studies ,26,29. T studies , whereas studies . By analRPA12 renders Pol I termination-deficient in vivo, as we observed significant rpa12Δ Pol I EC read-through of both previously defined sites of Pol I termination  method to probe for Pol I occupancy in WT yeast . In agreS. cerevisiae? How well-conserved are these termination sites in other eukaryotic species? Answering these questions will bring us closer to a fundamental understanding of Pol I, and the genomic region it transcribes. These high-resolution in vivo findings demonstrate that the A12.2 subunit of Pol I plays an important role in transcription elongation and termination. Furthermore, these data suggest that there may be a third, previously uncharacterized, transcription termination site for Pol I in yeast.Analysis of Pol I occupancy on the rDNA via NET-seq refines our understanding of the role for A12.2 in Pol I function in vivo, as well as the architecture of the yeast IGS. These data also suggest several new lines of inquiry. How does A12.2 moderate Pol I occupancy in a sequence-dependent manner? Which domain of A12.2 is responsible for this behavior? Does the promoter-proximal binding site represent a third site of Pol I termination and, if so, what purpose does it serve in NET-seq analyses demonstrate that the A12.2 subunit of Pol I plays an important role in transcription elongation and termination in living cells, consistent with previous findings in vitro. Furthermore, these data suggest that there may be a third, previously uncharacterized, transcription termination site for Pol I in yeast.