Abstract Recombinant adeno-associated viral vectors (rAAV) can achieve potent and durable transgene expression without integration in a broad range of tissue types, making them a popular choice for gene delivery in animal models and in clinical settings. In addition to therapeutic applications, rAAVs are a useful laboratory tool for delivering transgenes tailored to the researcher’s experimental needs and scientific goals in cultured cells. Some examples include exogenous reporter genes, overexpression cassettes, RNA interference, and CRISPR-based tools including those for genome-wide screens. rAAV transductions are less harmful to cells than electroporation or chemical transfection and moreover do not require any special equipment or expensive reagents to produce. Crude lysates or conditioned media containing rAAVs can be added directly to cultured cells without further purification to transduce many cell types – an underappreciated feature of rAAVs. Here, we provide protocols for basic transgene cassette cloning and demonstrate how to produce and apply crude rAAV preparations to cultured cells. As proof-of-principle, we demonstrate transduction of three cell types that have not yet been reported in rAAV applications. We discuss appropriate uses for crude rAAV preparations, the limitations of rAAVs for gene delivery, and considerations for capsid choice. The simplicity of production, exceedingly low cost, and often potent results make crude rAAV a primary choice for researchers to achieve effective DNA delivery. Summary Recombinant adeno-associated virus (rAAV) is widely used for clinical and preclinical gene delivery. An underappreciated use for rAAVs is the robust transduction of cultured cells without the need for purification. For researchers new to rAAV, we provide a protocol for transgene cassette cloning, crude vector production, and cell culture transduction.
Abstract As scientific projects and labs benefit from increasingly interdisciplinary expertise, students and trainees find themselves navigating a myriad of academic spaces, each with its own workplace culture and demographics. A clear example is the interdisciplinary field of optics and biological microscopy which bridges biology, physics and engineering. While Biology PhDs are now >50% women, men in physics and engineering fields still significantly outnumber women, resulting in an imbalance of gender representation among microscopists and other ‘tool innovators’ in the interdisciplinary field of biological microscopy and biomedical optics. In addition to the cultural and cognitive whiplash that results from disparate representation between fields such as Biology, Engineering, and Physics, indifference from institutional leaders to implement equity‐focused initiatives further contributes to cultures of exclusion, rather than belonging, for women. Here we elaborate on the motivation, structure, and outcomes of building a specific affinity‐based bootcamp as an intervention to create an inclusive, welcoming learning environment for women in optics. Considering the presence of nonbinary, trans and other gender minoritised scientists, we recognise that women are not the only gender group underrepresented in biological microscopy and biomedical optics; still, we focus our attention on women in this specific intervention to improve gender parity in biological microscopy and biomedical optics. We hope that these strategies exemplify concrete paths forward for increasing belonging in interdisciplinary fields, a key step towards improving and diversifying graduate education.
In eukaryotes, protein-coding genes expression involves the integration of cellular signals, chromatin modifications, and assembly of the transcription pre-initiation complex (PIC) that loads RNA polymerase II onto the promoter.PIC formation is initiated by recruitment of the TATA-binding protein (TBP) to the promoter by protein complexes containing TBP-associated factors (TAFs) that interact with activators, promoter DNA, and/or chromatin.Throughout evolution, these TAFs partitioned and specialized into two distinct coactivator complexes: the general transcription factor TFIID and the SAGA complex.Our structural studies on human TFIID (hTFIID) elucidated its molecular function as a chaperone for TBP deposition and initiation of PIC assembly.TFIID contains three large modules, A, B and C. Two of those, lobe A and B, are built on a core of histone fold-containing subunits and TAF5, with additional subunits conferring distinct arrangements of the modules within the full complex as well as distinct functions.In particular, lobe A includes TBP, which is kept in an inhibited state within TFIID by interaction with several TAFs.Following binding to downstream core promoter sequences, and with the help of TFIIA via its interaction with lobe B, TBP is ultimately deployed onto DNA, where it can initiate PIC assembly.The other TAFcontaining transcriptional coactivator, SAGA, has been implicated in a multitude of cellular pathways, including serving as a regulatory hub in transcription.It contains several functional modules: a core of scaffolding histone fold-containing TAFs that parallels that found in TFIID's lobe A; a TRRAP module that binds activators such as c-Myc or p53; a histone acetyltransferase that deposits H3K9ac and H3K14ac at promoters of active genes; and a deubiquitinase that removes H2BK120 ubiquitination from active gene bodies.Studies of yeast SAGA have revealed its modular organization and structural details for some of its modules.We sought to elucidate the architecture of human SAGA and possible functional implications of its conserved and distinct characteristics from its yeast counterpart.Our cryo-EM structure reveals unexpected features and a divergent architecture that have functional implications in transcription and splicing with relevance in genetic diseases and cancer.
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.
Recombinant adeno-associated viral vectors (rAAV) can achieve potent and durable transgene expression without integration in a broad range of tissue types, making them a popular choice for gene delivery in animal models and in clinical settings. In addition to therapeutic applications, rAAVs are a useful laboratory tool for delivering transgenes tailored to the researcher's experimental needs and scientific goals in cultured cells. Some examples include exogenous reporter genes, overexpression cassettes, RNA interference, and CRISPR-based tools, including those for genome-wide screens. rAAV transductions are less harmful to cells than electroporation or chemical transfection and do not require any special equipment or expensive reagents to produce. Crude lysates or conditioned media containing rAAVs can be added directly to cultured cells without further purification to transduce many cell types—an underappreciated feature of rAAVs. Here, we provide protocols for basic transgene cassette cloning and demonstrate how to produce and apply crude rAAV preparations to cultured cells. As proof of principle, we demonstrate the transduction of three cell types that have not yet been reported in rAAV applications: placental cells, myoblasts, and small intestinal organoids. We discuss appropriate uses for crude rAAV preparations, the limitations of rAAVs for gene delivery, and considerations for capsid choice. This protocol outlines a simple, low-cost, and effective method for researchers to achieve productive DNA delivery in cell culture using rAAV without the need for laborious titration and purification steps.
Abstract Human SAGA is an essential co-activator complex that regulates gene expression by interacting with enhancer-bound activators, recruiting transcriptional machinery, and modifying chromatin near promoters. Subunit variations and the metazoan-specific requirement of SAGA in development hinted at unique structural features of the human complex. Our 2.9 Å structure of human SAGA reveals intertwined functional modules flexibly connected to a core that distinctively integrates mammalian paralogs, incorporates U2 splicing subunits, and features a unique interface between the core and the activator-binding TRRAP. Our structure sheds light on unique roles and regulation of human coactivators with implications for transcription and splicing that have relevance in genetic diseases and cancer.
RNA polymerase II (RNAPII) transcription initiation is governed by the Pre-Initiation Complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNAPII, and Mediator. After initiation, RNAPII enzymes pause after transcribing less than 100 bases; precisely how RNAPII pausing is enforced and regulated remains unclear. To address specific mechanistic questions, we reconstituted human RNAPII promoter-proximal pausing in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pausing, and P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pausing, suggesting RNAPII pausing is an inherent PIC function. In agreement, pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP), and PRO-Seq experiments revealed widespread disruption of RNAPII pausing upon acute depletion (t=60 min) of TFIID subunits in human or Drosophila cells. These results establish a TFIID requirement for RNAPII pausing and suggest pause regulatory factors function directly or indirectly through TFIID.
