Super Elongation Complex Research Articles

Structural and functional insight into the effect of AFF4 dimerization on activation of HIV-1 proviral transcription

Super elongation complex (SEC) is a positive regulator of RNA polymerase II, which is required for HIV-1 proviral transcription. AFF1/4 is the scaffold protein that recruits other components of SEC and forms dimer depending on its THD domain (TPRL with Handle Region Dimerization Domain). Here we report the crystal structure of the human AFF4-THD at the resolution of 2.4 Å. The α4, α5, and α6 of one AFF4-THD mediate the formation of a dimer and pack tightly against the equivalent part of the second molecule in the dimer of AFF-THD. Mutagenesis analysis revealed that single mutations of either Phe1014 or Tyr1096 of AFF4 to alanine impair the formation of the AFF4 dimer. In addition, transactivation assay also indicated that Phe1014 and Tyr1096 of AFF4 are critical to the transactivation activity of AFF4. Interestingly, the corresponding residues Phe1063 and Tyr1145 in AFF1 have an effect on the transactivation of HIV-1 provirus. However, such mutations of AFF1/4 have no effect on the interaction of AFF1/4 with other subunits of the SEC. Together, our data demonstrated that the dimerization of AFF1/4 is essential to transactivation of HIV-1 provirus.

Integrated transcriptomic and epigenetic data analysis identifiesaberrant expression of genes in acute myeloid leukemia with MLL‑AF9 translocation.

Rearrangement of the mixed lineage leukemia (MLL; also known as lysine methyltransferase 2A) gene is a recurrent genomic aberration in acute myeloid leukemia (AML). MLLT3, super elongation complex subunit (AF9) is one of the most common MLL fusion partners in AML. The present study aimed to explore the aberrant expression of genes associated with the MLL-AF9 translocation and identified potential new targets for the therapy of AML with MLL-AF9 translocation. The transcriptomic and epigenetic datasets were downloaded from National Center of Biotechnology Information Gene Expression Omnibus (GEO) database. Differentially expressed genes were obtained from two independent datasets (GSE68643 and GSE73457). Gene Ontology biological process and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis was performed using the Database for Annotation, Visualization and Integrated Discovery. MLL-AF9-associated chromatin immunoprecipitation sequencing (ChIP-Seq) data was analyzed and identified binding sites for MLL-AF9 and wild type MLL (MLL WT). The ChIP-Seq of histone modification data was downloaded from the GEO database, including histone 3 lysine 4 trimethylation (H3K4me3), histone 3 lysine 79 dimethylation (H3K79me2) and histone 3 lysine 27 acetylation (H3K27ac), was used for comparing histone modification marks between the MLL-AF9 leukemia cells and normal hematopoietic cells at MLL-AF9 and MLL WT binding sites. The differentially expressed genes with the same trend in H3K79me2, H3K27ac and H3K4me3 alteration were identified as potential MLL-AF9 direct target genes. Upon validation using RNA-Seq data from the Therapeutically Applicable Research to Generate Effective Treatments AML project, eight potential direct target genes of MLL-AF9 were identified and further confirmed in MLL-AF9 mouse model using reverse transcription-quantitative polymerase chain reaction. These genes may have a critical role in AML with MLL-AF9 translocation.

An extra chromosome 9 derived from either a normal chromosome 9 or a derivative chromosome 9 in a patient with acute myeloid leukemia positive for t(9;11)(p21.3;q23.3): A case report.

Translocation (9;11)(p21.3;q23.3) is one of the most common lysine methyltransferase 2A (KMT2A)-rearrangements in de novo and therapy-related acute myeloid leukemia (AML). Numerous in vitro and in vivo studies have demonstrated that the KMT2A/MLLT3 super elongation complex subunit (MLLT3) fusion gene on the derivative chromosome 11 serves a crucial role in leukemogenesis. Trisomy 9 as a secondary chromosome change in patients with t(9;11) is relatively rare. The present study reported a unique case of AML with a chromosome 9 trisomy secondary to t(9;11)(p21.3;q23.3) through the cytogenetic analysis of leukemic blood and bone marrow. Further characterization with fluorescence in situ hybridization and array comparative genomic hybridization analysis revealed that this extra chromosome 9 was either a copy of normal chromosome 9 or a derivative chromosome 9. Conversely with the previously reported favorable outcome of AML patients with t(9;11)(p21.3;q23.3), in the present study, the cells with only translocation persisted, whereas the cells with an extra chromosome 9 disappeared following initial chemotherapy. With this unique case, the present study hypothesized that the extra chromosome 9 could serve a crucial role in AML disease progression and contribute to cellular sensitivity to chemotherapy.

