RNA Polymerase II Binding Research Articles

αKG-driven RNA polymerase II transcription of cyclin D1 licenses malic enzyme 2 to promote cell-cycle progression

Increased metabolic activity usually provides energy and nutrients for biomass synthesis and is indispensable for the progression of the cell cycle. Here, we find a role for α-ketoglutarate (αKG) generation in regulating cell-cycle gene transcription. A reduction in cellular αKG levels triggered by malic enzyme 2 (ME2) or isocitrate dehydrogenase 1 (IDH1) depletion leads to a pronounced arrest in G1 phase, while αKG supplementation promotes cell-cycle progression. Mechanistically, αKG directly binds to RNA polymerase II (RNAPII) and increases the level of RNAPII binding to the cyclin D1 gene promoter via promoting pre-initiation complex (PIC) assembly, consequently enhancing cyclin D1 transcription. Notably, αKG addition is sufficient to restore cyclin D1 expression in ME2- or IDH1-depleted cells, facilitating cell-cycle progression and proliferation in these cells. Therefore, our findings indicate a function of αKG in gene transcriptional regulation and cell-cycle control.

RNA polymerase II-associated proteins reveal pathways affected in VCP-related amyotrophic lateral sclerosis.

Valosin-containing protein (VCP) is a hexameric ATPase associated with diverse cellular activities. Genetic mutations in VCP are associated with several forms of muscular and neuronal degeneration, including amyotrophic lateral sclerosis (ALS). Moreover, VCP mediates UV-induced proteolysis of RNA polymerase II (RNAPII), but little is known about the effects of VCP mutations on the transcriptional machinery. Here, we used silica particle-assisted chromatin enrichment and mass spectrometry to study proteins co-localized with RNAPII in precursor neurons differentiated from VCP-mutant or control induced pluripotent stem cells. Remarkably, we observed diminished RNAPII binding of proteins involved in transcription elongation and mRNA splicing in mutant cells. One of these is SART3, a recycling factor of the splicing machinery, whose knockdown leads to perturbed intron retention in several ALS-associated genes. Additional reduced proteins are RBM45, EIF5A and RNF220, mutations in which are associated with various neurodegenerative disorders and are linked to TDP-43 aggregation. Conversely, we observed increased RNAPII binding of heat shock proteins such as HSPB1. Together, these findings shed light on how transcription and splicing machinery are impaired by VCP mutations, which might contribute to aberrant alternative splicing and proteinopathy in neurodegeneration.

The role of Toxoplasma TFIIS-like protein in the early stages of mRNA transcription

BackgroundThe mRNA transcription is a multistep process involving distinct sets of proteins associated with RNA polymerase II (RNAPII) through various stages. Recent studies have highlighted the role of RNAPII-associated proteins in facilitating the assembly of functional complexes in a crowded nuclear milieu. RNAPII dynamics and gene expression regulation have been primarily studied in model eukaryotes like yeasts and mammals and remain largely unchartered in protozoan parasites like Toxoplasma gondii, where considerable gene expression changes accompany stage differentiations. Here we report a key modulator of RNAPII activity, TFIIS in Toxoplasma gondii (TgTFIIS). MethodsA Pull-down assay demonstrated that TgTFIIS binds to RNAPII subunit TgRPB1. Truncation mutants of TFIIS help us define the regions critical for its binding to TgRPB1. Co-immunoprecipitation analysis confirmed the interaction between the native TgTFIIS and TgRPB1. Confocal microscopy revealed a predominantly nuclear localization. Native TgTFIIS was able to bind promoter DNA which was consistent with the CHIP results. ResultsTgTFIIS complements initiation defects in yeast mutants, and the regions implicated in RNAPII binding appeared essential for this function. Interestingly, the C-terminal zinc finger domain necessary for its potential elongation function is dispensable for TgRPB1 binding. TgTFIIS was found to be associated with the promoter region along with its association with the ORF on an RNAPII transcribed gene. ConclusionThe observations were in line with the potential role of TgTFIIS in early events of RNAPII transcription in addition to elongation. General significanceThe study elucidates the potential role of RNAPII-associated proteins in multiple steps of transcription.

