Structure of the human SAGA coactivator complex

The SAGA complex is a regulatory hub involved in gene regulation, chromatin modification, DNA damage repair and signaling. While structures of yeast SAGA (ySAGA) have been reported, there are noteworthy functional and compositional differences for this complex in metazoans. Here we present the cryogenic-electron microscopy (cryo-EM) structure of human SAGA (hSAGA) and show how the arrangement of distinct structural elements results in a globally divergent organization from that of yeast, with a different interface tethering the core module to the TRRAP subunit, resulting in a dramatically altered geometry of functional elements and with the integration of a metazoan-specific splicing module. Our hSAGA structure reveals the presence of an inositol hexakisphosphate (InsP6) binding site in TRRAP and an unusual property of its pseudo-(Ψ)PIKK. Finally, we map human disease mutations, thus providing the needed framework for structure-guided drug design of this important therapeutic target for human developmental diseases and cancer.

The SAGA core module is critical during Drosophila oogenesis and is broadly recruited to promoters

The Spt/Ada-Gcn5 Acetyltransferase (SAGA) coactivator complex has multiple modules with different enzymatic and non-enzymatic functions. How each module contributes to gene expression is not well understood. During Drosophila oogenesis, the enzymatic functions are not equally required, which may indicate that different genes require different enzymatic functions. An analogy for this phenomenon is the handyman principle: while a handyman has many tools, which tool he uses depends on what requires maintenance. Here we analyzed the role of the non-enzymatic core module during Drosophila oogenesis, which interacts with TBP. We show that depletion of SAGA-specific core subunits blocked egg chamber development at earlier stages than depletion of enzymatic subunits. These results, as well as additional genetic analyses, point to an interaction with TBP and suggest a differential role of SAGA modules at different promoter types. However, SAGA subunits co-occupied all promoter types of active genes in ChIP-seq and ChIP-nexus experiments, and the complex was not specifically associated with distinct promoter types in the ovary. The high-resolution genomic binding profiles were congruent with SAGA recruitment by activators upstream of the start site, and retention on chromatin by interactions with modified histones downstream of the start site. Our data illustrate that a distinct genetic requirement for specific components may conceal the fact that the entire complex is physically present and suggests that the biological context defines which module functions are critical.

Connection of core and tail Mediator modules restrains transcription from TFIID-dependent promoters

The Mediator coactivator complex is divided into four modules: head, middle, tail, and kinase. Deletion of the architectural subunit Med16 separates core Mediator (cMed), comprising the head, middle, and scaffold (Med14), from the tail. However, the direct global effects of tail/cMed disconnection are unclear. We find that rapid depletion of Med16 downregulates genes that require the SAGA complex for full expression, consistent with their reported tail dependence, but also moderately overactivates TFIID-dependent genes in a manner partly dependent on the separated tail, which remains associated with upstream activating sequences. Suppression of TBP dynamics via removal of the Mot1 ATPase partially restores normal transcriptional activity to Med16-depleted cells, suggesting that cMed/tail separation results in an imbalance in the levels of PIC formation at SAGA-requiring and TFIID-dependent genes. We propose that the preferential regulation of SAGA-requiring genes by tailed Mediator helps maintain a proper balance of transcription between these genes and those more dependent on TFIID.

SUMOylated non-canonical polycomb PRC1.6 complex as a prerequisite for recruitment of transcription factor RBPJ

Background Notch signaling controls cell fate decisions in many contexts during development and adult stem cell homeostasis and, when dysregulated, leads to carcinogenesis. The central transcription factor RBPJ assembles the Notch coactivator complex in the presence of Notch signaling, and represses Notch target gene expression in its absence. Results We identified L3MBTL2 and additional members of the non-canonical polycomb repressive PRC1.6 complex in DNA-bound RBPJ associated complexes and demonstrate that L3MBTL2 directly interacts with RBPJ. Depletion of RBPJ does not affect occupancy of PRC1.6 components at Notch target genes. Conversely, absence of L3MBTL2 reduces RBPJ occupancy at enhancers of Notch target genes. Since L3MBTL2 and additional members of the PRC1.6 are known to be SUMOylated, we investigated whether RBPJ uses SUMO-moieties as contact points. Indeed, we found that RBPJ binds to SUMO2/3 and that this interaction depends on a defined SUMO-interaction motif. Furthermore, we show that pharmacological inhibition of SUMOylation reduces RBPJ occupancy at Notch target genes. Conclusions We propose that the PRC1.6 complex and its conjugated SUMO-modifications provide a favorable environment for binding of RBPJ to Notch target genes.

The SAGA core module is critical during Drosophila oogenesis and is broadly recruited to promoters

The Spt/Ada-Gcn5 Acetyltransferase (SAGA) coactivator complex has multiple modules with different enzymatic and non-enzymatic functions. How each module contributes to gene activation in specific biological contexts is not well understood. Here we analyzed the role of the non-enzymatic core module during Drosophila oogenesis. We show that depletion of several SAGA-specific subunits belonging to the core module blocked egg chamber development during mid-oogenesis stages, resulting in stronger phenotypes than those obtained after depletion of SAGA’s histone acetyltransferase module or deubiquitination module. These results, as well as additional genetic analyses pointing to an interaction with TBP, suggested a differential role of SAGA modules at different promoter types. However, SAGA subunits co-occupied all promoter types of active genes in ChIP-seq and ChIP-nexus experiments. Thus, the SAGA complex appears to occupy promoters in its entirety, consistent with the strong biochemical integrity of the complex. The high-resolution genomic binding profiles are congruent with SAGA recruitment by activators upstream of the start site, and retention on chromatin by interactions with modified histones downstream of the start site. The stronger genetic requirement of the core module during oogenesis may therefore be explained through its interaction with TBP or its role in recruiting the enzymatic modules to the promoter. We propose the handyman principle, which posits that a distinct genetic requirement for specific components may conceal the fact that the entire complex is physically present.

