Methanococcus maripaludis, internal transcription termination sites (ioTTSs) were identified within 38% of operons. Higher transcript and protein abundances were found for genes upstream than downstream of ioTTSs. For representative operons, these differences were confirmed by northern blotting, qRT-PCR and western blotting, demonstrating that these ioTTS terminations were functional. Of special interest, mutation of ioTTSs in ribosomal protein (RP)-RNA polymerase (RNAP) operons not only elevated expression of the downstream RNAP genes but also decreased production of the assembled RNAP complex, slowed whole cell transcription and translation, and inhibited growth. Overexpression of the RNAP subunits with a shuttle vector generated the similar physiological effects. Therefore, ioTTS termination is a general and physiologically significant regulatory mechanism of the operon gene expression. Because the RP-RNAP operons are found to be widely distributed in archaeal species, this regulatory mechanism could be commonly employed in archaea.Genes organized within operons in prokaryotes benefit from coordinated expression. However, within many operons, genes are expressed at different levels, and the mechanisms for this remain obscure. By integrating PacBio-seq, dRNA-seq, Term-seq and Illumina-seq data of a representative archaeon   Clostridium cellulolyticum operon  and operons, but also revealed extensive transcription read-through at over 40% of the transcription termination sites (TTSs), which especially complicated the operon transcription profiles  operons also decreased the cellular RNAP complex abundances and the transcription and translation velocities, demonstrating a key role in controlling production of the RNAP complex. Given that similar RP-RNAP operons are ubiquitous in archaea, this work suggests that ioTTS termination could be a novel and potentially ancient mechanism for tuning differential expression of the operon genes in archaea.This study extends the above observations. The combination of PacBio-seq, dRNA-seq, Term-seq and Illumina-seq of the M. maripaludis and its derivatives were routinely cultured in anaerobic McF medium under a gas phase of N2/CO2 (80:20) as previously described (600). Unless indicated otherwise, 2.5 μg ml−1 puromycin was added for genetic selections and to maintain recombinant plasmids in M. maripaludis. E. coli strains were routinely grown in LB Miller broth at 37°C with shaking at 200 rpm. When needed, final concentrations of antibiotics and supplements used were ampicillin at 100 μg ml−1, kanamycin at 50 μg ml−1, isopropylthiogalactoside (IPTG) at 0.1 mM.Strains and plasmids used in this study are listed in escribed . As aCPSescribed ,22. For M. maripaludis mRNAs were sequenced on the PacBio platform as described previously  as described previously (E. coli Poly(A) Polymerase (New England Biolabs). rRNA was depleted using Ribo-Zero™ rRNA Removal Kit (Epicentre). The capped and tailed RNA was purified using AMPure beads and eluted in TE buffer, and the full-length primary transcripts were further enriched using streptavidin magnetic beads (New England Biolabs). Polyadenylation (A-tailing) ensured priming by the poly dT primer at the 3′ end of transcript and reverse transcription of the first cDNA strand. PolyG was then added to the cDNA 3′end for second-strand synthesis using Terminal Transferase . Unenriched full-length transcripts prepared without the capping reaction and streptavidin enrichment were used to construct a control library for verifying the positions and abundances of transcription start sites (TSSs) and transcription termination sites (TTSs) of operons. Library construction and PacBio-sequencing were performed at Frasergen Bioinformatics Technology Co., Ltd . As described previously /(TDER of the wild-type strain) (Dataset S2).Operons were identified based on the mapping of the PacBio reads in combination with data of the TSSs and TTSs that were previous identified at single-base resolution via dRNA-seq and Term-seq , respect+ membranes  by electroblotting and crosslinked to the membrane using UV. Next, membranes were prehybridized at 42°C in prehybridized buffer for 4 h, followed by hybridization for 12 h with 2–10 pmol of biotin-labeled DNA probes listed in Northern blot assays were performed as previously described . BrieflyTotal RNA was extracted from the mid-exponential cells as described above, and 500 ng of RNA was used to generate complementary DNAs using ReverTra Ace® qPCR RT Master Mix with gDNA Remover (Toyobo). Quantitative PCR amplifications were performed with Mastercycler eprealplex2  and a parallel amplification without RT reaction was used as a control to assess the absence of DNA in the RNA samples for each gene . The staM. maripaludis proteins in E. coli, the respective open reading frames (ORFs) for the His-tagged RpoP, Rpl37Ae, Rpl21e and AfuA and GST-tagged RpoF and AfuC proteins were cloned into the expression vector pET28a or pGEX 4T-1 via stepwise Gibson assembly using ClonExpress MultiS One Step Cloning Kit (Vazyme). The expression plasmids were then transformed into E. coli BL21(DE3)pLysS, which was cultured at 37°C in LB broth containing 50 μg ml−1 kanamycin or 100 μg ml−1 ampicilin. When the OD600 reached 0.6–0.8, 0.1 mM isopropyl-b-d-thiogalactoside (IPTG) was added. After 16 h at 22°C, cells were harvested by centrifugation and stored at -80°C. The harvested cells were resuspended in binding buffer  and lysed by sonication, and the supernatant was collected by centrifugation. The his-tagged or GST-tagged recombinant proteins were purified from the supernatant by passage through a His-Trap HP column or a GSTrap HP column  according to the manufacturer's protocol , 150 mM NaCl, 10 (w/v) glycerol, 0.05% (v/v) NP-40 detergent] and lysed by sonication. The cell lysate was centrifuged at 14,000 g for 15 min at 4°C, and proteins in the supernatant were separated on 15% SDS-PAGE and transferred to a nitrocellulose membrane. The antisera to the proteins were diluted 1:5000. A horseradish peroxidase (HRP)-linked secondary conjugate at a 1:5000 dilution was used for the immunoreaction. Immune-active bands were visualized by an Amersham ECL Prime western blot detection reagent . For quantifying the cellular content of a protein, cell-free extract of M. maripaludis at indicated amounts was electrophoresed on SDS-PAGE synchronously with loading the corresponding purified recombinant proteins from E. coli as references. Density of each protein band was quantified from photographs using ImageJ software, and the cellular abundance of a protein was calculated by comparison to the respective recombinant protein.Western blots were performed to determine the cellular protein abundances in the wild-type and genetically modified strains of M. maripaludis strains with ioTTS terminator mutations were constructed using the recently developed CRISPR-Cas9 genome-editing toolkit  sequence under control of constitutive M. maripaludis promoters, the plasmids for ioTTS terminator mutations  at a final concentration of 105 μCi/ml. Total RNA was extracted using TRIzolTM reagent (Invitrogen) and dissolved in 700 μl Ecoscint A . 