AFF1 acetylation by p300 temporally inhibits transcription during genotoxic stress response

Soon after exposure to genotoxic reagents, mammalian cells inhibit transcription to prevent collisions with repair machinery and to mount a proper DNA damage response. However, mechanisms underlying early transcriptional inhibition are poorly understood. In this report, we show that site-specific acetylation of super elongation complex (SEC) subunit AFF1 by p300 reduces its interaction with other SEC components and impairs P-TEFb-mediated C-terminal domain phosphorylation of RNA polymerase II both in vitro and in vivo. Reexpression of wild-type AFF1, but not an acetylation mimic mutant, restores SEC component recruitment and target gene expression in AFF1 knockdown cells. Physiologically, we show that, upon genotoxic exposure, p300-mediated AFF1 acetylation is dynamic and strongly correlated with concomitant global down-regulation of transcription-and that this can be reversed by overexpression of an acetylation-defective AFF1 mutant. Therefore, we describe a mechanism of dynamic transcriptional regulation involving p300-mediated acetylation of a key elongation factor during genotoxic stress.

Transcriptional addiction in mixed lineage leukemia: new avenues for target therapies.

Mixed lineage leukemia (MLL) is an aggressive and refractory blood cancer that predominantly occurs in pediatric patients and is often associated with poor prognosis and dismal outcomes. Thus far, no effective target therapy for the treatment of MLL leukemia is available. MLL leukemia is caused by the rearrangement of MLL genes at 11q23, which generates various MLL chimeric proteins that promote leukemogenesis through transcriptional misregulation of MLL target genes. Biochemical studies on MLL chimeras have identified that the most common partners exist in the superelongation complex (SEC) and DOT1L complex, which activate or sustain MLL target gene expression through processive transcription elongation. The results of these studies indicate a transcription-related mechanism for MLL leukemogenesis and maintenance. In this study, we first review the history of MLL leukemia and its related clinical features. Then, we discuss the biological functions of MLL and MLL chimeras, significant cooperating events, and transcriptional addiction mechanisms in MLL leukemia with an emphasis on potential and rational therapy development. Collectively, we believe that targeting the transcriptional addiction mediated by SEC and the DOT1L complex will provide new avenues for target therapies in MLL leukemia and serve as a novel paradigm for targeting transcriptional addiction in other cancers.

Abstract 3125: SOX2 controls the tumor initiation and development and acts as a prognostic predictor of oral squamous cell carcinoma

Abstract Background: Sex determining region Y-BOX2 (SOX2), a regulator of the self-renewal ability of stem cells, plays a compelling role in initiation and development of cancer. However, little is known about the role and network controlling of SOX2 in oral squamous cell carcinoma (OSCC) development. Method: In this study, we conducted a retrospective cohort of 229 OSCC patients from two independent Chinese centers from May 2003 to August 2012. SOX2 expression in these samples was determined by immunostaining. Univariate and multivariate analyses were performed. Then we generated SOX2-creERT2 mice that expressing an inducible Cre recombinase under the control of a minimal promoter of SOX2, along with a tdTomato reporter. These mice were exposed to low dose 4NQO in the drinking water either for 8, 12 or 16 weeks. We further investigate the network controlling of SOX2 by ChIP assay. Then we detected the expression of SOX2 in response to AFF4 knockdown and overexpression by western blot, respectively. Furthermore, we used wound-healing, transwell, sphere formation and limiting dilution assay to confirm whether SOX2 expression was regulated by AFF4. Results: In the multiple cohorts, high SOX2 protein expression was negatively correlated with the overall survival for OSCC patients. Impressively, we found chemotherapy only improved outcomes of patients with low SOX2 expression, but very limited effects to high SOX2 expression patients. Meanwhile low SOX2 expression patients had significantly better survival with rather than without chemotherapies. Lineage tracing study in vivo suggested that SOX2 positive cells play an important role in the carcinogenesis of OSCC as epithelial stem cells. Increasing SOX2 positive cells and their daughter cells was a major trigger in the initiation of OSCC. Mechanistically, we found SOX2 was regulated by AFF4, the core component of Super elongation complex (SEC) to promote the proliferation, migration and invasion of OSCC cells. SOX2 expression changed in parallel with AFF4 expression in response to depletion and overexpression of AFF4, respectively. More importantly, the inhibited proliferation, migration, invasion and ALDH activity induced by knockdown of AFF4 in OSCC cells were rescued by overexpression of SOX2. Conclusion: Collectively, our findings indicate SOX2 acts as a prognostic predictor and controls the tumor initiation and development in oral squamous cell carcinoma. SOX2 may serve as a potential target of therapies for patients with OSCC. Note: This abstract was not presented at the meeting. Citation Format: ZIHAO WEI, FENGYING YIN, PENG DENG, XUEFENG ZHANG, JING DENG, JING LI, LIANG XIE, QIANMING CHEN. SOX2 controls the tumor initiation and development and acts as a prognostic predictor of oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3125.

