Bidirectional cooperation between Ubtf1 and SL1 determines RNA Polymerase I promoter recognition in cell and is negatively affected in the UBTF-E210K neuroregression syndrome.

Transcription of the ~200 mouse and human ribosomal RNA genes (rDNA) by RNA Polymerase I (RPI/PolR1) accounts for 80% of total cellular RNA, around 35% of all nuclear RNA synthesis, and determines the cytoplasmic ribosome complement. It is therefore a major factor controlling cell growth and its misfunction has been implicated in hypertrophic and developmental disorders. Activation of each rDNA repeat requires nucleosome replacement by the architectural multi-HMGbox factor UBTF to create a 15kbp nucleosome free region (NFR). Formation of this NFR is also essential for recruitment of the TBP-TAF I factor SL1 and for preinitiation complex (PIC) formation at the gene and enhancer-associated promoters of the rDNA. However, these promoters show little sequence commonality and neither UBTF nor SL1 display significant DNA sequence binding specificity, making what drives PIC formation a mystery. Here we show that cooperation between SL1 and the longer UBTF1 splice variant generates the specificity required for rDNA promoter recognition in cell . We find that conditional deletion of the Taf1b subunit of SL1 causes a striking depletion UBTF at both rDNA promoters but not elsewhere across the rDNA. We also find that while both UBTF1 and -2 variants bind throughout the rDNA NFR, only UBTF1 is present with SL1 at the promoters. The data strongly suggest an induced-fit model of RPI promoter recognition in which UBTF1 plays an architectural role. Interestingly, a recurrent UBTF-E210K mutation and the cause of a pediatric neurodegeneration syndrome provides indirect support for this model. E210K knock-in cells show enhanced levels of the UBTF1 splice variant and a concomitant increase in active rDNA copies. In contrast, they also display reduced rDNA transcription and promoter recruitment of SL1. We suggest the underlying cause of the UBTF-E210K syndrome is therefore a reduction in cooperative UBTF1-SL1 promoter recruitment that may be partially compensated by enhanced rDNA activation.

The chemotherapeutic agent CX-5461 irreversibly blocks RNA polymerase I initiation and promoter release to cause nucleolar disruption, DNA damage and cell inviability

In the search for drugs to effectively treat cancer, the last 10 years have seen a resurgence of interest in targeting ribosome biogenesis. CX-5461 is a potential inhibitor of ribosomal RNA synthesis that is now showing promise in phase I trials as a chemotherapeutic agent for a range of malignancies. Here, we show that CX-5461 irreversibly inhibits ribosomal RNA transcription by arresting RNA polymerase I (RPI/Pol1/PolR1) in a transcription initiation complex. CX-5461 does not achieve this by preventing formation of the pre-initiation complex nor does it affect the promoter recruitment of the SL1 TBP complex or the HMGB-box upstream binding factor (UBF/UBTF). CX-5461 also does not prevent the subsequent recruitment of the initiation-competent RPI–Rrn3 complex. Rather, CX-5461 blocks promoter release of RPI–Rrn3, which remains irreversibly locked in the pre-initiation complex even after extensive drug removal. Unexpectedly, this results in an unproductive mode of RPI recruitment that correlates with the onset of nucleolar stress, inhibition of DNA replication, genome-wide DNA damage and cellular senescence. Our data demonstrate that the cytotoxicity of CX-5461 is at least in part the result of an irreversible inhibition of RPI transcription initiation and hence are of direct relevance to the design of improved strategies of chemotherapy.

FACT and Ash1 promote long-range and bidirectional nucleosome eviction at the HO promoter

The Saccharomyces cerevisiae HO gene is a model regulatory system with complex transcriptional regulation. Budding yeast divide asymmetrically and HO is expressed only in mother cells where a nucleosome eviction cascade along the promoter during the cell cycle enables activation. HO expression in daughter cells is inhibited by high concentration of Ash1 in daughters. To understand how Ash1 represses transcription, we used a myo4 mutation which boosts Ash1 accumulation in both mothers and daughters and show that Ash1 inhibits promoter recruitment of SWI/SNF and Gcn5. We show Ash1 is also required for the efficient nucleosome repopulation that occurs after eviction, and the strongest effects of Ash1 are seen when Ash1 has been degraded and at promoter locations distant from where Ash1 bound. Additionally, we defined a specific nucleosome/nucleosome-depleted region structure that restricts HO activation to one of two paralogous DNA-binding factors. We also show that nucleosome eviction occurs bidirectionally over a large distance. Significantly, eviction of the more distant nucleosomes is dependent upon the FACT histone chaperone, and FACT is recruited to these regions when eviction is beginning. These last observations, along with ChIP experiments involving the SBF factor, suggest a long-distance loop transiently forms at the HO promoter.