3H isotope incorporation was determined by liquid scintillation counting (Perkin Elmer). Relative overall transcription rate was calculated from the linear increase in cpm incorporation with time.The relative overall transcription rate was compared by determining the rate of 600 = 0.5. After 10 min at 37°C, cells were harvested, resuspended in Lysis Buffer, and lysed by sonication. Total protein in cell-free extracts (CFE) was quantified using a BCA protein assay kit (Thermo Scientific). Five microgram of CFE protein was western-blotted to quantify the puromycin incorporated using the monoclonal anti-puromycin antibody at a 1:10 000 dilution  and HRP-conjugated, anti-mice secondary antibody (Abmart) at a 1:2000 dilution.Puromycin incorporation was employed to determine the abundance of nascent proteins . PuromycM. maripaludis in the exponential growth phase at OD600 of ∼0.45 were harvested and resuspended in Lysis Buffer, lysed by sonication, and centrifuged at 12,000 g for 30 min at 4°C. The supernatant was incubated at 37°C for 30 min with and without treatment of DNase I (100 U) and RNase A (100 U). A total of 500 μg of the pretreated supernatant was fractionated through the size exclusion chromatography on a Superdex 200 10/300 GL column , and the Mr of each fraction was estimated by gel filtration molecular weight markers (Kit No.: MWGF1000 of Sigma-Aldrich). Finally, each fraction was collected for protein identification by western blotting.Cells of M. maripaludis. When integrated with previously published dRNA-seq data of transcription start sites (TSSs)  , Term-ses (TSSs) ,22, and n Figure . In totan Figure  and C. Tn Figure . Remarkan Figure  and C. Tn Figure , and then Figure .ioTTSs were found in 38% (157/410) of operons. The ioTTSs were characterized by their transcription differential expression ratios (TDERs) of the flanking genes or the transcript abundance of the upstream gene divided by that of the downstream gene. TDERs >1.5 were considered biologically significant, and TDERs >2 were considered as highly significant biologically (see below). For the operons containing ioTTSs, 72% (150/210) of the TDERs possessed mid- or high-significance Figure . Thus, mM. maripaludis, transcription termination depends upon the general transcription termination factor aCPSF1 and occurs at a cis-element with a polyuridine (polyU)motif  by growing at 22°C but not 37°C motif ,22. To dnot 37°C . Similarnot 37°C . These onot 37°C . In summd of TUs .MMP1635-1633 that encodes a thioredoxin/glutaredoxin homolog, DsrE-like protein, and a conserved hypothetical protein. A sharp decrease in transcription of MMP1634-1633 immediately downstream of the ioTTS was observed, and this decrease was greatly alleviated in the ▽aCPSF1 strain  uptake was selected. An ioTTS, assigned as ioTTSafuAB, was found in the IGR between afuA and afuB. While, no evidence for ioTSS or antisense RNA was detected in this operon. Illumina-seq and PacBio-seq detected a higher abundance of transcripts for afuA than afuBC  or a medium-strength terminator from MMP0229 (T2) into the IGRs of atpK-atpE and atpF-atpA reduced transcription of the ioTTS downstream genes and also disrupted the coordinated transcription of the ten genes in the operon  homologous to the eukaryotic RNAP II and composed of 12 subunits and encoded in five operons  genes, forming RP-RNAP operons. Because the levels of the RPs and RNAP proteins are different, differential transcription of the genes might be necessary for correct protein production. Interestingly, in these operons the RNAP genes are mostly located downstream of a RP gene, and an ioTTS separates them . Therefore, these results confirmed the role of the ioTTS in reducing both transcription and protein production of downstream genes within the RP-RNAP operon  and pMEV4(rpoF), respectively. Growth of the overexpression strains was also retarded and similar to those of the ioTTS terminator mutants . RNAP assembly in cell extracts was tested by size exclusion chromatography coupled to western blotting using antibodies against RpoA", RpoP and RpoD. Elution of the fully assembled RNAP with a predicted molecular weight (Mr) of ∼350 kDa was detected in fractions ∼9–10 ml from all tested strains  strains. In contrast, both overexpression strains contained ∼2-fold higher levels of free RpoP (eluting at 18–19 ml or ∼6 Kd), lower levels of free RpoD (eluting at 14.5–15 ml or ∼20 Kd) and potential RNAP assembly intermediates, presumably including RpoNPDL (eluting at 13.5–14.5 ml or ∼34 Kd)  Figure  and B.In conclusion, elevation of the levels of a RNAP subunit impaired the assembly of the RNAP complex, and termination at the ioTTS within the RP-RNAP operon was one mechanism to control the production of the appropriate levels of RNAP subunits.rpl37Ae-rpoP and rpl21e-rpoF gene organization among diverse archaea was investigated. Using the M. maripaludis rpoP as a probe, operons bearing rpl37Ae-rpoP were found in the Euryarchaeota and superphylum DPANN  are found in Euryarchaeota and DPANN species, while shorter operons consisting of 3–4 genes occur in Haloarchaea and Nanoarchaeota. Interestingly, in TACK species, the archaeal clade believed as the closest relatives of eukaryotes , the rpom Figure .rpl37Ae than that of rpoP was found in Thermococcus kodakarensis, T. onnurineus, Methanosarcina acetivorans, and Methanococcoides burtonii (Dataset S3). Thus, ioTTS termination may be common within this operon in the Euryarcheaota and awaits further examination in other archaea.Reexamination of the available transcriptomic data of archaea, higher levels of transcription of M. maripaludis  and the pMEV4-rpoP overexpression strain, instead decreased the cellular content of the complete RNAP complex and a potential assembly intermediate (Figure Clustering of functionally related genes in operons allows for coregulation and especially even gene expression in prokaryotes. However, achieving precise, and sometimes uneven, stoichiometries in protein complexes requires fine-tuning mechanisms. Translation efficiency, influenced by mRNA secondary structure and codon usage ,17,18, aipaludis ,22 but abacteria ,19,20, ieriments . In whiceriments ,58, and eriments . In the e Figure . Rpb10 ie Figure ,64. Recee Figure . AnalogoE. coli, ribosomal proteins account for up to 40% of the total cellular proteins, and multiple ribosomes can synchronically translate one transcript (M. maripaludis where the ratios of Rpl37Ae to RpoP and Rpl21e to RpoF are determined to be 14.3:1 and 4.3:1, respectively. While, changing the ratios by mutation of ioTTS terminators reduced transcription and translation velocities and also growth of M. maripaludis (Figures ioTTS termination in coordinating expression of the archaeal and bacterial RP-RNAP operons can exert an important role in physiology, representing a novel regulatory mode that not only ensures the proper levels of the suite of subunits that assemble into the complete RNAP complex but also in coordinating the production of the systems to couple translation and transcription. Coupled transcription and translation in prokaryotes is usually achieved by ribosomes loaded on the transcripts being transcribed, and the leading ribosome even binding to the processive RNAP . In actianscript ,67. ThusGiven the wide distribution of the RP-RNAP operons in archaea and presumably differential transcription of RNAP and RP subunit genes in the operons of some archaeal species  and dataIn conclusion, this work reports that ioTTS termination functions as a new regulatory mode in coordinating differential expression of genes in archaeal operons that encode protein complexes consisting of uneven stoichiometry of subunits or involving in different biological processes. The biological significance of this type of coordinated differential expression is particularly illustrated by the RP-RNAP operons, where disruption of this coordination reduced RNAP complex content and as a consequence transcription, translation, and growth. The ioTTS termination not only demonstrates the complexity of prokaryotic transcription regulation mechanisms but illustrates a novel mode of controlling the precise RNAP subunit content. Thus, ioTTS termination-controlled RP-RNAP operon expression is likely widely employed in prokaryotes.gkad575_Supplemental_FilesClick here for additional data file.