Structure of the super-elongation complex subunit AFF4 C-terminal homology domain reveals requirements for AFF homo- and heterodimerization

AF4/FMR2 family member 4 (AFF4) is the scaffold protein of the multisubunit super-elongation complex, which plays key roles in the release of RNA polymerase II from promoter-proximal pausing and in the transactivation of HIV-1 transcription. AFF4 consists of an intrinsically disordered N-terminal region that interacts with other super-elongation complex subunits and a C-terminal homology domain (CHD) that is conserved among AF4/FMR2 family proteins, including AFF1, AFF2, AFF3, and AFF4. Here, we solved the X-ray crystal structure of the CHD in human AFF4 (AFF4-CHD) to 2.2 Å resolution and characterized its biochemical properties. The structure disclosed that AFF4-CHD folds into a novel domain that consists of eight helices and is distantly related to tetratrico peptide repeat motifs. Our analyses further revealed that AFF4-CHD mediates the formation of an AFF4 homodimer or an AFF1-AFF4 heterodimer. Results from fluorescence anisotropy experiments suggested that AFF4-CHD interacts with both RNA and DNA in vitro Furthermore, we identified a surface loop region in AFF4-CHD as a substrate for the P-TEFb kinase cyclin-dependent kinase 9, which triggers release of polymerase II from promoter-proximal pausing sites. In conclusion, the AFF-CHD structure and biochemical analyses reported here reveal the molecular basis for the homo- and heterodimerization of AFF proteins and implicate the AFF4-CHD in nucleic acid interactions. The high conservation of the CHD among several other proteins suggests that our results are also relevant for understanding other CHD-containing proteins and their dimerization behavior.

Functional diversity of inhibitors tackling the differentiation blockage of MLL-rearranged leukemia

IntroductionThe chromosomal rearrangements of the mixed-lineage leukemia gene MLL (KMT2A) have been extensively characterized as a potent oncogenic driver in leukemia. For its oncogenic function, most MLL-fusion proteins exploit the multienzyme super elongation complex leading to elevated expression of MLL target genes. High expression of MLL target genes overwrites the normal hematopoietic differentiation program, resulting in undifferentiated blasts characterized by the capacity to self-renew. Although extensive resources devoted to increased understanding of therapeutic targets to overcome de-differentiation in ALL/AML, the inter-dependencies of targets are still not well described. The majority of inhibitors potentially interfering with MLL-fusion protein driven transformation have been characterized in individual studies, which so far hindered their direct cross-comparison.MethodsIn our study, we characterized head-to-head clinical stage inhibitors for BET, DHODH, DOT1L as well as two novel inhibitors for CDK9 and the Menin-MLL interaction with a focus on differentiation induction. We profiled those inhibitors for global gene expression effects in a large cell line panel and examined cellular responses such as inhibition of proliferation, apoptosis induction, cell cycle arrest, surface marker expression, morphological phenotype changes, and phagocytosis as functional differentiation readout. We also verified the combination potential of those inhibitors on proliferation and differentiation level.ResultsOur analysis revealed significant differences in differentiation induction and in modulating MLL-fusion target gene expression. We observed Menin-MLL and DOT1L inhibitors act very specifically on MLL-fused leukemia cell lines, whereas inhibitors of BET, DHODH and P-TEFb have strong effects beyond MLL-fusions. Significant differentiation effects were detected for Menin-MLL, DOT1L, and DHODH inhibitors, whereas BET and CDK9 inhibitors primarily induced apoptosis in AML/ALL cancer models. For the first time, we explored combination potential of the abovementioned inhibitors with regards to overcoming the differentiation blockage.ConclusionOur findings show substantial diversity in the molecular activities of those inhibitors and provide valuable insights into the further developmental potential as single agents or in combinations in MLL-fused leukemia.

