Inherited blood cancer predisposition through altered transcription elongation.

Despite advances in defining diverse somatic mutations that cause myeloid malignancies, a significant heritable component for these cancers remains largely unexplained. Here, we perform rare variant association studies in a large population cohort to identify inherited predisposition genes for these blood cancers. CTR9, which encodes a key component of the PAF1 transcription elongation complex, is among the significant genes identified. The risk variants found in the cases cause loss of function and result in a ∼10-fold increased odds of acquiring a myeloid malignancy. Partial CTR9 loss of function expands human hematopoietic stem cells (HSCs) by increased super elongation complex-mediated transcriptional activity, which thereby increases the expression of key regulators of HSC self-renewal. By following up on insights from a human genetic study examining inherited predisposition to the myeloid malignancies, we define a previously unknown antagonistic interaction between the PAF1 and super elongation complexes. These insights could enable targeted approaches for blood cancer prevention.

Targeting Processive Transcription Elongation via SEC Disruption for MYC-Induced Cancer Therapy.

The super elongation complex (SEC) is required for robust and productive transcription through release of RNA polymerase II (Pol II) with its P-TEFb module and promoting transcriptional processivity with its ELL2 subunit. Malfunction of SEC contributes to multiple human diseases including cancer. Here, we identify peptidomimetic lead compounds, KL-1 and its structural homolog KL-2, which disrupt the interaction between the SEC scaffolding protein AFF4 and P-TEFb, resulting in impaired release of Pol II from promoter-proximal pause sites and a reduced average rate of processive transcription elongation. SEC is required for induction of heat-shock genes and treating cells with KL-1 and KL-2 attenuates the heat-shock response from Drosophila to human. SEC inhibition downregulates MYC and MYC-dependent transcriptional programs in mammalian cells and delays tumor progression in a mouse xenograft model of MYC-driven cancer, indicating that small-molecule disruptors of SEC could be used for targeted therapy of MYC-induced cancer.

Therapeutic Targeting of MLL Degradation Pathways in MLL-Rearranged Leukemia.

Chromosomal translocations of the mixed-lineage leukemia (MLL) gene with various partner genes result in aggressive leukemia with dismal outcomes. Despite similar expression at the mRNA level from the wild-type and chimeric MLL alleles, the chimeric protein is more stable. We report that UBE2O functions in regulating the stability of wild-type MLL in response to interleukin-1 signaling. Targeting wild-type MLL degradation impedes MLL leukemia cell proliferation, and it downregulates a specific group of target genes of the MLL chimeras and their oncogenic cofactor, the super elongation complex. Pharmacologically inhibiting this pathway substantially delays progression, and it improves survival of murine leukemia through stabilizing wild-type MLL protein, which displaces the MLL chimera from some of its target genes and, therefore, relieves the cellular oncogenic addiction to MLL chimeras. Stabilization of MLL provides us with a paradigm in the development of therapies for aggressive MLL leukemia and perhaps for other cancers caused by translocations.

Transcriptional elongation control in developmental gene expression, aging, and disease.

The elongation stage of transcription by RNA polymerase II (RNA Pol II) is central to the regulation of gene expression in response to developmental and environmental cues in metazoan. Dysregulated transcriptional elongation has been associated with developmental defects as well as disease and aging processes. Decades of genetic and biochemical studies have painstakingly identified and characterized an ensemble of factors that regulate RNA Pol II elongation. This review summarizes recent findings taking advantage of genetic engineering techniques that probe functions of elongation factors in vivo. We propose a revised model of elongation control in this accelerating field by reconciling contradictory results from the earlier biochemical evidence and the recent in vivo studies. We discuss how elongation factors regulate promoter-proximal RNA Pol II pause release, transcriptional elongation rate and processivity, RNA Pol II stability and RNA processing, and how perturbation of these processes is associated with developmental disorders, neurodegenerative disease, cancer, and aging.

Structural visualization of de novo transcription initiation by Saccharomyces cerevisiae RNA polymerase II.