Phase separation provides a general mechanism for the formation of biomolecular condensates, and it plays a vital role in regulating diverse cellular processes, including gene expression. Although the role of transcription factors and coactivators in regulating transcription has long been understood, how phase separation is involved in this process is just beginning to be explored. In this review, we highlight recent advance in elucidating the molecular mechanisms and functions of transcriptional condensates in gene expression control. We discuss the different condensates formed at each stage of the transcription cycle and how they are dynamically regulated in response to diverse cellular and extracellular cues that cause rapid changes in gene expression. Furthermore, we present new findings regarding the dysregulation of transcription condensates and their implications in human diseases. The central dogma of molecular biology, initially stated by Francis CrickPol II transcription is a complex multi-step process. It starts with the assembly of a preinitiation complex (PIC) composed of Pol II, general transcription factors (GTFs) and the Mediator complex at a gene promoter region, where the two strands of DNA in the PIC are denatured to allow the template strand being positioned at the Pol II active center to initiate the RNA synthesis. Once the length of nascent RNA reaches to a critical length, Pol II escapes from the promoter region and forms an elongation complex to further extends the RNA chain until it travels to the end of the gene and terminates the transcription cycle –9 . OveAlthough much progress has been made in elucidating the key events during transcription, what is often overlooked is the fact that transcription takes place strictly in a spatially-defined mannerIn this review, we focus on the molecular mechanisms and functions of nuclear condensates in transcriptional regulation. We first summarize the condensates identified at different stages of the transcription cycle, followed by a discussion on the factors and pathways controlling the interplays between these separate condensates and how they are dynamically regulated in response to diverse cellular and extracellular cues that cause rapid changes in gene expression. Finally, we present new findings regarding the dysregulation of transcription condensates and their roles in human diseases.The biomolecules, including proteins and nucleic acids, must be strictly compartmentalized to elicit their corresponding functions at the correct time and space in cells. While forming various membrane-encapsulated organelles offers a means to compartmentalize and concentrate specific sets of molecules at discrete intracellular locations, many cellular compartments that are not bound by membranes also constitute an important mechanism to achieve spatiotemporal regulation of biological reactions ,11 . DeResearch into this question has revealed that these membraneless compartments, also called biomolecular condensates, are formed in part through phase separation, a physiochemical phenomenon that refers to the de-mixing process that forms dense and dilute phases from a homogeneous solution ,12 . ThExperimental evidence demonstrates that multivalent interactions are molecular driving forces underlying phase separation. Within the biomolecular condensates, multivalent molecules function as the scaffold to enable the assembly of phase-separated structures, whereas client components are often at a lower valency and are recruited to the condensates by directly binding to scaffolds ,13,14 Even before the era of phase separation, it has long been known that transcription occurs in a compartmentalized manner inside the nucleus. The nucleolus, for example, is a subnuclear compartment where ribosomal RNA genes are transcribed, processed, and assembled into ribosomal ribonucleoprotein complexesAccurate transcription requires the dynamic assembly of transcription machinery and myriads of regulatory factors at specific times and locations. Recent research suggests that functionally distinctive transcriptional condensates enriched with different sets of factors play a key role in regulating this spatial control . Since tSuper-enhancers are large clusters of enhancers that play an important role in driving the expression of genes involved in cell identity and developmentIn light of these unique features, it was proposed that the dense assemblies of the transcriptional machinery and regulatory factors at active super-enhancers are formed via phase separation and exist in the form of biomolecular condensatesAs the first step of gene transcription, transcription initiation is a complex process that involves the formation of a pre-initiation complex and the unwinding of the DNA double helix. Among the many proteins known to participate in the initiation process, Pol II lies at the core position because it serves as the scaffold for the pre-initiation complex, allowing other factors to interact with it and coordinate the initiation of transcription. This scaffolding ability of Pol II arises from its CTD, which is a tail-like extension from the body of Pol IIAccumulating evidence suggests a close link between CTD and phase separation in promoting efficient transcription initiation. On one hand, CTD has been suggested to facilitate phase separation through interactions between CTDs and with other activatorsAfter escaping the promoter, Pol II does not immediately advance into the productive elongation phase to synthesize the full-length transcript. Instead, it is initially paused, where a significant portion of Pol II molecules either stop forward translocation or turn to premature termination. This can be caused by a variety of factors, including intrinsic regulatory mechanisms and extrinsic obstacles, such as the presence of inhibitory molecules or physical barriers on the template DNASeveral recent studies have highlighted the importance of phase separation in regulating Pol II transcription elongation. As a cyclin partner required for the catalytic activity of CDK9, CycT1 tightly associates with CDK9 through its N-terminal region that is well-folded into structural domain. However, CycT1 also contains an extended C-terminal region that is largely unstructured and intrinsically disordered, with its precise role in regulating P-TEFb activity being unknown for a long time. Recently, it was reported that CycT1 plays a crucial role in driving the assembly of transcription factors-enriched condensates for transcriptional elongation controlInside the cells, P-TEFb activity is also regulated by a complex network of factors that associate with it and regulate its activity in response to different stimuli and cellular conditions. It is known that the majority of P-TEFb is sequestered in the 7SK snRNP, in which the CDK9 kinase activity is inhibited by HEXIM1 to prevent its activation. In addition to residing in the 7SK snRNP, P-TEFb also exists in either the BRD4-containing complex or the super elongation complex (SEC), wherein the P-TEFb is catalytically active to stimulate Pol II elongation. The SEC represents one of the most active P-TEFb-containing complexes, which comprises Pol II elongation factors eleven-nineteen Lys-rich leukemia (ELL) proteins, P-TEFb, and several frequent mixed lineage leukemia (MLL) translocation partnersEmerging evidence also revealed a fundamental role of phase separation in regulating the transcriptional events occurred at the end of a transcription cycle. In Arabidopsis, FCA is an RNA-binding protein associated with RNA 3’-end processing factors. A recent study has shown that FCA forms phase-separated nuclear bodies with the help of the coiled-coil protein FLL2In addition to the transcriptional condensates described above, many nuclear condensates or membraneless organelles are also involved in gene expression control. For instance, a slew of studies has shown that condensation of key heterochromatin-associated factors, such as HP1 and MeCP2, underpins the formation of heterochromatin and transcriptional silencing ‒60 . DeIt has become increasingly clear that Pol II can be incorporated into transcriptional condensates with different components and, correspondingly, can have distinct functions depending on the specific components that are present. However, transcription is also known as a dynamic process that involves the coordination of multiple