Fused in sarcoma silences HIV gene transcription and maintains viral latency through suppressing AFF4 gene activation

BackgroundThe human immunodeficiency virus (HIV) cell reservoir is currently a main obstacle towards complete eradication of the virus. This infected pool is refractory to anti-viral therapy and harbors integrated proviruses that are transcriptionally repressed but replication competent. As transcription silencing is key for establishing the HIV reservoir, significant efforts have been made to understand the mechanism that regulate HIV gene transcription, and the role of the elongation machinery in promoting this step. However, while the role of the super elongation complex (SEC) in enhancing transcription activation of HIV is well established, the function of SEC in modulating viral latency is less defined and its cell partners are yet to be identified.ResultsIn this study we identify fused in sarcoma (FUS) as a partner of AFF4 in cells. FUS inhibits the activation of HIV transcription by AFF4 and ELL2, and silences overall HIV gene transcription. Concordantly, depletion of FUS elevates the occupancy of AFF4 and Cdk9 on the viral promoter and activates HIV gene transcription. Live cell imaging demonstrates that FUS co-localizes with AFF4 within nuclear punctuated condensates, which are disrupted upon treating cells with aliphatic alcohol. In HIV infected cells, knockout of FUS delays the gradual entry of HIV into latency, and similarly promotes viral activation in a T cell latency model that is treated with JQ1. Finally, effects of FUS on HIV gene transcription are also exhibited genome wide, where FUS mainly occupies gene promoters at transcription starting sites, while its knockdown leads to an increase in AFF4 and Cdk9 occupancy on gene promoters of FUS affected genes.ConclusionsTowards eliminating the HIV infected reservoir, understanding the mechanisms by which the virus persists in the face of therapy is important. Our observations show that FUS regulates both HIV and global gene transcription and modulates viral latency, thus can potentially serve as a target for future therapy that sets to reactivate HIV from its latent state.

ELL2 Is Downregulated and Associated with Galactose-Deficient IgA1 in IgA Nephropathy.

Background Galactose-deficient IgA1 (Gd-IgA1) is an important causal factor in IgA nephropathy; however, the underlying mechanism for the production of Gd-IgA1 is unknown. The elongation factor for RNA polymerase II (ELL2), which encoded a key component of the superelongation complex (SEC), drives secretory-specific Ig mRNA production. Methods We enrolled 21 patients with IgAN, 18 healthy controls, and 20 patients with non-IgAN glomerulonephritis. The differential expression of ELL2 was compared using publically available data from Gene Expression Omnibus (GEO) datasets. The relationship between ELL2 expressions and galactose-deficient IgA1 (Gd-IgA1) levels in serum were also studied. At last, the results were validated by shELL2 treatment experiment. Results We found that the number of CD19+ B cells was increased in IgAN patients compared to healthy controls. The expression level of ELL2 in patients with IgAN was significantly lower than that of healthy control and disease control. Consistent with present results, the lower ELL2 expression in IgAN patients was observed in microarray expression profiles from GEO datasets. Pearson correlation analysis showed that ELL2 expression negatively correlated with Gd-IgA1 levels. Furthermore, in an in vitro experiment, we found that loss of ELL2 function in human B lymphoma DAKIKI cells, an IgA1-producing cell line, increased the levels of Gd-IgA1, which confirmed that ELL2 modulated the levels of Gd-IgA1. Conclusion Our findings implied that decreased ELL2 expression was negatively correlated with the numbers of B cells and aberrant glycosylation of IgA1 in IgAN.

Rewiring the Epigenetic Networks in MLL-Rearranged Leukemias: Epigenetic Dysregulation and Pharmacological Interventions.