Previous structural studies of the initiation-elongation transition of RNA polymerase II (pol II) transcription have relied on the use of synthetic oligonucleotides, often artificially discontinuous to capture pol II in the initiating state. Here, we report multiple structures of initiation complexes converted de novo from a 33-subunit yeast pre-initiation complex (PIC) through catalytic activities and subsequently stalled at different template positions. We determine that PICs in the initially transcribing complex (ITC) can synthesize a transcript of ∼26 nucleotides before transitioning to an elongation complex (EC) as determined by the loss of general transcription factors (GTFs). Unexpectedly, transition to an EC was greatly accelerated when an ITC encountered a downstream EC stalled at promoter proximal regions and resulted in a collided head-to-end dimeric EC complex. Our structural analysis reveals a dynamic state of TFIIH, the largest of GTFs, in PIC/ITC with distinct functional consequences at multiple steps on the pathway to elongation.

Pre-termination Transcription Complex: Structure and Function.

Rho is a general transcription termination factor playing essential roles in RNA polymerase (RNAP) recycling, gene regulation, and genomic stability in most bacteria. Traditional models of transcription termination postulate that hexameric Rho loads onto RNA prior to contacting RNAP and then translocates along the transcript in pursuit of the moving RNAP to pull RNA from it. Here, we report the cryoelectron microscopy (cryo-EM) structures of two termination process intermediates. Prior to interacting with RNA, Rho forms a specific "pre-termination complex" (PTC) with RNAP and elongation factors NusA and NusG, which stabilize the PTC. RNA exiting RNAP interacts with NusA before entering the central channel of Rho from the distal C-terminal side of the ring. We map the principal interactions in the PTC and demonstrate their critical role in termination. Our results support a mechanism in which the formation of a persistent PTC is a prerequisite for termination.

Allosteric transcription stimulation by RNA polymerase II super elongation complex.

The super elongation complex (SEC) contains the positive transcription elongation factor b (P-TEFb) and the subcomplex ELL2-EAF1, which stimulates RNA polymerase II (RNA Pol II) elongation. Here, we report the cryoelectron microscopy (cryo-EM) structure of ELL2-EAF1 bound to a RNA Pol II elongation complex at 2.8 Å resolution. The ELL2-EAF1 dimerization module directly binds the RNA Pol II lobe domain, explaining how SEC delivers P-TEFb to RNA Pol II. The same site on the lobe also binds the initiation factor TFIIF, consistent with SEC binding only after the transition from transcription initiation to elongation. Structure-guided functional analysis shows that the stimulation of RNA elongation requires the dimerization module and the ELL2 linker that tethers the module to the RNA Pol II protrusion. Our results show that SEC stimulates elongation allosterically and indicate that this stimulation involves stabilization of a closed conformation of the RNA Pol II active center cleft.

Acute perturbation strategies in interrogating RNA polymerase II elongation factor function in gene expression.

The regulation of gene expression catalyzed by RNA polymerase II (Pol II) requires a host of accessory factors to ensure cell growth, differentiation, and survival under environmental stress. Here, using the auxin-inducible degradation (AID) system to study transcriptional activities of the bromodomain and extraterminal domain (BET) and super elongation complex (SEC) families, we found that the CDK9-containing BRD4 complex is required for the release of Pol II from promoter-proximal pausing for most genes, while the CDK9-containing SEC is required for activated transcription in the heat shock response. By using both the proteolysis targeting chimera (PROTAC) dBET6 and the AID system, we found that dBET6 treatment results in two major effects: increased pausing due to BRD4 loss, and reduced enhancer activity attributable to BRD2 loss. In the heat shock response, while auxin-mediated depletion of the AFF4 subunit of the SEC has a more severe defect than AFF1 depletion, simultaneous depletion of AFF1 and AFF4 leads to a stronger attenuation of the heat shock response, similar to treatment with the SEC inhibitor KL-1, suggesting a possible redundancy among SEC family members. This study highlights the usefulness of orthogonal acute depletion/inhibition strategies to identify distinct and redundant biological functions among Pol II elongation factor paralogs.

