Polymerase II Research Articles

Structural basis of exoribonuclease-mediated mRNA transcription termination.

Efficient termination is required for robust gene transcription. Eukaryotic organisms use a conserved exoribonuclease-mediated mechanism to terminate the mRNA transcription by RNA polymerase II (PolII)1-5. Here we report two cryogenic electron microscopy structures of Saccharomyces cerevisiae PolII pre-termination transcription complexes bound to the 5'-to-3' exoribonuclease Rat1 and its partner Rai1. Our structures show that Rat1 displaces the elongation factor Spt5 to dock at the PolII stalk domain. Rat1 shields the RNA exit channel of PolII, guides the nascent RNA towards its active centre and stacks three nucleotides at the 5' terminus of the nascent RNA. The structures further show that Rat1 rotates towards PolII as it shortens RNA. Our results provide the structural mechanism for the Rat1-mediated termination of mRNA transcription by PolII in yeast and the exoribonuclease-mediated termination of mRNA transcription in other eukaryotes.

Abstract 1699: Defining the impact of aberrant transcription on the chromatin structure

Abstract Genome organization in humans is governed by two major mechanisms: loop extrusion by cohesin and CTCF and the spatial compartmentalization of the chromosomes. These folding mechanisms have been shown to regulate several genomic functions, including gene expression. However, how transcription might regulate the chromatin structure remains a subject of many ongoing investigations. In this study, we use the Integrator protein complex as a tool to understand the impact of aberrant transcription on the 3D structure of the genome. During transcription initiation, RNA Polymerase-II (Pol-II) often pauses proximally to the promoter before further elongating transcripts. The Integrator protein complex regulates this checkpoint by its endonuclease and phosphatase activity. This protein complex is essential in premature transcription termination in hundreds of protein-coding genes. It also facilitates the synthesis of non-coding (ncRNA), such as enhancer RNAs (eRNAs) and long non-coding RNAs (lncRNAs), which have been shown to support loop extrusion domains, and their absence may disrupt these domains. Using HiC sequencing, we found that depleting Integrator proteins can interfere with forming and maintaining loop domains. This study aims to be the first to decipher the relationship between chromosome structure and Integrator protein activity and address our very little knowledge of how misregulation of transcription termination may influence the folding of chromosomes. Citation Format: Naiju Thomas, Timothy E. Reznicek, Erez Lieberman Aiden, M. Jordan Rowley, Eric Wagner, Guy Nir. Defining the impact of aberrant transcription on the chromatin structure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1699.

Emerging roles and mechanisms of lncRNAs in fruit and vegetables.

With the development of genome sequencing technologies, many long non-coding RNAs (lncRNAs) have been identified in fruit and vegetables. lncRNAs are primarily transcribed and spliced by RNA polymerase II (PolII) or plant-specific PolIV/V, and exhibit limited evolutionary conservation. lncRNAs intricately regulate various aspects of fruit and vegetables, including pigment accumulation, reproductive tissue development, fruit ripening, and responses to biotic and abiotic stresses, through diverse mechanisms such as gene expression modulation, interaction with hormones and transcription factors, microRNA regulation, and involvement in alternative splicing. This review presents a comprehensive overview of lncRNA classification, basic characteristics, and, most importantly, recent advances in understanding their functions and regulatory mechanisms.

Evolutionary history of a cold-adapted limnephilid caddisfly: Effects of climate change and topography on genetic structure