different phases. Hence, it raises an important question of how Pol II is moved between different condensates as transcription progresses from one stage to another.Emerging evidence suggests that phosphorylation of Pol II CTD has a switch-like function in determining the partitioning of Pol II into different condensates. During the initiation of transcription at Pol II promoters, the assembly of the pre-initiation complex begins with the recruitment of Pol II containing an unphosphorylated CTD ,63 . ReIn addition to CTD phosphorylation, RNA has also been suggested to play a role in regulating the formation of condensates during transcription. For example, the enhancer and promoter-associated RNAs produced in the early stages of transcription were recently proposed to facilitate transcriptional condensate formationOn the other hand, high levels of RNA produced from gene transcription can provide negative feedback control on transcriptional condensate formation. Transcription is well-known to be stochastic in nature, switching between active and inactive states periodicallyTranscription factors are classified into general transcription factors and gene-specific transcription factors, which provide ubiquitous and diverse mechanisms for gene regulation, respectively. Likewise, some transcriptional condensates, such as those discussed above, are indispensable for transcription and paly no part in specific gene regulation, whereas others may only engage in the transcription of a specific set of genes. These condensates act as regulatory compartments, turning the expression of their target genes on or off in response to various signaling pathways or environmental cues. Recent advances have provided an unprecedented view of how these conditional transcriptional condensates act in cells to enable rapid gene expression control .FigureIn response to heat shock, cells elicit a complex transcriptional change to adapt to stress and prevent damage. This process is mediated by a group of transcription factors known as heat shock factors (HSFs), which upregulates a transcriptional program consisting of chaperone genes to maintain protein homeostasis and support cellular function ,75 . ReOn the other hand, heat shock also leads to the suppression of global transcription, which is mainly caused by the disruption of Pol II′s progression beyond the initiation stage –81 . ItCells are constantly exposed to various types of genotoxic insults that cause DNA damage and genomic instability. These insults arise from a variety of sources, including exposure to radiation, chemotherapy drugs, as well as many endogenous metabolic side products. To cope with these threats, cells have developed a surveillance mechanism, known as DNA damage response (DDR), to promote DNA repair and protect genomic integrity ,84 . InBesides transcriptional condensates, many nuclear condensates formed via phase separation are also integral to the maintenance of genome integrity –88 , buTranscriptional condensates can also be dynamically modulated to promote transcriptional silencing in response to DNA damage. For example, CycT1 can concentrate Pol II and various transcription factors into a membraneless compartment to stimulate robust transcription elongationHyperosmotic stress is a physiologic dysfunction that is caused by a sudden change in the solute concentration around a cell, leading to altered movement of water across the cell membrane. It usually occurs when cells are exposed to an environment that has a higher concentration of solutes than the cells themselves, and consequently, causing water loss and cell shrinkageRecent studies have provided novel insights into the role of transcriptional condensates in modulating rapid response to hyperosmotic stress. Adapting to osmotic stress is crucial for plant growth and crop yield, as it helps plants maintain proper hydration and ion balance in changing environments. SEU, a transcription regulator, is essential for plant growth and developmentIn mammalian cells, hyperosmotic stress has been shown to activate YAP signaling, which is a key effector of the Hippo pathway that activates various cellular functions ,95 . LiSignaling factors in the nucleus have been found to form biomolecular condensates to facilitate signal transductions in various cellular processesSteroid hormones regulate various cellular processes through their effects on gene transcription, which involves the binding of hormones to their corresponding receptors and the coordination of genome-wide transcriptional programs. A growing body of evidence has suggested that phase separation extensively engages in hormone-activated gene expression, including that mediated by estrogen and androgen receptors. In breast cancer cells, acute activation of 17β-estradiol (E2) responsive enhancers requires the recruitment of a large protein complex, known as MegaTrans complex, in an estrogen receptor α (ERα) and enhancer RNA-dependent mannerIn addition to the processes mentioned above, biomolecular condensates are involved in many other signaling pathways, including Wnt, TGF-β, and JAK/STATCancer cells often exhibit altered gene expression patterns that support their uncontrolled proliferation and survival, which leads to the question of whether transcriptional condensates are hijacked by cancer cells to fulfill this purpose. Recent studies have provided important clues on how abnormal formation, composition, and altered biophysical properties of transcriptional condensates contribute to the development and progression of cancer .FigureUbiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) is a specific histone H3K27 demethylase whose function is linked to diverse cellular processesThe eleven-nineteen-leukemia (ENL) is a chromatin reader that primarily recognizes H3K27ac through its YEATS domain. While the function of ENL in driving oncogenic gene expression programs has been well established, recent findings provided even deeper insights into the role of ENL and its phase separation in oncogenesis. The YEATS domain of ENL is a hotspot containing recurrent mutations found in many cancers, such as Wilms tumor and AMLOther types of genetic alteration, especially those occurring in transcription factors, are also implicated in human pathologies associated with aberrant condensate formation. For instance, disease-associated repeat expansions in transcription factors have been shown to alter the biophysical properties of transcriptional condensates, which perturb the condensate composition and lead to transcriptional changes of target genes in disease-relevant cellsIn this review, we have mainly focused on the role of phase separation in Pol II-mediated transcription, ranging from the basal transcriptional condensates that are involved in regulating each stage of the transcription cycle to the conditional transcriptional condensates that are swiftly formed and regulated to enable rapid transcriptional response. We also highlight the fundamental principles and molecular mechanisms that underlie the dynamical changes in these transcriptional condensates and how their dysregulation is linked to many human diseases, pointing therapeutic strategies that target transcriptional condensates as potential treatments for these diseases.Despite the rapid progress in the field of phase separation since its first description in 2009, many knowledge gaps remain to be filled to fully understand the role of phase separation in transcription control. For example, transcriptional condensates involved in transcription initiation and elongation have been well-studied, however, whether or not phase separation contributes to other aspects of transcription, such as termination, remains unclear. Given that transcription termination requires coordinated actions of myriads of factors, it is likely that phase separation is also involved in this process. Nevertheless, future studies are required to experimentally confirm this point.Defining the composition of transcriptional condensate is another outstanding question that needs to be addressed. While key drivers are important for transcriptional condensate formation, other components present in transcriptional condensate are also essential for transcriptional regulation. These “client” proteins can provide multiple layers of regulation on condensate formation, dynamics, and dissolution, therefore greatly expanding the functional flexibility and diversity of transcriptional condensates for gene expression control. However, compared to the drivers of transcriptional condensate that have been extensively studied, we know surprisingly little about client proteins. This is partly due to the lack of appropriate approaches to faithfully capture weak and multivalent interactions within phase-separated compartments. As such, innovative purification and proteomic strategies capable of mapping the composition of phase-separated condensates are urgently needed. The development of such approaches will provide invaluable tools for gaining a better appreciation of transcriptional control by phase separation mechanism.Finally, although the concept of phase separation has transformed our understanding of gene transcription control, there are still controversies regarding the biological functions of transcriptional condensates. For example, further study is needed to comprehensively define the functional importance of transcriptional condensates at the organism level. The role of phase separation in transcriptional activation should also be rigorously explored, as recent findings argue that phase separation has no effect or may even suppress transcription ,112 . I