NELF Regulates a Promoter-Proximal Step Distinct from RNA Pol II Pause-Release.

RNA polymerase II (RNA Pol II) is generally paused at promoter-proximal regions in most metazoans, and based on in vitro studies, this function has been attributed to the negative elongation factor (NELF). Here, we show that upon rapid depletion of NELF, RNA Pol II fails to be released into gene bodies, stopping instead around the +1 nucleosomal dyad-associated region. The transition to the 2nd pause region is independent of positive transcription elongation factor P-TEFb. During the heat shock response, RNA Pol II is rapidly released from pausing at heat shock-induced genes, while most genes are paused and transcriptionally downregulated. Both of these aspects of the heat shock response remain intact upon NELF loss. We find that NELF depletion results in global loss of cap-binding complex from chromatin without global reduction of nascent transcript 5' cap stability. Thus, our studies implicate NELF functioning in early elongation complexes distinct from RNA Pol II pause-release.

Structural basis of transition from initiation to elongation in de novo viral RNA-dependent RNA polymerases.

De novo viral RNA-dependent RNA polymerases (RdRPs) utilize their priming element (PE) to facilitate accurate initiation. Upon transition to elongation, the PE has to retreat from the active site to give room to the template-product RNA duplex. However, PE conformational change upon this transition and the role of PE at elongation both remain elusive. Here, we report crystal structures of RdRP elongation complex (EC) from dengue virus serotype 2 (DENV2), demonstrating a dramatic refolding of PE that allows establishment of interactions with the RNA duplex backbone approved to be essential for EC stability. Enzymology data from both DENV2 and hepatitis C virus (HCV) RdRPs suggest that critical transition of the refolding likely occurs after synthesis of a 4- to 5-nucleotide (nt) product together providing a key basis in understanding viral RdRP transition from initiation to elongation.

MLL-AF9 regulates transcriptional initiation in mixed lineage leukemic cells.

Mixed lineage leukemia-fusion proteins (MLL-FPs) are believed to maintain gene activation and induce MLL through aberrantly stimulating transcriptional elongation, but the underlying mechanisms are incompletely understood. Here, we show that both MLL1 and AF9, one of the major fusion partners of MLL1, mainly occupy promoters and distal intergenic regions, exhibiting chromatin occupancy patterns resembling that of RNA polymerase II in HEL, a human erythroleukemia cell line without MLL1 rearrangement. MLL1 and AF9 only coregulate over a dozen genes despite of their co-occupancy on thousands of genes. They do not interact with each other, and their chromatin occupancy is also independent of each other. Moreover, AF9 deficiency in HEL cells decreases global TBP occupancy while decreases CDK9 occupancy on a small number of genes, suggesting an accessory role of AF9 in CDK9 recruitment and a possible major role in transcriptional initiation via initiation factor recruitment. Importantly, MLL1 and MLL-AF9 occupy promoters and distal intergenic regions, exhibiting identical chromatin occupancy patterns in MLL cells, and MLL-AF9 deficiency decreased occupancy of TBP and TFIIE on major target genes of MLL-AF9 in iMA9, a murine acute myeloid leukemia cell line inducibly expressing MLL-AF9, suggesting that it can also regulate initiation. These results suggest that there is no difference between MLL1 and MLL-AF9 with respect to location and size of occupancy sites, contrary to what people have believed, and that MLL-AF9 may also regulate transcriptional initiation in addition to widely believed elongation.

SEC: A core hub during cell fate alteration.