EP400NL is required for cMyc-mediated PD-L1 gene activation by forming a transcriptional coactivator complex

EP400 is an ATP-dependent chromatin remodeling enzyme that has been implicated in DNA double-strand break repair and transcription regulation including Myc-dependent gene expression. We previously showed that the N-terminal domain of EP400 increases the efficacy of chemotherapeutic drugs against cancer cells. As the EP400 N-terminal-Like (EP400NL) gene resides next to the EP400 gene locus prompted us to investigate whether EP400NL also plays a similar role in epigenetic transcriptional regulation to the full-length EP400 protein. We found that EP400NL forms a human NuA4-like chromatin remodelling complex that lacks both the TIP60 histone acetyltransferase and EP400 ATPase. However, this EP400NL complex displays H2A.Z deposition activity on a chromatin template comparable to the human NuA4 complex, suggesting another associated ATPase such as BRG1 or RuvBL1/RuvBL2 catalyses the reaction. We also demonstrated that the transcriptional coactivator function of EP400NL is required for cMyc and IFNγ-mediated PD-L1 gene activation. Collectively, our studies show that EP400NL plays a role as a transcription coactivator for cMyc-mediated gene expression and provides a potential target to modulate PD-L1 expression in cancer immunotherapy.

SUMOylated Non-canonical Polycomb PRC1.6 Complex as a Prerequisite for Recruitment of Transcription Factor RBPJ

BackgroundNotch signaling controls cell fate decisions in many contexts during development and adult stem cell homeostasis and, when dysregulated, leads to carcinogenesis. The central transcription factor RBPJ assembles the Notch coactivator complex in the presence of Notch signalling, and represses Notch target gene expression in its absence.ResultsWe identified L3MBTL2 and additional members of the non-canonical polycomb repressive PRC1.6 complex in DNA-bound RBPJ associated complexes and demonstrate that L3MBTL2 directly interacts with RBPJ. Depletion of RBPJ does not affect occupancy of PRC1.6 components at Notch target genes. Conversely, absence of L3MBTL2 reduces RBPJ occupancy at enhancers of Notch target genes. Since L3MBTL2 and additional members of the PRC1.6 are known to be SUMOylated, we investigated whether RBPJ uses SUMO-moieties as contact points. Indeed, we found that RBPJ binds to SUMO2/3 and that this interaction depends on a defined SUMO-interaction motif. Furthermore, we show that pharmacological inhibition of SUMOylation reduces RBPJ occupancy at Notch target genes.ConclusionsWe propose that the PRC1.6 complex and its conjugated SUMO-modifications provide a scaffold that is recognized by RBPJ and promotes its recruitment to Notch target genes.

Connection of core and tail Mediator modules restrains transcription from TFIID-dependent promoters

The Mediator coactivator complex is divided into four modules: head, middle, tail, and kinase. Deletion of the architectural subunit Med16 separates core Mediator (cMed), comprising the head, middle, and scaffold (Med14), from the tail. However, the direct global effects of tail/cMed disconnection are unclear. We find that rapid depletion of Med16 downregulates genes that require the SAGA complex for full expression, consistent with their reported tail dependence, but also moderately overactivates TFIID-dependent genes in a manner partly dependent on the separated tail, which remains associated with upstream activating sequences. Suppression of TBP dynamics via removal of the Mot1 ATPase partially restores normal transcriptional activity to Med16-depleted cells, suggesting that cMed/tail separation results in an imbalance in the levels of PIC formation at SAGA-requiring and TFIID-dependent genes. We suggest that the preferential regulation of SAGA-requiring genes by tailed Mediator helps maintain a proper balance of transcription between these genes and those more dependent on TFIID.

An ALYREF-MYCN coactivator complex drives neuroblastoma tumorigenesis through effects on USP3 and MYCN stability

To achieve the very high oncoprotein levels required to drive the malignant state cancer cells utilise the ubiquitin proteasome system to upregulate transcription factor levels. Here our analyses identify ALYREF, expressed from the most common genetic copy number variation in neuroblastoma, chromosome 17q21-ter gain as a key regulator of MYCN protein turnover. We show strong co-operativity between ALYREF and MYCN from transgenic models of neuroblastoma in vitro and in vivo. The two proteins form a nuclear coactivator complex which stimulates transcription of the ubiquitin specific peptidase 3, USP3. We show that increased USP3 levels reduce K-48- and K-63-linked ubiquitination of MYCN, thus driving up MYCN protein stability. In the MYCN-ALYREF-USP3 signal, ALYREF is required for MYCN effects on the malignant phenotype and that of USP3 on MYCN stability. This data defines a MYCN oncoprotein dependency state which provides a rationale for future pharmacological studies.