Biology subjects

Knoll, E. R.

Publications and source records attributed to Knoll, E. R..

3 recordsLinked to original sources

bioRxiv2019

Kin28 depletion increases association of TFIID subunits Taf1 and Taf4 with promoters in Saccharomyces cerevisiae

In eukaryotes, transcription of mRNA-encoding genes by RNA polymerase II (Pol II) begins with assembly of the pre-initiation complex (PIC), comprising Pol II and the general transcription factors. Although the pathway of PIC assembly is well established, the mechanism of assembly and the dynamics of PIC components are not fully understood. For example, only recently has it been shown in yeast that the Mediator complex, which assists in pre-initiation complex formation at promoters of essentially all genes transcribed by Pol II, normally occupies promoters only transiently. This was inferred from studies showing that inhibiting Pol II promoter escape by depleting or inactivating Kin28 resulted in increased promoter occupancy by Mediator, as measured by chromatin immunoprecipitation (ChIP). Here we show that two subunits of TFIID, Taf1 and Taf4, similarly show increased occupancy as measured by ChIP upon depletion or inactivation of Kin28. In contrast, TBP occupancy is unaffected by depletion of Kin28, thus revealing an uncoupling of Taf and TBP occupancy during the transcription cycle. Increased Taf1 occupancy upon Kin28 depletion is suppressed by depletion of TBP, while depletion of TBP in the presence of Kin28 has little effect on Taf1 occupancy. Taf1 occupancy relative to TBP is higher at TFIID-dominated promoters and promoters having consensus TATA elements than at SAGA-dominated promoters and promoters lacking consensus TATA elements, consistent with prior work, and the increase in Taf occupancy upon depletion of Kin28 is more pronounced at TFIID-dominated promoters. Our results support the suggestion, based on recent structural studies, that TFIID may not remain bound to gene promoters through the transcription initiation cycle.\n\nAuthor SummaryTranscription of mRNA-encoding genes by RNA polymerase II (Pol II) begins when the pre-initiation complex, a large complex comprising Pol II and several general transcription factors, including the TATA-binding protein (TBP)-containing TFIID complex, assembles at gene promoters. Although the major steps in the pathway of PIC assembly have been identified, the mechanism of assembly in vivo and the dynamics of PIC components are not fully understood. In this work we have used a yeast strain that is engineered to allow inhibition of promoter escape by Pol II by administration of a chemical, in order to \"freeze\" the assembled PIC and thus determine whether this condition increases the promoter occupancy of TBP and two TBP-associated factors (Tafs) that are components of TFIID. This approach was used recently to demonstrate that the Mediator complex, which facilitates PIC assembly, normally binds only transiently to gene promoters. We find that Tafs, like Mediator, show increased occupancy when Pol II promoter escape is inhibited, whereas TBP binding is constant. These results imply that binding of TBP and Tafs is uncoupled during the transcription cycle, and that Taf occupancy is at least partially interrupted upon Pol II promoter escape.

genetics↗

bioRxiv2017

Role of the pre-initiation complex in Mediator recruitment and dynamics

The Mediator complex functions in eukaryotic transcription by stimulating the cooperative assembly of a pre-initiation complex (PIC) and recruitment of RNA Polymerase II (Pol II) for gene activation. The core Mediator complex is organized into head, middle, and tail modules, and in budding yeast (Saccharomyces cerevisiae), Mediator recruitment has generally been ascribed to sequence-specific activators engaging the tail module triad of Med2-Med3-Med15 at upstream activating sequences (UASs). We show that med2{Delta} med3{Delta} med15{Delta} yeast are viable and that Mediator lacking Med2-Med3-Med15 is associated with active promoters genome-wide. To test whether Mediator might alternatively be recruited via interactions with the PIC, we examined Mediator association genome-wide after depleting PIC components. We found that depletion of Taf1, Rpb3, and TBP profoundly affected Mediator association at active gene promoters, with TBP being critical for transit of Mediator from UAS to promoter, while Pol II and Taf1 stabilize Mediator association at proximal promoters.

genetics↗

bioRxiv2017

The Mediator co-activator complex regulates Ty1 retromobility via alternative transcription start site selection

The Ty1 retrotransposons present in the genome of Saccharomyces cerevisiae belong to the large class of mobile genetic elements that replicate via an RNA intermediary and constitute a significant portion of most eukaryotic genomes. The retromobility of Ty1 is regulated by numerous host factors, including several subunits of the Mediator transcriptional co-activator complex. In spite of its known function in the nucleus, previous studies have implicated Mediator in the regulation of post-translational steps in Ty1 retromobility. To resolve this paradox, we systematically examined the effects of deleting nonessential Mediator subunits on the frequency of Ty1 retromobility and levels of retromobility intermediates. Our findings reveal that loss of distinct Mediator subunits alters Ty1 retromobility positively or negatively over a >10,000-fold range by regulating the ratio of an internal transcript, Ty1i, to the genomic Ty1 transcript. Ty1i RNA encodes a dominant negative inhibitor of Ty1 retromobility that blocks virus-like particle maturation and cDNA synthesis. These results resolve the conundrum of Mediator exerting sweeping control of Ty1 retromobility with only minor effects on the levels of Ty1 genomic RNA and the capsid protein, Gag. Since the majority of characterized intrinsic and extrinsic regulators of Ty1 retromobility alter a post-translational step(s), Mediator could play a central role in integrating signals that influence Ty1i expression to modulate retromobility.\n\nAuthor SummaryRetrotransposons are mobile genetic elements that copy their RNA genomes into DNA and insert the DNA copies into the host genome. These elements contribute to genome instability, control of host gene expression and adaptation to changing environments. Retrotransposons depend on numerous host factors for their own propagation and control. The retrovirus-like retrotransposon, Ty1, in the yeast Saccharomyces cerevisiae has been an invaluable model for retrotransposon research, and hundreds of host factors that regulate Ty1 retrotransposition have been identified. Non-essential subunits of the Mediator transcriptional co-activator complex have been identified as one set of host factors implicated in Ty1 regulation. Here, we report a systematic investigation of the effects of loss of these non-essential subunits of Mediator on Ty1 retrotransposition. Our findings reveal a heretofore unknown mechanism by which Mediator influences the balance between transcription from two promoters in Ty1 to modulate expression of an autoinhibitory transcript known as Ty1i RNA. Our results provide new insights into host control of retrotransposon activity via promoter choice and elucidate a novel mechanism by which the Mediator co-activator governs this choice.

molecular biology↗