First Report of Botrytis cinerea Causing Leaf Spot on Strawberry in Florida.

During the 2020-2021 Florida strawberry season (October to April), strawberry (Fragaria × ananassa) plants showing leaf spots were observed on samples submitted to the Diagnostic Clinic at the University of Florida's Gulf Coast Research and Education Center. Disease incidence was up to 5% and observed on four different farms in Plant City, FL on cultivars SensationTM Florida127 and Florida Brilliance. All the samples were submitted early in the season (November) and shared the same nursery source in California. Symptoms consisted of circular or irregular lesions with purple or brown halos, eventually developing leaf blight with sporulation at the center on advanced lesions. Diseased tissues (0.5 mm2) were surface disinfested with 10% bleach solution for 90 s, rinsed twice in sterile deionized water, and plated on general isolation medium (Amiri et al. 2018). Plates were incubated at 25°C and a 12-h photoperiod. A fungus producing white mycelia with sparse sporulation of Botrytis-like spores was consistently isolated. Isolates were single-spored and grown on HA medium to induce sporulation (Leroch et al. 2013). Three isolates (20-291, 20-293, and 20-295) were selected for identification and pathogenicity assays. Resulting cultures on HA had profuse sporulation resembling gray mold. Conidia (n=50) were round to ellipsoid ranging from 9 to 14.6 μm long (Avg=10.8, SD=1.3) and 6.3 to 9.5 μm wide (Avg=7.7, Sd=0.7). No sclerotia formation was observed on GI and HA medium. Based on morphology, the pathogen was tentatively identified as Botrytis cinerea (Hong et al. 2001; Jarvis 1977). DNA was extracted from the same three isolates using the FastDNA kit (MP Biomedicals, Solon, OH), and the heat shock protein (HSP60), RNA polymerase II-binding (RPB2), and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) genes were amplified (Staats et al. 2004). Sequences were deposited in GenBank (accession nos. MZ288746 - MZ288754). BLASTn searches revealed that isolates were 100% identical to B. cinerea reported causing leaf spot on strawberry in California; accession numbers MK919494 (HSP60, 996/996 bp), MK919495 (RPB2, 1131/1131 bp), and MK919496 (G3PDH, 877/877 bp). To test for pathogenicity, four one-month-old plants of 'Florida Brilliance' were used per isolate and control treatment. Spores were harvested from two-week-old cultures grown on HA medium, and the suspension adjusted to 106 spores/mL in a solution of 0.1% of Tween 20. Plants were spray inoculated until run-off and kept inside clear plastic boxes for 48 h. Control plants were sprayed with sterile deionized water. Afterward, plants were kept in a misting table in the greenhouse with a water regime of 3 s every 10 min during the day. Disease incidence was evaluated weekly, and the experiment repeated once. Two weeks after inoculation, leaf spots were observed in all inoculated plants, while controls remained healthy. Fungi morphologically identical to the original isolates were re-isolated from the diseased tissues. To our knowledge, this is the first report of B. cinerea causing leaf spot on strawberry in Florida. This disease was recently reported in California (Mansouripour and Holmes 2020), which is where the transplants originated from. Considering the disease was observed early in the Florida season, it is likely that it was introduced with transplants from the nursery. This pathogen is also the causal agent of Botrytis fruit rot, which is considered a major disease of strawberry, and a previous study has shown that populations resistant to multiple fungicides are introduced with transplants (Mertely et al. 2018, Oliveira et al. 2018). While Botrytis leaf spot is currently considered rare and of minor significance (Mansouripour and Holmes 2020), it could contribute to the spread of fungicide resistance to from nursery to strawberry fruit production fields. Efforts should be implemented to monitor its occurrence and spread considering the high variability and fungicide resistance profile of this pathogen.