RNA Polymerase II (Pol II) is a multi-subunit complex that undergoes covalent modifications as transcription proceeds through genes and enhancers. Rate-limiting steps of transcription control Pol II recruitment, site and degree of initiation, pausing duration, productive elongation, nascent transcript processing, transcription termination, and Pol II recycling. Here, we develop Precision Run-On coupled to Immuno-Precipitation sequencing (PRO-IP-seq), which double-selects nascent RNAs and transcription complexes, and track phosphorylation of Pol II C-terminal domain (CTD) at nucleotide-resolution. We uncover precise positional control of Pol II CTD phosphorylation as transcription proceeds from the initiating nucleotide (+1 nt), through early (+18 to +30 nt) and late (+31 to +60 nt) promoter-proximal pause, and into productive elongation. Pol II CTD is predominantly unphosphorylated from initiation until the early pause-region, whereas serine-2- and serine-5-phosphorylations are preferentially deposited in the later pause-region. Upon pause-release, serine-7-phosphorylation rapidly increases and dominates over the region where Pol II assembles elongation factors and accelerates to its full elongational speed. Interestingly, tracking CTD modifications upon heat-induced transcriptional reprogramming demonstrates that Pol II with phosphorylated CTD remains paused on thousands of heat-repressed genes. These results uncover dynamic Pol II regulation at rate-limiting steps of transcription and provide a nucleotide-resolution technique for tracking composition of engaged transcription complexes. Tracking molecular modifications of engaged Pol II complexes is important for studying transcriptional regulation. Here, the authors combine run-on sequencing (PRO-seq) and immunoprecipitation, revealing dynamics of Pol II CTD phosphorylation at nucleotide-resolution. These steps of transcription can be modulated by the action of transcription and elongation factors, chromatin remodelers and other co-factors, architectural proteins, and RNA-binding proteins (reviewed in refs. 3). Additionally, transcription-regulating RNA-species and their associated RNA-binding factors can form ‘RNA clouds’ over clusters of active genes and their distal regulatory elements (reviewed in4).Regulation of transcription defines cell type-specific gene expression programs and coordinates cellular responses in health and disease. In each cell type, a distinct set of genes and enhancers are transcribed by RNA Polymerase II (Pol II) complexes. To complete a full cycle of transcription, chromatin and Pol II must undergo a series of steps: (i) chromatin opening, (ii) assembly of the Pre-Initiation Complex (PIC), (iii) initiation of transcription, (iv) promoter-proximal Pol II pausing, (v) promoter-proximal Pol II pause-release, (vi) productive elongation, (vii) co-transcriptional RNA processing, (viii) transcript cleavage, (ix) transcription termination, and (x) recycling of Pol II  and form a protean platform for regulatory interactions (reviewed in refs. 6). The initiating Pol II is mainly unphosphorylated, but CDK7-mediated phosphorylation of serine-5 of the CTD disrupts Pol II interaction with TATA box Binding Protein (TBP) and the Mediator, allowing Pol II to escape the PIC11. After initiation, Pol II rapidly synthetizes 20–60 nucleotides (nts) of RNA, and pauses16. The promoter-proximal Pol II pause has emerged as a major rate-limiting step20 via which genome-wide transcription programs can be coordinated26. However, the CTD-modifications and regulatory signals along the promoter-proximal pause-region remain enigmatic. Multiple laboratories have shown that the release of Pol II from the promoter-proximal pause requires Positive Transcription Elongation Factor b (P-TEFb), the CDK9 subunit of which phosphorylates Negative Elongation Factor (NELF), transcription elongation factor SPT5 and serine-2 of Pol II CTD (reviewed in28). The release of phosphorylated NELF exposes a binding site for Polymerase Associated Factor 1 (PAF1), which converts Pol II to an elongation complex30. After pause-release, elongation factors and histone chaperones assemble on Pol II, and topoisomerase gains its full activity, preparing the transcription complex to progress through the array of nucleosomes and other obstacles including intragenic enhancers along the gene34.As Pol II proceeds through the transcription cycle, its phosphorylation state changes dramatically. In particular, the C-terminal domain (CTD) of the largest subunit in Pol II consists of Y15 labels active sites of nascent transcripts with biotinylated nucleotides, which allows affinity purification of nascent transcripts away from the non-nascent RNA that covers the chromatin, and identification of the precise genomic positions and orientations of engaged transcription complexes. The IP portion of the PRO-IP-seq protocol36 selects Pol II complexes based on molecular modifications or composition using native immunoprecipitation. Here, we describe the biochemical principle of the PRO-IP-seq methodology and demonstrate how it tracks regulatory changes of the engaged transcription machinery at nucleotide-resolution. Taken together: The Pol II CTD remains unphosphorylated until the early promoter-proximal pause-region. Phosphorylation on serines 2 and 5 of the CTD occur preferentially at the late pause-region, between +31 nt to +60 nt from the initiation. Serine-7-phosphorylation of Pol II CTD dominates from the pause-release through to the +5 nucleosome, a region where Pol II assembles elongation factors and gains speed (reviewed in ref. 37). Our results confirm serine-2-phosphorylation as the most distinctive CTD mark of active elongation; however, serine-5-phosphorylation is also prevalent throughout the gene’s body and correlated with higher transcriptional activity of a gene. Finally, tracking changes in Pol II CTD upon rapid transcriptional reprogramming by heat shock reveal that the CTD of paused Pol II can be, and often is, phosphorylated at serines 2 and 5.To track molecular modifications of engaged Pol II complexes at high specificity and nucleotide-resolution, we develop and describe Precision Run-On coupled to Immunoprecipitation sequencing (PRO-IP-seq). The PRO-seq portion of the protocol15. To couple run-on reaction to immunoselection of Pol II populations, we performed the run-on reaction on chromatin38. Chromatin was prepared from three biological replicates of non-stressed (NHS) and 30 min at 42 °C heat shocked (HS30) human K562 cells and fragmented to <80 base pairs (bp) using light sonication and DNase I , and as a positive control, we examined total nascent (PRO-seq) RNAs (noAb). In this strategy, the same chromatin and labeled nascent RNAs served as starting material for all antibody pulldowns and controls in a condition. After the immunoprecipitation, the nascent transcripts were purified and barcoded, and samples re-pooled to ensure identical handling  strategy  and the active sites of transcription. The Transcription Start Sites (TSSs) derived from the reference genomes generally report the longest form of each mRNA isoform, which does not necessarily coincide with the