RNA Pol II Length and Disorder Enable Cooperative Scaling of Transcriptional Bursting

RNA Polymerase II contains a disordered C-terminal domain (CTD) whose length enigmatically correlates with genome size. The CTD is crucial to eukaryotic transcription, yet the functional and evolutionary relevance of this variation remains unclear. Here, we use smFISH, live imaging, and RNA-seq to investigate how CTD length and disorder influence transcription. We find that length modulates the size and frequency of transcriptional bursting. Disorder is highly conserved and mediates CTD-CTD interactions, an ability we show is separable from protein sequence and necessary for efficient transcription. We build a data-driven quantitative model, simulations of which recapitulate experiments and support CTD length promotes initial polymerase recruitment to the promoter but slows down its release from it, and that CTD-CTD interactions enable promoter recruitment of multiple polymerases. Our results reveal how these tunable parameters provide access to a range of transcriptional activity, offering a new perspective for the mechanistic significance of CTD length and disorder in transcription across eukaryotes.

Multiple Evolutionarily Conserved Domains of Cap2 Are Required for Promoter Recruitment and Iron Homeostasis Gene Regulation

ABSTRACTIron is required for growth and metabolism by virtually all organisms. The human fungal pathogenCandida albicanshas evolved multiple strategies to acquire iron. The Cap2/Hap43 transcriptional regulator, essential for robust virulence ofC. albicans, controls iron homeostasis gene expression by promoter binding and repression of iron utilization genes. The expression of iron uptake genes is also dependent on Cap2, although Cap2 was not recruited to its promoters. Cap2, bearing the conserved bipartite HAP4L-bZIP domain, also contains multiple blocks of amino acids that form the highly conserved carboxyl-terminal region. In this study, we sought to identify the requirements of the different domains for Cap2 function. We constructed a series of mutants bearing either point mutations or deletions in the conserved domains and examined Cap2 activity. Deletion of the highly conserved extreme C-terminal region did not impair expression of Cap2 mutant protein but impaired cell growth and expression of iron homeostasis genes under iron-depleted conditions. Mutations in the amino-terminal HAP4L and basic leucine zipper (bZIP) domains also impaired growth and gene expression. Furthermore, chromatin immunoprecipitation (ChIP) assays showed that the HAP4L domain and the bZIP domain are both essential for Cap2 recruitment toACO1andCYC1promoters. Unexpectedly, the C-terminal conserved region was also essential for Cap2 promoter recruitment. Thus, our results suggest that Cap2 employs multiple evolutionarily conserved domains, including the C-terminal domain for its transcriptional activity.IMPORTANCEIron is an essential micronutrient for living cells.Candida albicans, the predominant human fungal pathogen, thrives under diverse environments with vastly different iron levels in the mammalian host. Therefore, to tightly control iron homeostasis,C. albicanshas evolved a set of transcriptional regulators that cooperate to either upregulate or downregulate transcription of iron uptake genes or iron utilization genes. Cap2/Hap43, a critical transcriptional regulator, contains multiple conserved protein domains. In this study, we carried out mutational analyses to identify the functional roles of the conserved protein domains in Cap2. Our results show that the bZIP, HAP4L, and the C-terminal domain are each required for Cap2 transcriptional activity. Thus, Cap2 employs multiple, disparate protein domains for regulation of iron homeostasis inC. albicans.

Set1/COMPASS and Mediator are repurposed to promote epigenetic transcriptional memory

In yeast and humans, previous experiences can lead to epigenetic transcriptional memory: repressed genes that exhibit mitotically heritable changes in chromatin structure and promoter recruitment of poised RNA polymerase II preinitiation complex (RNAPII PIC), which enhances future reactivation. Here, we show that INO1 memory in yeast is initiated by binding of the Sfl1 transcription factor to the cis-acting Memory Recruitment Sequence, targeting INO1 to the nuclear periphery. Memory requires a remodeled form of the Set1/COMPASS methyltransferase lacking Spp1, which dimethylates histone H3 lysine 4 (H3K4me2). H3K4me2 recruits the SET3C complex, which plays an essential role in maintaining this mark. Finally, while active INO1 is associated with Cdk8- Mediator, during memory, Cdk8+ Mediator recruits poised RNAPII PIC lacking the Kin28 CTD kinase. Aspects of this mechanism are generalizable to yeast and conserved in human cells. Thus, COMPASS and Mediator are repurposed to promote epigenetic transcriptional poising by a highly conserved mechanism.