Diurnal RNAPII-tethered chromatin interactions are associated with rhythmic gene expression in rice

BackgroundThe daily cycling of plant physiological processes is speculated to arise from the coordinated rhythms of gene expression. However, the dynamics of diurnal 3D genome architecture and their potential functions underlying the rhythmic gene expression remain unclear.ResultsHere, we reveal the genome-wide rhythmic occupancy of RNA polymerase II (RNAPII), which precedes mRNA accumulation by approximately 2 h. Rhythmic RNAPII binding dynamically correlates with RNAPII-mediated chromatin architecture remodeling at the genomic level of chromatin interactions, spatial clusters, and chromatin connectivity maps, which are associated with the circadian rhythm of gene expression. Rhythmically expressed genes within the same peak phases of expression are preferentially tethered by RNAPII for coordinated transcription. RNAPII-associated chromatin spatial clusters (CSCs) show high plasticity during the circadian cycle, and rhythmically expressed genes in the morning phase and non-rhythmically expressed genes in the evening phase tend to be enriched in RNAPII-associated CSCs to orchestrate expression. Core circadian clock genes are associated with RNAPII-mediated highly connected chromatin connectivity networks in the morning in contrast to the scattered, sporadic spatial chromatin connectivity in the evening; this indicates that they are transcribed within physical proximity to each other during the AM circadian window and are located in discrete “transcriptional factory” foci in the evening, linking chromatin architecture to coordinated transcription outputs.ConclusionOur findings uncover fundamental diurnal genome folding principles in plants and reveal a distinct higher-order chromosome organization that is crucial for coordinating diurnal dynamics of transcriptional regulation.

Altered Enhancer Activity Represses CEBPE Expression in MLL -Rearranged Leukemia

Mixed lineage leukemia (MLL)-rearranged leukemia is characterized by the presence of MLL fusion proteins resulting from chromosomal translocation between MLL and its partner genes. The resultant fusion proteins can confer aberrant epigenetic patterns at gene regulatory elements to disrupt transcriptional program, and ultimately leads to differentiation blockage at early hematopoietic progenitor stage. CCAAT enhancer binding proteins (C/EBPs) are key transcription factors involved in myeloid lineage commitment and maturation, among which CEBPE is crucial in terminal differentiation of granulocytes/macrophages. We found that Cebpe is markedly downregulated in murine hematopoietic stem/progenitor cells (HSPCs) expressing MLL-EEN, which could be associated with differentiation blockage in MLL -rearranged leukemia. However, how CEBPE is epigenetically dysregulated by MLL fusion proteins remains unclear. In murine granulocytes, we demonstrated long-range chromatin interaction between Cebpe promoter and enhancer by chromatin conformation capture (3C) assay, conferring the strong expression of Cebpe . Strikingly, we noticed gain of DNA methylation at Cebpe promoter in MLL-EEN expressing HSPCs, suggesting the disruption of chromatin interaction through aberrant epigenetic alterations resulted in Cebpe repression. Interestingly, CEBPE expression was also found significantly lower in human MLL acute myeloid leukemia (AML) cell lines (HL60, NB4, U937 and Kasumi-1), when compared to non-MLL AML cell lines (ML-2, MV4:11, Monomac-6 and THP-1). DNA hypermethylation was observed at both promoter and predicated enhancer regions in MLL AML cells, which were absent in non-MLL AML cells. In addition, reduction of H3K27ac enrichment at the predicted enhancer was found in MLL AML cells. Analysis of the public available RNA Polymerase II (RNAPII) ChIP-seq data demonstrated RNAPII binding at both CEBPE promoter and enhancers in non-MLL AML cell lines, but it was absent at the enhancer region in MLL AML cells. These results suggest a common epigenetic mechanism which disrupts the CEBPE enhancer-promoter interaction in MLL -rearranged leukemia. Besides, treatment of MLL AML cell lines with DNA methylation inhibitor 5'-aza-cytidine (5-aza-C) demonstrated induction of CEBPE and myeloid marker CD11b . While all -trans retinoid acid failed to trigger cell differentiation in ML-2 AML cells, co-treatment with 5-aza-C resulted in further induction of CEBPE and CD11b, compared to 5-aza-C treatment per se . Collectively, our work shows that aberrant DNA methylation mediated by MLL fusion proteins represses CEBPE expression through altering its enhancer activity, which subsequently leads to cell differentiation blockage in MLL AML cells. This study provides novel insights on the aberrant epigenetic regulation in MLL -rearranged leukemia and facilitates the development of differentiation therapy for leukemia. DisclosuresNo relevant conflicts of interest to declare.

Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription

In eukaryotes, RNA polymerase II (RNApII) transcribes messenger RNA from template DNA. Decades of experiments have identified the proteins needed for transcription activation, initiation complex assembly, and productive elongation. However, the dynamics of recruitment of these proteins to transcription complexes, and of the transitions between these steps, are poorly understood. We used multiwavelength single-molecule fluorescence microscopy to directly image and quantitate these dynamics in a budding yeast nuclear extract that reconstitutes activator-dependent transcription in vitro. A strong activator (Gal4-VP16) greatly stimulated reversible binding of individual RNApII molecules to template DNA. Binding of labeled elongation factor Spt4/5 to DNA typically followed RNApII binding, was NTP dependent, and was correlated with association of mRNA binding protein Hek2, demonstrating specificity of Spt4/5 binding to elongation complexes. Quantitative kinetic modeling shows that only a fraction of RNApII binding events are productive and implies a rate-limiting step, probably associated with recruitment of general transcription factors, needed to assemble a transcription-competent preinitiation complex at the promoter. Spt4/5 association with transcription complexes was slowly reversible, with DNA-bound RNApII molecules sometimes binding and releasing Spt4/5 multiple times. The average Spt4/5 residence time was of similar magnitude to the time required to transcribe an average length yeast gene. These dynamics suggest that a single Spt4/5 molecule remains associated during a typical transcription event, yet can dissociate from RNApII to allow disassembly of abnormally long-lived (i.e., stalled) elongation complexes.

First Report of Botrytis cinerea Causing Leaf Spot on Strawberry in California

On 29 October 2018, a leaf spot disease was observed on strawberry leaves (Fragaria × ananassa Duch., cultivar Portola) on organically grown plants in high plastic tunnels in Santa Maria, California. Symptoms consisted of circular lesions measuring 2 to 3 cm in diameter, concentric rings with a darker brown outer ring and fungal sporulation in the center of the lesion. The diseased leaf sample was surface sterilized in 10% sodium hypochlorite for 1 min, rinsed thrice in sterile water, and placed on potato dextrose agar (PDA) after drying. The resulting culture produced conidia that were hyaline and round to ellipsoid (8 to 14 × 5 to 8 µm, n = 50) and sclerotia that were hard, dark, round to oblong, and measured 2 to 12 × 2 to 9 mm (n = 50). Based on its morphological characteristics in culture, the isolate was putatively identified as Botrytis cinerea (Hong et al. 2001; Jarvis 1977). The isolate produced an average of 133 sclerotia per Petri dish (100 × 15 mm; n = 3) with PDA, an uncommon feature of B. cinerea isolates from strawberry. Koch’s postulates were performed using two inoculation methods: (i) Botrytis agar plug on leaf; and (ii) conidial suspension on leaf. Leaf inoculations used a spore suspension containing 1 × 10⁶ conidia/ml and were replicated three times. Leaf inoculations using an agar plug or spore suspension were allowed to dry, misted with water, and covered with a plastic bag to maintain continuous moisture. Agar-plug inoculated leaves produced symptoms in 4 days, whereas the spore-suspension inoculations produced symptoms in 9 days. Leaf symptoms matched those observed in the field. The pathogen was reisolated from symptomatic tissue, and its morphological features matched those of the original culture. In both inoculation methods, the noninoculated controls did not result in any disease symptoms. Cross pathogenicity to leaves using an isolate of B. cinerea collected from fruit was successful in causing foliar disease, but symptoms lacked concentric rings. To determine the fungal species, total genomic DNA of five subcultures of the original field isolate was extracted using a DNeasy Plant Mini Kit (Qiagen). The heat shock protein (HSP60) (Hsp60for+/Hsp60 rev+ primers), RNA polymerase II-binding (RPB2) (RPB2for+/RPB2 rev+ primers), and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) (G3PDHfor+/G3PDH rev+ primers) genes were amplified (Staats et al. 2004). The PCR amplicons were purified and sequenced. The sequences were processed using BLAST in the National Center for Biotechnology, and results showed 100% homology with the HSP60 (MH796663), RPB2 (MH479932), and G3PDH (MH479930) sequence for isolates of B. cinerea. The sequences were deposited in GenBank with accession numbers MK919494 (HSP60), MK919495 (RPB2), and MK919496 (G3PDH). B. cinerea is a common pathogen of strawberry fruit and can cause gray mold, postharvest rot, and dry crown rot but has not been reported as causing a leaf spot. This disease is rare and considered of minor significance to strawberry production. However, it could be of greater significance in protected culture where humidity is higher. To our knowledge, this is the first report of B. cinerea causing strawberry leaf spot in California or elsewhere.