most prominent nucleotide that initiates the transcription. To precisely map the Transcription Start Nucleotide  at each gene, we used the 5'-ends of nascent RNAs that associate with Pol II in our PRO-IP-seq data Fig. . The 5'-Visualizing Pol II CTD modifications across individual genes uncovered high enrichment of Pol II with unphosphorylated CTD at the promoter-proximal pause region Fig. . Serine-5). Consequently, the availability of unphosphorylated CTD could be affected by modifications beyond serine-2, serine-5, and serine-7 phosphorylation. To ensure appropriate comparison of Pol II CTD state, we also analyzed the Pol II CTD modifications relative to the total level of transcription . These analyses showed highly similar patterns and densities of Pol II CTD modifications as detected when comparing phosphorylations relative to unphosphorylated CTD . Overall, PRO-IP-seq identified modifications at engaged Pol II . However, mNET-seq selects Pol II associated RNAs, not all of which are nascent, giving rise to various signal peaks that are not present in run-on based assays (reviewed in1). Accordingly, PRO-IP-seq did not contain spurious spikes, which are present in various mNET-seq datasets, with or without the use of empigen , unphosphorylated CTD was the primary form of paused Pol II detected across the pause region . To investigate Pol II CTD phosphorylation with respect to the nucleosomes, we overlayed MNase-seq to the relative enrichments of serine-2- and serine-7-phosphorylations . At thousands of heat-repressed genes, Pol II accumulates at the promoter-proximal region, while heat-activated genes overcome the global repression by recruiting potent trans-activators that trigger the release of paused Pol II into elongation. One of these potent trans-activators is Heat Shock Factor 1 (HSF1) that can release paused Pol II at chaperone, co-chaperone, and polyubiquitin genes57 (reviewed in ref. 58) via binding to proximal and distal regulatory elements59. To address how heat shock changes Pol II CTD modifications genome-wide, we utilized PRO-IP-seq to track Pol II CTD phosphorylations at (1) heat-induced genes where paused Pol II is efficiently released into elongation, and (2) heat-repressed genes where the release of paused Pol II is inhibited.Heat shock triggers an instant reprogramming of transcriptionHSP70 and HSP90 loci, both contacting a strongly heat-repressed histone locus , and structures of the PIC, as well as pause and elongation complexes are available in elegant detail in vitro (reviewed in ref. 63). We have lacked efficient tools to monitor nucleotide resolution changes in Pol II complexes in vivo. Multiple techniques use immuno-selection of chromatin-bound Pol II, some of which sequence the associated RNAs to track RNA synthesis (reviewed in ref. 1). These techniques include Pol II ChIP-seq64, NET-seq46 and mNET-seq47 that have uncovered serine-2-phosphorylation of Pol II CTD to enrich at the 3'-ends of genes while serine-5- and serine-7-phosphorylations dominate at genes’ 5’-regions (reviewed in ref. 5). None of the previous techniques combine nascent transcript labeling and selection of transcription machineries in distinct molecular states to achieve the sensitivity to map the nucleotide-resolution changes in Pol II CTD code. The PRO-IP-seq technique described here (1) labels the active sites of transcription, (2) generates chromatin fragments with a single engaged transcription complex, (3) immuno-selects Pol II by its modifications, (4) purifies the nascent transcripts, (5) maps the active sites of transcription and (6) the precise +1 nts from nascent transcripts, and (7) uses normalization strategy that does not constrain samples to the same total transcript count . Particularly, the CDK9 mediated phosphorylation of SPT5 has been identified as the key trigger to release paused Pol II70. Besides functioning as a signaling platform, CTD phosphorylation has been suggested to aid Pol II escape from a phase-separated domain or condensate around the promoter71. NELF has been reported to form condensates when overexpressed in heat stressed cells, and reduced release of NELF coincides with heat-repression72. Coupling nucleotide-resolution changes in Pol II composition to the progression of RNA synthesis through the rate-limiting steps of transcription should be highly informative in clarifying the regulatory logic of Pol II pause-release, including the functional consequences of P-TEFb, as well as serine-2, NELF and SPT5 phosphorylation.Serine-5, and to a lower extent serine-2, of the CTD was phosphorylated within the pause-region, primarily between +31 to +60 nt from the initiating nucleotide Fig.  and c. S37). We found serine-7-phosphorylation to rapidly increase around the pause-release, peaking at the +1 nucleosome, and remaining high until the end of the MNase-accessible region, +1000 nt from the initiation , gaining nearly its full elongation speed (2–5 kb/min) after +1000 nt from the TSS . The spike-in wild-type MEFs24 originated from Prof. Susan Lindquist laboratory, MIT, Boston, USA, and were cultured in DMEM (Gibco). RPMI and DMEM were supplemented with 10% fetal calf serum, 2 mM L-glutamate, 100 μg ml−1 streptomycin, and 100 U ml−1 penicillin. Both cell lines were maintained at 37 °C with 5% CO2 and 90% humidity. Both cell lines were tested to be mycoplasma free, and to display morphology, proliferation rate and transcriptional profile characteristic to the respective cell line. The heat shocks in K562 cells were instantly provoked by resuspending 30 million pelleted cells in pre-conditioned pre-warmed media (42 °C) and maintaining the cells at 42 °C in a water bath for 30 min. 30 million non-heat shocked cells were resuspended in 37 °C pre-conditioned pre-warmed media and maintained at 37 °C for 30 min. To avoid provoking transcriptional changes by freshly added media, the cells were expanded 24 h prior to the treatments. The pre-conditioned media was collected from excess cells of the same culture prior to the experiments75.Human K562 erythroleukemia cells, originating from ATCC and obtained2, 0.2 mM EDTA, 1 mM DTT), complemented with 20 units SUPERaseIn RNase Inhibitor (Life Technologies # AM2694) and 1× cOmplete EDTA-free Protease Inhibitor Cocktail (Roche cat nr. 11873580001). The samples were vortexed for 30 s and centrifuged at 12 500 G at 4 °C for 30 min. The chromatin pellet  at −80 °C.Cells were collected by transferring the cell suspension from 37 °C or 42 °C into eight times of the volume of ice-cold PBS. The cells were pelleted by centrifugation at 4 °C and washed with ice cold PBS. The cell pellets were resuspended in 1 mL ice-cold NUN buffer  compared to human (2.9 × 109 bp) and the hypotriploid genome of K562 cells , the spike-in was estimated to corresponds to 0.5% of total chromatin per run-on reaction. The sonicated chromatin was fragmented to <80 bp with high concentration RNase-free DNase I for 5 min at 37 °C water bath .Chromatin from 30 million cells was sonicated for 5 min at 4 °C using Bioruptor (Diagenode) with 30 s on / 30 s off intervals and high throughput settings. Per each sample of 30 million K562 cells, sonicated chromatin from 250 000 MEFs was added as spike-in material. The same batch of spike-in was utilized for all the samples. Given the smaller genome size of mouse , 40 units SUPERaseIn RNase Inhibitor, 1% sarcosyl] was prepared and pre-warmed at 37 °C water bath. Directly after the 5-min DNase I treatment, pre-warmed 2x run-on mix was added, and the run-on reaction conducted by maintaining the samples at 37 °C for an additional 5 min.2x run-on mix . The run-on chromatin was pre-cleared with pre-washed protein G beads , and each sample divided into distinct tubes for antibody pulldowns see Fig. . The antThe samples were diluted in TRIzol LS and chloroform, homogenized by vortexing, incubated 1 min on ice and centrifuged 18 000 G, 4 °C for 5 min. The aqueous layer, containing total RNA in the chromatin-Pol II complexes, was collected, and the RNA pelleted with EtOH using glycoblue  as a marker of the precipitate. The RNA pellet was washed with 70% EtOH, air-dried, and diluted in RNase-free water. The RNA was base hydrolyzed with NaOH for 5 min and the base hydrolysis terminated with Tris-HCl pH 6.8. NoAb sample was passed through P-30 columns  to remove free biotinylated nucleotides. The nascent RNAs, containing biotin-nt at the 3’-end, were