Leukemias driven by chromosomal translocation of the mixed-lineage leukemia gene (MLL or KMT2A) are highly prevalent in pediatric oncology. The poor survival rate and lack of an effective targeted therapy for patients with MLL-rearranged (MLL-r) leukemias emphasize an urgent need for improved knowledge and novel therapeutic approaches for these malignancies. The resulting chimeric products of MLL gene rearrangements, i.e., MLL-fusion proteins (MLL-FPs), are capable of transforming hematopoietic stem/progenitor cells (HSPCs) into leukemic blasts. The ability of MLL-FPs to reprogram HSPCs toward leukemia requires the involvement of multiple chromatin effectors, including the histone 3 lysine 79 methyltransferase DOT1L, the chromatin epigenetic reader BRD4, and the super elongation complex. These epigenetic regulators constitute a complicated network that dictates maintenance of the leukemia program, and therefore represent an important cluster of therapeutic opportunities. In this review, we will discuss the role of MLL and its fusion partners in normal HSPCs and hematopoiesis, including the links between chromatin effectors, epigenetic landscapes, and leukemia development, and summarize current approaches to therapeutic targeting of MLL-r leukemias.

SAT-037 Functional Interplay between Distinct P-TEFb Complexes Following Endothelin or α1-Adrenergic Receptor Activation in Rat Cardiomyocytes

Heart disease is characterized by the remodelling of cardiac tissue following prolonged stress on the heart, such as myocardial infarction and chronic hypertension. The heart initially undergoes hypertrophy of the individual contractile unit, the cardiomyocyte, in order to preserve cardiac function. However, prolonged stress can lead to fibrosis, cardiomyocyte death, and subsequent heart failure. Cardiac remodelling is mediated predominantly through neurohormonal activation of G protein-coupled receptors (GPCRs) expressed by cardiomyocytes and other cell types in the heart. Current therapeutic approaches target implicated GPCRs to slow disease progression by inhibiting signalling pathways leading to remodelling. While this approach benefits a portion of patients, other therapeutic approaches are required to improve disease prognosis (1). A potential target downstream of GPCR activation is the transcription regulator positive transcription elongation factor b (P-TEFb). Hormonal activation of multiple GPCRs, such as the α1-adrenergic receptor (α1AR) by epinephrine or the endothelin receptor (ETR) by endothelin-1, leads to P-TEFb-dependent cardiomyocyte hypertrophy, a hallmark of cardiac remodelling (2). Active P-TEFb is recruited to chromatin to regulate gene expression as a constituent of two complexes, either through interactions with the Super Elongation Complex (SEC) or the bromodomain and extra-terminal protein Brd4. Small molecule inhibition of Brd4 prevents hypertrophy and activation of inflammatory and profibrotic genes in cardiomyocytes (3). On the other hand, the role of the SEC in regulating cardiomyocyte gene expression has not been examined. Furthermore, the signalling mechanisms leading to activation of either complex and subsequent gene expression changes are not known. We hypothesized that activation and interplay between distinct P-TEFb complexes differs following activation of different GPCRs implicated in heart failure. We assessed the role of Brd4 and the SEC downstream of the endogenous ETR and α1AR stimulated with endothelin-1 or phenylephrine, respectively, in primary neonatal rat cardiomyocytes. Although P-TEFb activity is required downstream of both receptors to drive gene expression changes and cardiomyocyte hypertrophy, we find evidence for receptor-specific differences in the functions of the SEC and the Brd4 complex. We are currently investigating signalling mechanisms regulating each complex downstream of the ETR and α1AR. Understanding how mechanisms downstream of various hormones differentially regulate transcription has clinical implications as therapies targeting transcriptional machinery are being explored for heart failure. (1) Wang et al., Circ Res. 2018; 123:716-35 (2) Sano et al., Nat Med. 2002; 8:1310-7 (3) Duan et al., Sci Transl Med; 9(390)

Discovery of a Selective Inhibitor for the YEATS Domains of ENL/AF9

Eleven-nineteen leukemia (ENL) contains an epigenetic reader domain (YEATS domain) that recognizes lysine acylation on histone 3 and facilitates transcription initiation and elongation through its interactions with the super elongation complex (SEC) and the histone methyl transferase DOT1L. Although it has been known for its role as a fusion protein in mixed lineage leukemia (MLL), overexpression of native ENL, and thus dysregulation of downstream genes in acute myeloid leukemia (AML), has recently been implicated as a driver of disease that is reliant on the epigenetic reader activity of the YEATS domain. We developed a peptide displacement assay (histone 3 tail with acylated lysine) and screened a small-molecule library totaling more than 24,000 compounds for their propensity to disrupt the YEATS domain–histone peptide binding. Among these, we identified a first-in-class dual inhibitor of ENL (Kd = 745 ± 45 nM) and its paralog AF9 (Kd = 523 ± 53 nM) and performed “SAR by catalog” with the aim of starting the development of a chemical probe for ENL.