Nuclear Ndp52 - a Putative Transcription Regulator

NDP52/CALCOCO2 has been well characterised as an autophagy receptor, however a population of the protein exists in the nucleus. Moreover, NDP52 shares significant homology to a transcription co-activator CoCoA. We have recently described the activation of Myosin VI by NDP52 in RNA Polymerase II (RNAPII)-dependent transcription. Although depletion of NDP52 is known to reduce the steady-state levels of mRNA in cells, the molecular role of NDP52 in the nucleus is not clear. We are using cell biology combined with biophysical techniques to understand the functions of NDP52 in nucleus. Using SEC-MALS, Microscale Thermophoresis and Electron Microscopy, we can show that NDP52 is an elongated dimer of approximately 110 kDa that is capable of binding double-stranded DNA in vitro with high affinity. This interaction can also be observed in cells through Chromatin Immunoprecipitation (ChIP). We observed NDP52 bound to promoter regions of several oestrogen-responsive genes. When we knock-down NDP52 in these cells, we see dysregulation of known gene-targets of the oestrogen receptor, as well as of those relating to apoptosis, DNA repair and recruitment of RNAPII. Our proteomics data now suggest that this regulation might not only be sustained through direct interactions with DNA, but also through protein-protein interactions with binding partners of NDP52 in the nucleus. Overall, we propose that NDP52 may be functioning as a transcription regulator in human cells. To test this idea, we have started using super-resolution imaging - PALM-STORM, combined with Cluster analysis software, to image NDP52 at single-molecule level in combination with RNAPII and other candidate binding partners.

The Chromatin Remodeler Isw1 Prevents CAG Repeat Expansions During Transcription in Saccharomyces cerevisiae.

CAG/CTG trinucleotide repeats are unstable sequences that are difficult to replicate, repair, and transcribe due to their structure-forming nature. CAG repeats strongly position nucleosomes; however, little is known about the chromatin remodeling needed to prevent repeat instability. In a Saccharomyces cerevisiae model system with CAG repeats carried on a YAC, we discovered that the chromatin remodeler Isw1 is required to prevent CAG repeat expansions during transcription. CAG repeat expansions in the absence of Isw1 were dependent on both transcription-coupled repair (TCR) and base-excision repair (BER). Furthermore, isw1∆ mutants are sensitive to methyl methanesulfonate (MMS) and exhibit synergistic MMS sensitivity when combined with BER or TCR pathway mutants. We conclude that CAG expansions in the isw1∆ mutant occur during a transcription-coupled excision repair process that involves both TCR and BER pathways. We observed increased RNA polymerase II (RNAPII) occupancy at the CAG repeat when transcription of the repeat was induced, but RNAPII binding did not change in isw1∆ mutants, ruling out a role for Isw1 remodeling in RNAPII progression. However, nucleosome occupancy over a transcribed CAG tract was altered in isw1∆ mutants. Based on the known role of Isw1 in the reestablishment of nucleosomal spacing after transcription, we suggest that a defect in this function allows DNA structures to form within repetitive DNA tracts, resulting in inappropriate excision repair and repeat-length changes. These results establish a new function for Isw1 in directly maintaining the chromatin structure at the CAG repeat, thereby limiting expansions that can occur during transcription-coupled excision repair.