purified from the non-nascent RNAs using Streptavidin Dynabeads . The bead-nascent-RNA-complexes were washed with PRO-seq wash buffers 1 , 2  and 3 , after which the RNA was isolated with TRIzol and chloroform, and precipitated with EtOH.53. An example 3'-adapter is shown below, indicating the sample-specific in-line barcode inside the square brackets.The nascent RNA pellet was diluted in barcoded 3'-adapters, and intermolecular interactions were disrupted at 65 °C for 20 sec. The barcoded adapters were ligated to the nascent transcripts over night at 25 °C using T4 RNA Ligase I  in 10 µl reaction volume and 5 µM final 3'-adapter concentration. The sample-specific barcoded 3'-adapters used in this study contain a constant G at the ligation site (5'-end of the adapter), followed by a 6-nt in-line barcode, and an inverted T at the 3'-end to prevent adapter concatemers[rArUrCrArCrG]rCrGrArUrGrUrGrArUrCrGrUrCrGrGrArCrUrGrUrArGrArArCrUrCrUrGrArArC/3InvdT/rGThe unligated adapters were removed by binding the nascent transcripts to Streptavidin Dynabeads and washing the beads with PRO-seq wash buffers 1 and 3. During the washes, the samples originating from a run-on reaction were combined in a single new tube Fig. .53. The sequence of the 5'-adapter is shown below, indicating the UMI bolded inside the square brackets.The 5'-decapping and 5'-hydroxyrepair were conducted on-beads. The bead-nascent-RNA-complexes were diluted in RNA 5' Pyrophosphorylase mix  and incubated at 37 °C for 45 min. The 5'-hydroxyrepair was subsequently conducted by adding T4 polynucleotide kinase mix  and maintaining the samples at 37 °C for additional 45 min. The bead-nascent-RNA-complexes were washed with PRO-seq buffers 1 and 3. The nascent transcripts were isolated from the beads with TRIzol and chloroform, and precipitated with EtOH. The air-dried RNA was diluted in 5'-adapter, and the sample incubated at 65 °C for 20 s. The UMI-containing 5'-adapters were ligated to the nascent transcripts with T4 RNA Ligase I over night at 25 °C in 20 µl reaction volume and 2.5 µM final 5'-adapter concentration. The 5'-adapter contained a constant C at the ligation site (3'-end of the adapter), an adjacent 6-nt UMI, and an inverted T at the 5'-end to prevent adapter concatemers[NrNrNrNrNrN]rC/5InvddT/CCTTGGCACCCGAGAATTCCAThe unligated adapters were removed by binding the nascent transcripts to Streptavidin Dynabeads and washing the bead-nascent-RNA-complexes with PRO-seq wash buffers 1, 2 and 3. The nascent transcripts were isolated with TRIzol and chloroform, and precipitated with EtOH.The pellet of nascent transcripts was diluted in a mix of RP1-primer and dNTPs, and intermolecular interactions were disrupted at 65 °C for 20 s. Reverse transcription was performed with Super Script III reverse transcriptase  in 25 µl reaction volume containing 2.5 µM RP1-primer, 625 µM dNTPs and 20 units SUPERaseIn RNA inhibitors. The reverse-transcription was conducted using the following settings: 15 min at 45 °C, 40 min at 50 °C, 10 min at 55 °C, 15 min at 70 °C. The reverse-transcribed samples were test-amplified (as described in 44) and visualized on a 6% PAGE. The samples were then amplified in a total of 12 cycles using Phusion Polymerase (in-house) in 1x HF buffer , 1 M betaine, 0.25 mM dNTPs, 0.2 µM RP1 primer and 0.2 µM RPI-n index primer. The amplified samples were purified with 1.6x Ampure XP beads . The samples were analyzed with BioAnalyzer and sequenced with Illumina HiSeq2500  or NovoSeq6000  using either SE75 or PE150 settings. RP1 and RPI-n primers are Illumina small RNA TruSeq design .76. Here, the PRO-IP-seq samples originating from one run-on reaction were barcoded with an in-line hexamer in the 3’-adapter53. In the computational analyses, reads in a pool of samples were first separated using fastx barcode splitter (http://hannonlab.cshl.edu/fastx_toolkit/), and when paired-end sequencing was used, correct pairing of reads ensured with fastq_pair . The adapters sequences  were then removed and the 6-nt UMI in the 5’-adapter was used to collapse PCR duplicates into a single read using fastp77. The 7-nucleotide sequences of barcode+C and UMI + C were removed and the Read 1 reverse complemented with fastx toolkit. To avoid mapping human reads to the mouse spike-in genome, we first used high sequencing depth PRO-seq data (80 million uniquely mapping reds) that did not contain foreign DNA24, and aligned it to mm10 using Bowtie278. Regions of mm10 where cross-mapping from human nascent transcriptome occurred were masked with maskFasta (bedtools package79). Then, PRO-IP-seq datasets were mapped to the masked mm10 spike-in genome, and uniquely mapped reads used to calculate nfspike-in. The PRO-IP-seq reads were subsequently mapped to the human genome (hg19) with Bowtie2. Mapped reads were processed from bam files to bed, bedgraph and bigwig files76 using samtools80, bedtools79, and bedGraphToBigWig (https://www.encodeproject.org/software/bedgraphtobigwig/). The complete PRO-IP-seq datasets with raw (fastq) and processed (bigWig) files are available via Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/) with accession code GSE200269.The computational analyses of PRO-IP-seq largely follows the established analyses pipelines of PRO-seq5). This RPM normalization, however, assumes samples to have the same level of total transcription, and it skews analyses when transcriptional activity changes genome-wide, occurring for example during heat shock53. We, therefore, refined the normalization strategy by deriving normalization factors (nfs) from reads mapping to spike-in genome81 (nfspike-in), and to ends of long (>150 kb) genes (nflongGE) where heat-triggered changes in transcription had not proceeded during the 30-min heat shock53. The nfspike-in was counted against the unphosphorylated CTD from the same run-on reaction (nfspike-in = spike-inCountsample/spike-inCountunPhCTD). HS30 sample pool was then adjusted against the NHS pool using the ends of long genes (endCountunPhCTD_HS30/endCountunPhCTD_NHS). In this strategy, a designed control sample (here unPhCTD in NHS condition) gets multiplied with 1. Finally, all the samples are RPM normalized using sequencing depth from the control sample  and POINT-seq82 (GSE159326) datasets were visualized using an in-house tool  and Integrative Genomics Viewer83. The mNET-seq and POINT-seq data were downloaded as normalized bigWig files and visualized using the genome version indicated at the site of download (hg19 or hg38). The scale of y-axis in PRO-IP-seq is comparable to sequencing depth-normalized control sample (nf-cRPM explained above). In each browser image, the scale is indicated in the above most track, and is the same and linear for all tracks in the browser image. The signal on the plus strand is shown on a positive scale, the signal on the minus strand is shown on a negative scale. The Hi-C data84 was visualized using 3D Genome Browser85 at 25 kb resolution and the Pol II ChIA-PET data86 (GSE33664) was downloaded in hg19 mapped bed-format from ucsc genome browser (https://genome.ucsc.edu/cgi-bin/hgGateway) and visualized as connected blocks with IGV.PRO-IP-seq , mNET-seqhttps://github.com/andrelmartins/bigWig/). The normalized count of engaged Pol II was queried at defined genomic loci, using a query window of 1 nt, 5 nts, or a 1/50th fraction of the gene body. The queried genomic sites and query windows are indicated in respective figure and figure legend. The average intensities are show with solid lines, the shaded areas display 12.5–87.5% confidence interval.The composite profiles of factor intensities from ChIP-seq, enzymatic footprints from MNase-seq and DNase I-seq, and engaged Pol II complexes from PRO-IP-seq, were generated using bigWig package . Then, the count of engaged Pol II with a given CTD modification was divided with the count of unphosphorylated Pol II CTD at each nucleotide. This ratio was set to log2-scale to center the signal to 0  was selected for normalization baseline (obtaining nf 1). This antibody was used across all the replicates  reports the density of Pol II along the gene body. To compare gene transcription in PRO-IP-seq versus PRO-seq data, we combined the normalized gbRPK signals from different antibodies in each condition (sum CTD antibodies = unPh + Ser2Ph + Ser5Ph + Ser7Ph). The mRNA expression of respective gene was counted from existing mRNA-seq data in K562 cells87, remapped to hg19 as previously described88 .Transcriptional activity for each gene was measured as previously described53. In brief, the 5’-nucleotide from each read was retained and reported as a bed file. Next, the region −100–+400 from annotated TSS was queried to find the genomic coordinate with highest count of 5’-nts. The +1 nt was identified from each PRO-IP-seq dataset reported here. Since the +1 nts were highly similar between the PRO-IP-seq datasets -seq reads as previously described53. In brief, the 3’-nucleotide from each read was retained in a bed file. Next, the region −100 to +400 from annotated TSS was queried to find the genomic coordinate with highest count of 3'-nts. The distance of the pause nucleotide from the +1 nt was counted for each gene and plotted as a histogram for the distinct PRO-IP-seq datasets.The exact pause nucleotide was called from the 3'-ends of PRO(-IP)-seq reads as previously described53 with minor modifications: Cells were lysed in buffer C , and protein concentration in the soluble fraction was measured using Qubit. 