Pol II pause release is a rate-limiting step in gene transcription, influencing various cell fate alterations. Numerous proteins orchestrate Pol II pause release, thereby playing pivotal roles in the intricate process of cellular fate modulation. Super elongation complex (SEC), a large assembly comprising diverse protein components, has garnered attention due to its emerging significance in orchestrating physiological and pathological cellular identity changes by regulating the transcription of crucial genes. Consequently, SEC emerges as a noteworthy functional complex capable of modulating cell fate alterations. Therefore, a comprehensive review is warranted to systematically summarize the core roles of SEC in different types of cell fate alterations. This review focuses on elucidating the current understanding of the structural and functional basis of SEC. Additionally, we discuss the intricate regulatory mechanisms governing SEC in various models of cell fate alteration, encompassing both physiological and pathological contexts. Furthermore, leveraging the existing knowledge of SEC, we propose some insightful directions for future research, aiming to enhance our mechanistic and functional comprehension of SEC within the diverse landscape of cell fate alterations.

The super elongation complex (SEC) mediates phase transition of SPT5 during transcriptional pause release.

Release of promoter-proximally paused RNA Pol II into elongation is a tightly regulated and rate-limiting step in metazoan gene transcription. However, the biophysical mechanism underlying pause release remains unclear. Here, we demonstrate that the pausing and elongation regulator SPT5 undergoes phase transition during transcriptional pause release. SPT5 per se is prone to form clusters. The disordered domain in SPT5 is required for pause release and gene activation. During early elongation, the super elongation complex (SEC) induces SPT5 transition into elongation droplets. Depletion of SEC increases SPT5 pausing clusters. Furthermore, disease-associated SEC mutations impair phase properties of elongation droplets and transcription. Our study suggests that SEC-mediated SPT5 phase transition might be essential for pause release and early elongation and that aberrant phase properties could contribute to transcription abnormality in diseases.

MED26-containing Mediator may orchestrate multiple transcription processes through organization of nuclear bodies.

Mediator is a coregulatory complex that plays essential roles in multiple processes of transcription regulation. One of the human Mediator subunits, MED26, has a role in recruitment of the super elongation complex (SEC) to polyadenylated genes and little elongation complex (LEC) to non-polyadenylated genes, including small nuclear RNAs (snRNAs) and replication-dependent histone (RDH) genes. MED26-containing Mediator plays a role in 3' Pol II pausing at the proximal region of transcript end sites in RDH genes through recruitment of Cajal bodies (CBs) to histone locus bodies (HLBs). This finding suggests that Mediator is involved in the association of CBs with HLBs to facilitate 3' Pol II pausing and subsequent 3'-end processing by supplying 3'-end processing factors from CBs. Thus, we argue the possibility that Mediator is involved in the organization of nuclear bodies to orchestrate multiple processes of gene transcription.

In transcription antitermination by Qλ, NusA induces refolding of Qλ to form a nozzle that extends the RNA polymerase RNA-exit channel.

Lambdoid bacteriophage Q proteins are transcription antipausing and antitermination factors that enable RNA polymerase (RNAP) to read through pause and termination sites. Q proteins load onto RNAP engaged in promoter-proximal pausing at a Q binding element (QBE) and adjacent sigma-dependent pause element to yield a Q-loading complex, and they translocate with RNAP as a pausing-deficient, termination-deficient Q-loaded complex. In previous work, we showed that the Q protein of bacteriophage 21 (Q21) functions by forming a nozzle that narrows and extends the RNAP RNA-exit channel, preventing formation of pause and termination RNA hairpins. Here, we report atomic structures of four states on the pathway of antitermination by the Q protein of bacteriophage λ (Qλ), a Q protein that shows no sequence similarity to Q21 and that, unlike Q21, requires the transcription elongation factor NusA for efficient antipausing and antitermination. We report structures of Qλ, the Qλ-QBE complex, the NusA-free pre-engaged Qλ-loading complex, and the NusA-containing engaged Qλ-loading complex. The results show that Qλ, like Q21, forms a nozzle that narrows and extends the RNAP RNA-exit channel, preventing formation of RNA hairpins. However, the results show that Qλ has no three-dimensional structural similarity to Q21, employs a different mechanism of QBE recognition than Q21, and employs a more complex process for loading onto RNAP than Q21, involving recruitment of Qλ to form a pre-engaged loading complex, followed by NusA-facilitated refolding of Qλ to form an engaged loading complex. The results establish that Qλ and Q21 are not structural homologs and are solely functional analogs.