Small-Activating RNA Can Change Nucleosome Positioning in Human Fibroblasts

RNA activation (RNAa) is a mechanism of positive gene expression regulation mediated by small-activating RNAs (saRNAs), which target gene promoters and have been used as tools to manipulate gene expression. Studies have shown that RNAa is associated with epigenetic modifications at promoter regions; however, it is unclear whether these modifications are the cause or a consequence of RNAa. In this study, we examined changes in nucleosome repositioning and the involvement of RNA polymerase II (RNAPII) in this process. We screened saRNAs for OCT4 (POU5F1), SOX2, and NANOG, and identified several novel saRNAs. We found that nucleosome positioning was altered after saRNA treatment and that the formation of nucleosome-depleted regions (NDRs) contributed to RNAa at sites of RNAPII binding, such as the TATA box, CpG islands (CGIs), proximal enhancers, and proximal promoters. Moreover, RNAPII appeared to be bound specifically to NDRs. These results suggested that changes in nucleosome positions resulted from RNAa. We thus propose a hypothesis that targeting promoter regions using exogenous saRNAs can induce the formation of NDRs, exposing regulatory binding sites to recruit RNAPII, a key component of preinitiation complex, and leading to increased initiation of transcription.

A conserved Mediator–CDK8 kinase module association regulates Mediator–RNA polymerase II interaction

The CDK8 kinase module (CKM) is a conserved, dissociable Mediator subcomplex whose component subunits were genetically linked to the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and individually recognized as transcriptional repressors before Mediator was identified as a preeminent complex in eukaryotic transcription regulation. We used macromolecular electron microscopy and biochemistry to investigate the subunit organization, structure, and Mediator interaction of the Saccharomyces cerevisiae CKM. We found that interaction of the CKM with Mediator’s Middle module interferes with CTD-dependent RNAPII binding to a previously unknown Middle module CTD-binding site targeted early on in a multi-step holoenzyme formation process. Taken together, our results reveal the basis for CKM repression, clarify the origin of the connection between CKM subunits and the CTD, and suggest that a combination of competitive interactions and conformational changes that facilitate holoenzyme formation underlie the Mediator mechanism.

A role for CTCF and cohesin in subtelomere chromatin organization, TERRA transcription, and telomere end protection.

The contribution of human subtelomeric DNA and chromatin organization to telomere integrity and chromosome end protection is not yet understood in molecular detail. Here, we show by ChIP-Seq that most human subtelomeres contain a CTCF- and cohesin-binding site within ∼1-2 kb of the TTAGGG repeat tract and adjacent to a CpG-islands implicated in TERRA transcription control. ChIP-Seq also revealed that RNA polymerase II (RNAPII) was enriched at sites adjacent to the CTCF sites and extending towards the telomere repeat tracts. Mutation of CTCF-binding sites in plasmid-borne promoters reduced transcriptional activity in an orientation-dependent manner. Depletion of CTCF by shRNA led to a decrease in TERRA transcription, and a loss of cohesin and RNAPII binding to the subtelomeres. Depletion of either CTCF or cohesin subunit Rad21 caused telomere-induced DNA damage foci (TIF) formation, and destabilized TRF1 and TRF2 binding to the TTAGGG proximal subtelomere DNA. These findings indicate that CTCF and cohesin are integral components of most human subtelomeres, and important for the regulation of TERRA transcription and telomere end protection.

The N-terminal Region of Yeast TFIIB Contains Two Adjacent Functional Domains Involved in Stable RNA Polymerase II Binding and Transcription Start Site Selection

The general transcription factor IIB (TFIIB) is required for accurate and efficient transcription of protein-coding genes by RNA polymerase II (RNAPII). To define functional domains in the highly conserved N-terminal region of TFIIB, we have analyzed 14 site-directed substitution mutants of yeast TFIIB for their ability to support cell viability, transcription in vitro, accurate start site selection in vitro and in vivo, and to form stable complexes with purified RNAPII in vitro. Mutations impairing the formation of stable TFIIB.RNAPII complexes mapped to the zinc ribbon fold, whereas mutations conferring downstream shifts in transcription start site selection were identified at multiple positions within a highly conserved homology block adjacent and C-terminal to the zinc ribbon. These results demonstrate that the N-terminal region of yeast TFIIB contains two separable and adjacent functional domains involved in stable RNAPII binding and transcription start site selection, suggesting that downstream shifts in transcription start site selection do not result from impairment of stable TFIIB.RNAPII binding. We discuss models for yeast start site selection in which TFIIB may affect the ability of preinitiation complexes to interact with downstream DNA or to affect start site recognition by a scanning polymerase.