20 μg of total soluble protein was loaded into SDS-PAGE gel and transferred to nitrocellulose membrane . The membranes were incubated with primary antibodies against Pol II RPB1, either the N-terminal domain , used in 1:200 dilution, or the CTD , each used in 1:1000 dilution. β-tubulin  in 1:5000 dilution was visualized as a control for loading. The secondary antibodies were HRP conjugated from Pierce  or Invitrogen (anti-rat A10549), used in dilutions recommended by manufacture, and the blots were developed using an enhanced chemiluminescence method . The original scans of the films are available in Mendeley (https://data.mendeley.com/datasets/s44mkg6jmb/1).Analyses of Pol II protein levels were conducted as previously describedFurther information on research design is available in the Supplementary informationPeer Review FileReporting SummarySource Data


Comparative genomics has recently provided unprecedented insights into the biology and evolution of the fungal lineage. In the postgenomics era, a major research interest focuses now on detailing the functions of fungal genomes, i.e. how genomic information manifests into complex phenotypes. Emerging evidence across diverse eukaryotes has revealed that the organization of DNA within the nucleus is critically important. Here, we discuss the current knowledge on the fungal genome organization, from the association of chromosomes within the nucleus to topological structures at individual genes and the genetic factors required for this hierarchical organization. Chromosome conformation capture followed by high-throughput sequencing (Hi-C) has elucidated how fungal genomes are globally organized in Rabl configuration, in which centromere or telomere bundles are associated with opposite faces of the nuclear envelope. Further, fungal genomes are regionally organized into topologically associated domain-like (TAD-like) chromatin structures. We discuss how chromatin organization impacts the proper function of DNA-templated processes across the fungal genome. Nevertheless, this view is limited to a few fungal taxa given the paucity of fungal Hi-C experiments. We advocate for exploring genome organization across diverse fungal lineages to ensure the future understanding of the impact of nuclear organization on fungal genome function. Within the nucleus, fungal genomes are organized hierarchically, from the association of chromosomes to topological structures at individual genes, and this organization directly impacts genome function. In eukaryotes, the DNA within the nucleus is organized as chromatin, a dynamic complex formed by DNA in association with proteins  space of the nucleus to structurally organize and package DNA, while also enabling precise gene expression  in size divided into 16 chromosomes, while the fission yeast S. pombe has a 13 Mb genome divided over only three chromosomes  or chromosomes], the soil-borne plant pathogen Verticillium dahliae (36 Mb divided into eight chromosomes), the human pathogens Aspergillus fumigatus (28 Mb across eight chromosomes), and Cryptococcus neoformans (19 Mb across five chromosomes), the wheat head blight fungus Fusarium graminearum (36 Mb across four chromosomes), and the grass endophyte Epichloë festucae (35 Mb across seven chromosomes)  that are embedded within different chromosomes and contain in planta expressed genes   being the SET domain-containing subunit with histone methyltransferase catalytic activity  to directly recruit the DNA methyltransferase DIM-2 for methylation on the fifth intracyclical atom of cytosine bases (5mC)   or PAS (PRC2 Accessory Subunit) abolishes subtelomeric H3K27me2/3 in N. crassa, while the conserved protein EPR-1 is known to “read” the H3K27me2/3 mark for gene repression; in support, deletion of the EPR-1 homolog in F. graminearum, BP1, phenocopies loss of KMT6 and BP1 directly binds DNA to stabilize nucleosomes for enhancing transcriptional repression , which is catalyzed by the SET-domain containing histone methyltransferase ASH1  onto the chromosome ends (Szostak and Blackburn Telomeric Repeat-binding Factor 1), TRF2 (Telomeric Repeat-binding Factor 2), and POT1 (Protection Of Telomeres protein 1) bind the telomeric repeats while TIN2 (TERF1-Interacting Nuclear factor 2), TPP1 , and RAP1 (Repressor Activator Protein 1) mediate protein intracomplex interactions  to regulate telomere length, while TBF1 (TTAGGG Binding Factor 1), a yeast ortholog of human TRF1, binds to single stranded telomeric DNA to antagonize improper telomere lengthening , RAP1, RIF1, POT1, TPZ1 (Telomeres Protection protein in SchiZosaccharomyces pombe 1), and CCQ1 (Coiled-Coil Quantitatively enriched protein 1) for protecting telomeric DNA and silencing subtelomeric chromatin 2–3(TG)1–6], while S. pombe telomeres have [TTACAG2–5]   that are often found upstream of genes: most are small, but a subset of these intergenic ACRs are large (>2000 bp), presumably for multiple transcription factors to control of expression of ACR-proximal genes; these open chromatin regions are enriched with acetylation of H3K27 to possibly “open” the chromatin  chromatin fibers, a beads on a string conformation that in turn may aggregate further into 30 nm chromatin fibers Bernardi . PresumaStructural Maintenance of Chromosomes (SMC)-1, SMC-3, Scc1, and Scc3 proteins form the core machinery for chromatin looping  are among the most prevalent 3D structures in metazoan genomes  globules are readily visible in Hi-C datasets  are 3D chromosomal structures that regionally organize the genome by subdividing chromatin compartments  named for the aggregation of several ∼40 kb euchromatic globules into larger, compact structures ∼250 kb in size that could be interpreted as a large chromatin aggregates analogous to metazoan TADs  expressed genes occur at, or in proximity to, TAD-like boundaries in V. dahliae and E. festucae, suggesting that TAD-like structures in these fungi are necessary for proper gene expression. Additional evidence from yeasts suggests that TAD-like structures are critical for other genome functions apart from gene expression, such as repressing recombination or promoting genome evolution   at the nuclear envelope , all of which contain a LEM  domain capable of binding chromatin at the nuclear envelope  domains in their primary structures. In S. pombe, Lem2 facilitates the anchoring of heterochromatic regions to the nuclear envelope, where the chromatin silencing machinery, including histone deacetylase complexes targeted to the telomeres, are recruited for chromatin repression  “A” and heterochromatic (silent) “B” compartments. This compartmentalization of chromatin is readily observed as a “checkerboard” pattern in Hi-C contact maps Fig. . The funnts Fig.  telomeres associate nonrandomly in four to eight foci at the nuclear membrane opposite the centromere bundle , N. crassa  N. crassa strain devoid of H3K27me2/3 can have microscopic colocalization of centromeres and telomeres when facultative heterochromatin is compromised  strain that loses subtelomeric H3K27me2/3 exhibits genome disorder consistent with compromised subtelomere interactions despite internal H3K27me2/3 enrichment being maintained -based experiments, which revolutionized the field of nuclear genome organization (Dekker et al. fuad021_Supplemental_FilesClick here for additional data file.