Biochemical characterization of Mycobacterial RNA polymerases.

Tuberculosis is caused by the bacterium Mycobacterium tuberculosis (Mtb). While eukaryotic species employ several specialized RNA polymerases (Pols) to fulfill the RNA synthesis requirements of the cell, bacterial species use a single RNA polymerase (RNAP). To contribute to the foundational understanding of how Mtb and the related non-pathogenic mycobacterial species, Mycobacterium smegmatis (Msm), perform the essential function of RNA synthesis, we performed a series of in vitro transcription experiments to define the unique enzymatic properties of Mtb and Msm RNAPs. In this study, we characterize the mechanism of nucleotide addition used by these bacterial RNAPs with comparisons to previously characterized eukaryotic Pols I, II, and III. We show that Mtb RNAP and Msm RNAP demonstrate similar enzymatic properties and nucleotide addition kinetics to each other but diverge significantly from eukaryotic Pols. We also show that Mtb RNAP and Msm RNAP uniquely bind a nucleotide analog with significantly higher affinity than canonical nucleotides, in contrast to eukaryotic RNA polymerase II. This affinity for analogs may reveal a vulnerability for selective inhibition of the pathogenic bacterial enzyme.IMPORTANCETuberculosis, caused by the bacterium Mycobacterium tuberculosis (Mtb), remains a severe global health threat. The World Health Organization (WHO) has reported that tuberculosis is second only to COVID-19 as the most lethal infection worldwide, with more annual deaths than HIV and AIDS (WHO.int). The first-line treatment for tuberculosis, Rifampin (or Rifampicin), specifically targets the Mtb RNA polymerase. This drug has been used for decades, leading to increased numbers of multi-drug-resistant infections (Stephanie, et al). To effectively treat tuberculosis, there is an urgent need for new therapeutics that selectively target vulnerabilities of the bacteria and not the host. Characterization of the differences between Mtb enzymes and host enzymes is critical to inform these ongoing drug design efforts.

Molecular Structures of Transcribing RNA Polymerase I.

RNA polymerase I (Pol I) is a 14-subunit enzyme that solely synthesizes pre-ribosomal RNA. Recently, the crystal structure of apo Pol I gave unprecedented insight into its molecular architecture. Here, we present three cryo-EM structures of elongating Pol I, two at 4.0 Å and one at 4.6 Å resolution, and a Pol I open complex at 3.8 Å resolution. Two modules in Pol I mediate the narrowing of the DNA-binding cleft by closing the clamp domain. The DNA is bound by the clamp head and by the protrusion domain, allowing visualization of the upstream and downstream DNA duplexes in one of the elongation complexes. During formation of the Pol I elongation complex, the bridge helix progressively folds, while the A12.2 C-terminal domain is displaced from the active site. Our results reveal the conformational changes associated with elongation complex formation and provide additional insight into the Pol I transcription cycle.

Purification of synchronized Escherichia coli transcription elongation complexes by reversible immobilization on magnetic beads.