The distribution of organisms is influenced by complex factors such as the phylogenetic evolutionary histories of species, the physiological and ecological characteristics of organisms, climate, and geographical and geohistorical features. In this study, we focused on a caddisfly, Asynarchus sachalinensis (Trichoptera: Limnephilidae), which has adapted to cold habitats. From phylogeographic analyses based on the mitochondrial DNA (mtDNA) cytochrome c oxidase subunit I (COI) and 16S rRNA regions and the nuclear DNA (nDNA) 18S rRNA, 28S rRNA, carbamoyl-phosphate synthetase (CAD), elongation factor-1 alpha (EF1-α), and RNA polymerase II (POLII) regions, two distinct genetic clades were detected. Clade I was shown to be widely distributed from Sakhalin to Honshu, whereas Clade II was only distributed within Honshu. The distributions of these clades overlapped in Honshu. The habitats were located at relatively lower altitudes for Clade I and higher altitudes for Clade II. The divergence time of these clades was estimated to be during the Pleistocene, indicating that repeated climatic changes facilitated distributional shifts. Haplotype network and demographic analyses based on the mtDNA COI region showed contrasting genetic structures in the two clades. It was indicated that the population sizes of Clade I had expanded rapidly in a recent period, whereas Clade II had maintained stable population sizes. The habitats of Clade II were typically isolated and scattered at high altitudes, resulting in restricted migration and dispersal because of their discontinuous “Sky Island” habitats. The habitats of Clade I were located at relatively low altitudes, and it was assumed that the populations were continuous, which resulted in a higher frequency of migration and dispersal between populations. Thus, differences in the spatial scale of the adapted habitats of each clade may have resulted in different patterns of population connectivity and fragmentation associated with repeated climatic changes during the Pleistocene. Our study provided new insight into the distributional patterns of cold-adapted aquatic insects in the Japanese Archipelago. Furthermore, the distributional shifts predicted by ecological niche modeling under future climatic change conditions were different for each clade. Therefore, different principles are required in the assessment of each clade to predict temporal changes in their distributions.

The HOS15-HDA9 complex associates with HYL1 to modulate miRNA expression in response to ABA signaling.

The regulation of microRNA (miRNA) biogenesis is crucial for maintaining plant homeostasis under biotic and abiotic stress. The crosstalk between the RNA polymerase II (Pol-II) complex and the miRNA processing machinery has emerged as a central hub modulating transcription and co-transcriptional processing of primary miRNA transcripts (pri-miRNAs). However, it remains unclear how miRNA-specific transcriptional regulators recognize MIRNA loci. Here, we show that the Arabidopsis (Arabidopsis thaliana) HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE15 (HOS15)-HISTONE DEACETYLASE9 (HDA9) complex is a conditional suppressor of miRNA biogenesis, particularly in response to ABA. When treated with ABA, hos15/hda9 mutants show enhanced transcription of pri-miRNAs that is accompanied by increased processing, leading to over-accumulation of a set of mature miRNAs. Moreover, upon recognition of the nascent pri-miRNAs, the ABA-induced recruitment of the HOS15-HDA9 complex to MIRNA loci is guided by HYPONASTIC LEAVES 1 (HYL1). The HYL1-dependent recruitment of the HOS15-HDA9 complex to MIRNA loci suppresses expression of MIRNAs and processing of pri-miRNA. Most importantly, our findings indicate that nascent pri-miRNAs serve as scaffolds for recruiting transcriptional regulators, specifically to MIRNA loci. This indicates that RNA molecules can act as regulators of their own expression by causing a negative feedback loop that turns off their transcription, providing a self-buffering system.

TEAD1 regulates cell proliferation through a pocket-independent transcription repression mechanism.