Reiterative Enrichment and Authentication of CRISPRi Targets (REACT) identifies the proteasome as a key contributor to HIV-1 latency.

The establishment of HIV-1 latency gives rise to persistent chronic infection that requires life-long treatment. To reverse latency for viral eradiation, the HIV-1 Tat protein and its associated ELL2-containing Super Elongation Complexes (ELL2-SECs) are essential to activate HIV-1 transcription. Despite efforts to identify effective latency-reversing agents (LRA), avenues for exposing latent HIV-1 remain inadequate, prompting the need to identify novel LRA targets. Here, by conducting a CRISPR interference-based screen to reiteratively enrich loss-of-function genotypes that increase HIV-1 transcription in latently infected CD4+ T cells, we have discovered a key role of the proteasome in maintaining viral latency. Downregulating or inhibiting the proteasome promotes Tat-transactivation in cell line models. Furthermore, the FDA-approved proteasome inhibitors bortezomib and carfilzomib strongly synergize with existing LRAs to reactivate HIV-1 in CD4+ T cells from antiretroviral therapy-suppressed individuals without inducing cell activation or proliferation. Mechanistically, downregulating/inhibiting the proteasome elevates the levels of ELL2 and ELL2-SECs to enable Tat-transactivation, indicating the proteasome-ELL2 axis as a key regulator of HIV-1 latency and promising target for therapeutic intervention.

RNA Polymerase II-Dependent Transcription Initiated by Selectivity Factor 1: A Central Mechanism Used by MLL Fusion Proteins in Leukemic Transformation.

Cancer cells transcribe RNAs in a characteristic manner in order to maintain their oncogenic potentials. In eukaryotes, RNA is polymerized by three distinct RNA polymerases, RNA polymerase I, II, and III (RNAP1, RNAP2, and RNAP3, respectively). The transcriptional machinery that initiates each transcription reaction has been purified and characterized. Selectivity factor 1 (SL1) is the complex responsible for RNAP1 pre-initiation complex formation. However, whether it plays any role in RNAP2-dependent transcription remains unclear. Our group previously found that SL1 specifically associates with AF4 family proteins. AF4 family proteins form the AEP complex with ENL family proteins and the P-TEFb elongation factor. Similar complexes have been independently characterized by several different laboratories and are often referred to as super elongation complex. The involvement of AEP in RNAP2-dependent transcription indicates that SL1 must play an important role in RNAP2-dependent transcription. To date, this role of SL1 has not been appreciated. In leukemia, AF4 and ENL family genes are frequently rearranged to form chimeric fusion genes with MLL. The resultant MLL fusion genes produce chimeric MLL fusion proteins comprising MLL and AEP components. The MLL portion functions as a targeting module, which specifically binds chromatin containing di-/tri-methylated histone H3 lysine 36 and non-methylated CpGs. This type of chromatin is enriched at the promoters of transcriptionally active genes which allows MLL fusion proteins to selectively bind to transcriptionally-active/CpG-rich gene promoters. The fusion partner portion, which recruits other AEP components and SL1, is responsible for activation of RNAP2-dependent transcription. Consequently, MLL fusion proteins constitutively activate the transcription of previously-transcribed MLL target genes. Structure/function analysis has shown that the ability of MLL fusion proteins to transform hematopoietic progenitors depends on the recruitment of AEP and SL1. Thus, the AEP/SL1-mediated gene activation pathway appears to be the central mechanism of MLL fusion-mediated transcriptional activation. However, the molecular mechanism by which SL1 activates RNAP2-dependent transcription remains largely unclear. This review aims to cover recent discoveries of the mechanism of transcriptional activation by MLL fusion proteins and to introduce novel roles of SL1 in RNAP2-dependent transcription by discussing how the RNAP1 machinery may be involved in RNAP2-dependent gene regulation.