Synchronized transcription elongation complexes (TECs) are a fundamental tool for in vitro studies of transcription and RNA folding. Transcription elongation can be synchronized by omitting one or more nucleoside triphosphates from an in vitro transcription reaction so that RNA polymerase can only transcribe to the first occurrence of the omitted nucleotide(s) in the coding DNA strand. This approach was developed over four decades ago and has been applied extensively in biochemical investigations of RNA polymerase enzymes but has not been optimized for RNA-centric assays. In this work, we describe the development of a system for isolating synchronized TECs from an in vitro transcription reaction. Our approach uses a custom 5' leader sequence, called capture sequence 3-structure cassette 1 (C3-SC1), to reversibly capture synchronized TECs on magnetic beads. We first show, using electrophoretic mobility shift and high-resolution in vitro transcription assays, that complexes isolated by this procedure, called C3-SC1TECs, are >95% pure, >98% active, highly synchronous (94% of complexes chase in <15s upon addition of saturating nucleoside triphosphates), and compatible with solid-phase transcription; the yield of this purification is ∼8%. We then show that C3-SC1TECs perturb, but do not interfere with, the function of ZTP (5-aminoimidazole-4-carboxamide riboside 5'-triphosphate)-sensing and ppGpp (guanosine-3',5'-bisdiphosphate)-sensing transcriptional riboswitches. For both riboswitches, transcription using C3-SC1TECs improved the efficiency of transcription termination in the absence of ligand but did not inhibit ligand-induced transcription antitermination. Given these properties, C3-SC1TECs will likely be useful for developing biochemical and biophysical RNA assays that require high-performance, quantitative bacterial in vitro transcription.

AFF4 globally affects the release of paused RNA polymerase II in HEL cells.

P-TEFb, a heterodimer of the kinase CDK9 and Cyclin T1, is a critical regulator of promoter-proximal pause release of Pol II in metazoans. It is capable of forming three larger complexes, including the super elongation complex (SEC), the BRD4/P-TEFb complex and the 7SK snRNP. In the SEC or the BRD4/P-TEFb complex, P-TEFb is enzymatically active, while in the 7SK snRNP, its activity is inhibited. The SEC consists of AFF1 or 4, ENL or AF9, ELL1, 2 or 3 and EAF1 or 2 in addition to P-TEFb, the only subunit with catalytic activity, and the noncatalytic subunits have been found to be able to regulate pause release through P-TEFb. We and others recently found that AFF1, ENL and AF9 are capable of regulating transcriptional initiation, but it is unknown yet whether AFF4 is also capable of doing so. With respect to the gene regulation selectivity of the SEC and the BRD4/P-TEFb complex, one recent study showed that in human DLD-1 cells, the SEC only regulates pause release of heat shock (HS) genes, whereas the BRD4/P-TEFb complex regulates pause release of the rest of the genes. However, it is unclear whether those mechanisms are general. In this study for the purpose of further understanding the role of AFF4 in transcriptional regulation, we found that AFF4 knockdown by RNA interference in human HEL cells decreased not only cellular level but also global chromatin occupancy of CTD serine 2 phosphorylated Pol II. Direct target genes of AFF4 were identified by RNA-seq and CUT&Tag. Notably, we found by ChIP-seq and PRO-seq that AFF4 loss also increased promoter-proximal pause of Pol II on several hundred HS and thousands of non-HS genes. Mechanistically, AFF4 promotes pause release likely by facilitating the binding of P-TEFb to Pol II. These results suggest that extent of the impact of AFF4 on pause release is likely to be context-dependent or cell-type dependent.

Preparation of E. coli RNA polymerase transcription elongation complexes by selective photoelution from magnetic beads.

In vitro studies of transcription frequently require the preparation of defined elongation complexes. Defined transcription elongation complexes (TECs) are typically prepared by constructing an artificial transcription bubble from synthetic oligonucleotides and RNA polymerase. This approach is optimal for diverse applications but is sensitive to nucleic acid length and sequence and is not compatible with systems where promoter-directed initiation or extensive transcription elongation is crucial. To complement scaffold-directed approaches for TEC assembly, I have developed a method for preparing promoter-initiated Escherichia coli TECs using a purification strategy called selective photoelution. This approach combines TEC-dependent sequestration of a biotin-triethylene glycol transcription stall site with photoreversible DNA immobilization to enrich TECs from an in vitro transcription reaction. I show that selective photoelution can be used to purify TECs that contain a 273-bp DNA template and 194-nt structured RNA. Selective photoelution is a straightforward and robust procedure that, in the systems assessed here, generates precisely positioned TECs with >95% purity and >30% yield. TECs prepared by selective photoelution can contain complex nucleic acid sequences and will therefore likely be useful for investigating RNA structure and function in the context of RNA polymerases.