The Expression and Roles of the Super Elongation Complex in Mouse Cochlear Lgr5+ Progenitor Cells

The super elongation complex (SEC) has been reported to play a key role in the proliferation and differentiation of mouse embryonic stem cells. However, the expression pattern and function of the SEC in the inner ear has not been investigated. Here, we studied the inner ear expression pattern of three key SEC components, AFF1, AFF4, and ELL3, and found that these three proteins are all expressed in both cochlear hair cells (HCs)and supporting cells (SCs). We also cultured Lgr5+ inner ear progenitors in vitro for sphere-forming assays and differentiation assays in the presence of the SEC inhibitor flavopiridol. We found that flavopiridol treatment decreased the proliferation ability of Lgr5+ progenitors, while the differentiation ability of Lgr5+ progenitors was not affected. Our results suggest that the SEC might play important roles in regulating inner ear progenitors and thus regulating HC regeneration. Therefore, it will be very meaningful to further investigate the detailed roles of the SEC signaling pathway in the inner ear in vivo in order to develop effective treatments for sensorineural hearing loss.

The Expression and Roles of the Super Elongation Complex in Mouse Cochlear Lgr5+ Progenitor Cells

Background: The super elongation complex (SEC) has been reported to play a key role in the proliferation and differentiation of mouse embryonic stem cells. However, the expression pattern and function of the SEC in the inner ear has not been investigated.Methods: Here, we measured the expression of the three key SEC subunits AFF1, AFF4, and ELL3. We also used Lgr5-EGFP-Ires-CreERT2 mice to isolate the Lgr5+ progenitors via flow cytometry to evaluate the effect of the SEC on the proliferation and differentiation ability of Lgr5+ progenitors.Results: We studied the inner ear expression pattern of three key SEC components, AFF1, AFF4 and ELL3, and found that these three proteins are all expressed in both cochlear hair cells and supporting cells. We also cultured Lgr5+ inner ear progenitors in vitro for sphere-forming assays and differentiation assays in the presence of the SEC inhibitor flavopiridol. We found that flavopiridol treatment decreased the proliferation ability of Lgr5+ progenitors, while the differentiation ability of Lgr5+ progenitors was not affected.Conclusions: Our results suggest that the SEC might play important roles in regulating inner ear progenitors and thus regulating hair cell regeneration. Therefore, it will be very meaningful to further investigate the detailed roles of the SEC signaling pathway in the inner ear in vivo in order to develop effective treatments for sensorineural hearing loss.

DIPG-34. SUPER ELONGATION COMPLEX AS A TARGETABLE DEPENDENCY IN H3K27M+ DIFFUSE MIDLINE GLIOMA

Mutations in the histone 3 gene (H3K27M) are the eponymous driver in diffuse intrinsic pontine gliomas (DIPGs) and other diffuse midline gliomas (DMGs), aggressive pediatric brain tumors for which no curative therapy currently exists. To identify specific epigenetic dependencies within the context of the H3K27M mutation, we performed an shRNA screen targeting 408 genes classified as epigenetic/chromatin-associated molecules in patient-derived DMG cultures. This identified AFF4, a component of the super elongation complex (SEC), as necessary for DMG cells to maintain growth and self-renewal. We hypothesized that aberrant SEC expression occurs as a consequence of the H3K27M mutation and that this dysregulated SEC signaling overcomes repressive transcriptional regulation in order to suppresses differentiation and promote self-renewal of DMG tumor stem cells. We interrogated the role of AFF4 in DMG using an shRNA lentiviral approach. We demonstrate a significant decrease in in vitro clonogenicity and stem cell maintenance following AFF4 depletion. We employed RNA-seq-based gene set enrichment analysis to delineate differentiation programs under SEC regulatory control. Finally, we sought to determine whether CDK9, the catalytic subunit of the SEC, represents a therapeutic vulnerability in DMG. Using RNA polymerase II ChIP-seq, we demonstrate that CDK9 pharmacologic inhibition restores regulatory Pol II pausing, promotes cellular differentiation, and leads to potent anti-tumor effect both in vitro and in patient-derived xenograft models. These studies present a biologic rationale for the translational exploration of CDK9 inhibition as a promising therapeutic approach.