Multivalent Role of Human TFIID in Recruiting Elongation Components at the Promoter-Proximal Region for Transcriptional Control

Despite substantial progress in our understanding of the players involved and the regulatory mechanisms controlling the initiation and elongation steps of transcription, little is known about the recruitment of elongation factors at promoter-proximal regions for the initiation-to-elongation transition. Here, we show evidence that human TFIID, which initiates pre-initiation complex (PIC) assembly, contributes to regulating the recruitment of super-elongation complex (SEC) components at the promoter-proximal region through interactions among selective TAF and SEC components. Invitro direct interactions, coupled with cell-based assays, identified an important poly-Ser domain within SEC components that are involved in their interaction with TFIID. DNA template-based recruitment assays, using purified components, further show a direct role for poly-Ser domain-dependent TFIID interaction in recruiting SEC components on target DNA. Consistently, ChIP and RNA analyses have shown the importance of this mechanism in TFIID-dependent SEC recruitment and target gene expression within mammalian cells.

Structural mechanism for HIV-1 TAR loop recognition by Tat and the super elongation complex

Promoter-proximal pausing by RNA polymerase II (Pol II) is a key regulatory step in human immunodeficiency virus-1 (HIV-1) transcription and thus in the reversal of HIV latency. By binding to the nascent transactivating response region (TAR) RNA, HIV-1 Tat recruits the human super elongation complex (SEC) to the promoter and releases paused Pol II. Structural studies of TAR interactions have been largely focused on interactions between the TAR bulge and the arginine-rich motif (ARM) of Tat. Here, the crystal structure of the TAR loop in complex with Tat and the SEC core was determined at a 3.5-Å resolution. The bound TAR loop is stabilized by cross-loop hydrogen bonds. It makes structure-specific contacts with the side chains of the Cyclin T1 Tat-TAR recognition motif (TRM) and the zinc-coordinating loop of Tat. The TAR loop phosphate backbone forms electrostatic and VDW interactions with positively charged side chains of the CycT1 TRM. Mutational analysis showed that these interactions contribute importantly to binding affinity. The Tat ARM was present in the crystallized construct; however, it was not visualized in the electron density, and the TAR bulge was not formed in the RNA construct used in crystallization. Binding assays showed that TAR bulge-Tat ARM interactions contribute less to TAR binding affinity than TAR loop interactions with the CycT1 TRM and Tat core. Thus, the TAR loop evolved to make high-affinity interactions with the TRM while Tat has three roles: scaffolding and stabilizing the TRM, making specific interactions through its zinc-coordinating loop, and making electrostatic interactions through its ARM.

The Super Elongation Complex May Be Targetable in MYC-Driven Tumors

Abstract Identified small-molecule inhibitors of the super elongation complex (SEC) disrupt transcriptional elongation.

An ultra-high affinity ligand of HIV-1 TAR reveals the RNA structure recognized by P-TEFb.

The HIV-1 trans-activator protein Tat binds the trans-activation response element (TAR) to facilitate recruitment of the super elongation complex (SEC) to enhance transcription of the integrated pro-viral genome. The Tat–TAR interaction is critical for viral replication and the emergence of the virus from the latent state, therefore, inhibiting this interaction has long been pursued to discover new anti-viral or latency reversal agents. However, discovering active compounds that directly target RNA with high affinity and selectivity remains a significant challenge; limiting pre-clinical development. Here, we report the rational design of a macrocyclic peptide mimic of the arginine rich motif of Tat, which binds to TAR with low pM affinity and 100-fold selectivity against closely homologous RNAs. Despite these unprecedented binding properties, the new ligand (JB181) only moderately inhibits Tat-dependent reactivation in cells and recruitment of positive transcription elongation factor (P-TEFb) to TAR. The NMR structure of the JB181–TAR complex revealed that the ligand induces a structure in the TAR loop that closely mimics the P-TEFb/Tat1:57/AFF4/TAR complex. These results strongly suggest that high-affinity ligands which bind the UCU bulge are not likely to inhibit recruitment of the SEC and suggest that targeting of the TAR loop will be an essential feature of effective Tat inhibitors.

MYC in elongation and repression.

Transcription elongation supported by the super elongation complex, and histone H3 lysine 9 methylation and gene repression by G9a mediate the oncogenic function of MYC.