Inhibition of the Super Elongation Complex Suppresses Herpes Simplex Virus Immediate Early Gene Expression, Lytic Infection, and Reactivation from Latency

ABSTRACT Induction of herpes simplex virus (HSV) immediate early (IE) gene transcription promotes the initiation of lytic infection and reactivation from latency in sensory neurons. IE genes are transcribed by the cellular RNA polymerase II (RNAPII) and regulated by multiple transcription factors and coactivators. The HCF-1 cellular coactivator plays a central role in driving IE expression at multiple stages through interactions with transcription factors, chromatin modulation complexes, and transcription elongation components, including the active super elongation complex/P-TEFb (SEC-P-TEFb). Here, we demonstrate that the SEC occupies the promoters of HSV IE genes during the initiation of lytic infection and during reactivation from latency. Specific inhibitors of the SEC suppress viral IE expression and block the spread of HSV infection. Significantly, these inhibitors also block the initiation of viral reactivation from latency in sensory ganglia. The potent suppression of IE gene expression by SEC inhibitors indicates that transcriptional elongation represents a determining rate-limiting stage in HSV IE gene transcription and that the SEC plays a critical role in driving productive elongation during both phases of the viral life cycle. Most importantly, this supports the model that signal-mediated induction of SEC-P-TEFb levels can promote reactivation of a population of poised latent genomes. IMPORTANCE HSV infections can cause pathologies ranging from recurrent lesions to significant ocular disease. Initiation of lytic infection and reactivation from latency in sensory neurons are dependent on the induced expression of the viral immediate early genes. Transcription of these genes is controlled at multiple levels, including modulation of the chromatin state of the viral genome and appropriate recruitment of transcription factors and coactivators. Following initiation of transcription, IE genes are subject to a key regulatory stage in which transcriptional elongation rates are controlled by the activity of the super elongation complex. Inhibition of the SEC blocks both lytic infection and reactivation from latency in sensory neurons. In addition to providing insights into the mechanisms controlling viral infection and reactivation, inhibitors of critical components such as the SEC may represent novel antivirals.

Competition between PAF1 and MLL1/COMPASS confers the opposing function of LEDGF/p75 in HIV latency and proviral reactivation

Transcriptional status determines the HIV replicative state in infected patients. However, the transcriptional mechanisms for proviral replication control remain unclear. In this study, we show that, apart from its function in HIV integration, LEDGF/p75 differentially regulates HIV transcription in latency and proviral reactivation. During latency, LEDGF/p75 suppresses proviral transcription via promoter-proximal pausing of RNA polymerase II (Pol II) by recruiting PAF1 complex to the provirus. Following latency reversal, MLL1 complex competitively displaces PAF1 from the provirus through casein kinase II (CKII)–dependent association with LEDGF/p75. Depleting or pharmacologically inhibiting CKII prevents PAF1 dissociation and abrogates the recruitment of both MLL1 and Super Elongation Complex (SEC) to the provirus, thereby impairing transcriptional reactivation for latency reversal. These findings, therefore, provide a mechanistic understanding of how LEDGF/p75 coordinates its distinct regulatory functions at different stages of the post-integrated HIV life cycles. Targeting these mechanisms may have a therapeutic potential to eradicate HIV infection.

ENL initiates multivalent phase separation of the super elongation complex (SEC) in controlling rapid transcriptional activation

Release of paused RNA polymerase II (Pol II) requires incorporation of the positive transcription elongation factor b (P-TEFb) into the super elongation complex (SEC), thus resulting in rapid yet synchronous transcriptional activation. However, the mechanism underlying dynamic transition of P-TEFb from inactive to active state remains unclear. Here, we found that the SEC components are able to compartmentalize and concentrate P-TEFb via liquid-liquid phase separation from the soluble inactive HEXIM1 containing the P-TEFb complex. Specifically, ENL or its intrinsically disordered region is sufficient to initiate the liquid droplet formation of SEC. AFF4 functions together with ENL in fluidizing SEC droplets. SEC droplets are fast and dynamically formed upon serum exposure and required for rapid transcriptional induction. We also found that the fusion of ENL with MLL can boost SEC phase separation. In summary, our results suggest a critical role of multivalent phase separation of SEC in controlling transcriptional pause release.

Super elongation complex as a targetable dependency in diffuse midline glioma

Mutations in the histone 3 gene (H3K27M) are the eponymous drivers in diffuse intrinsic pontine gliomas (DIPGs) and other diffuse midline gliomas (DMGs), aggressive pediatric brain cancers for which no curative therapy currently exists. The salient molecular consequence of these recurrent oncohistones is a global loss of repressive H3K27me3 residues and broad epigenetic dysregulation. In order to identify specific, therapeutically targetable epigenetic dependencies within this disease context, we performed an shRNA screen targeting 408 genes classified as epigenetic/chromatin-associated molecules in patient-derived DMG cultures. This approach identified AFF4, the scaffold protein of the super elongation complex (SEC), as a previously-undescribed dependency in DMG. Interrogation of SEC function demonstrated a key role for maintaining DMG cell viability and clonogenic potential while promoting self-renewal of DMG tumor stem cells. Small-molecule inhibition of the SEC with the highly-specific, clinically relevant CDK9 inhibitors atuveciclib and AZD4573 restores regulatory RNA polymerase II pausing, promotes cellular differentiation, and leads to potent anti-tumor effect both in vitro and in patient-derived xenograft models. These studies present a biologic rationale for translational exploration of CDK9 inhibition as a promising therapeutic approach in a disease which currently has no effective medical therapies.