The Hippo-TEAD pathway regulates cellular proliferation and function. The existing paradigm is that TEAD co-activators, YAP and TAZ, and co-repressor, VGLL4, bind to the pocket region of TEAD1 to enable transcriptional activation or repressive function. Here we demonstrate a pocket-independent transcription repression mechanism whereby TEAD1 controls cell proliferation in both non-malignant mature differentiated cells and in malignant cell models. TEAD1 overexpression can repress tumor cell proliferation in distinct cancer cell lines. In pancreatic β cells, conditional knockout of TEAD1 led to a cell-autonomous increase in proliferation. Genome-wide analysis of TEAD1 functional targets via transcriptomic profiling and cistromic analysis revealed distinct modes of target genes, with one class of targets directly repressed by TEAD1. We further demonstrate that TEAD1 controls target gene transcription in a motif-dependent and orientation-independent manner. Mechanistically, we show that TEAD1 has a pocket region-independent, direct repressive function via interfering with RNA polymerase II (POLII) binding to target promoters. Our study reveals that TEAD1 target genes constitute a mutually restricted regulatory loop to control cell proliferation and uncovers a novel direct repression mechanism involved in its transcriptional control that could be leveraged in future studies to modulate cell proliferation in tumors and potentially enhance the proliferation of normal mature cells.

ERK-mediated NELF-A phosphorylation promotes transcription elongation of immediate-early genes by releasing promoter-proximal pausing of RNA polymerase II

Growth factor-induced, ERK-mediated induction of immediate-early genes (IEGs) is crucial for cell growth and tumorigenesis. Although IEG expression is mainly regulated at the level of transcription elongation by RNA polymerase-II (Pol-II) promoter-proximal pausing and its release, the role of ERK in this process remains unknown. Here, we identified negative elongation factor (NELF)-A as an ERK substrate. Upon growth factor stimulation, ERK phosphorylates NELF-A, which dissociates NELF from paused Pol-II at the promoter-proximal regions of IEGs, allowing Pol-II to resume elongation and produce full-length transcripts. Furthermore, we found that in cancer cells, PP2A efficiently dephosphorylates NELF-A, thereby preventing aberrant IEG expression induced by ERK-activating oncogenes. However, when PP2A inhibitor proteins are overexpressed, as is frequently observed in cancers, decreased PP2A activity combined with oncogene-mediated ERK activation conspire to induce NELF-A phosphorylation and IEG upregulation, resulting in tumor progression. Our data delineate previously unexplored roles of ERK and PP2A inhibitor proteins in carcinogenesis.

VIP152 is a selective CDK9 inhibitor with pre-clinical in vitro and in vivo efficacy in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is effectively treated with targeted therapies including Bruton tyrosine kinase inhibitors and BCL2 antagonists. When these become ineffective, treatment options are limited. Positive transcription elongation factor complex (P-TEFb), a heterodimeric protein complex composed of cyclin dependent kinase 9 (CDK9) and cyclin T1, functions to regulate short half-life transcripts by phosphorylation of RNA Polymerase II (POLII). These transcripts are frequently dysregulated in hematologic malignancies; however, therapies targeting inhibition of P-TEFb have not yet achieved approval for cancer treatment. VIP152 kinome profiling revealed CDK9 as the main enzyme inhibited at 100 nM, with over a 10-fold increase in potency compared with other inhibitors currently in development for this target. VIP152 induced cell death in CLL cell lines and primary patient samples. Transcriptome analysis revealed inhibition of RNA degradation through the AU-Rich Element (ARE) dysregulation. Mechanistically, VIP152 inhibits the assembly of P-TEFb onto the transcription machinery and disturbs binding partners. Finally, immune competent mice engrafted with CLL-like cells of Eµ-MTCP1 over-expressing mice and treated with VIP152 demonstrated reduced disease burden and improvement in overall survival compared to vehicle-treated mice. These data suggest that VIP152 is a highly selective inhibitor of CDK9 that represents an attractive new therapy for CLL.

Histone Acetyltransferases p300 and CBP Coordinate Distinct Chromatin Remodeling Programs in Vascular Smooth Muscle Plasticity.

Vascular smooth muscle cell (VSMC) phenotypic switching contributes to cardiovascular diseases. Epigenetic regulation is emerging as a key regulatory mechanism, with the methylcytosine dioxygenase TET2 acting as a master regulator of smooth muscle cell phenotype. The histone acetyl-transferases p300 and CREB-binding protein (CBP) are highly homologous and often considered to be interchangeable, and their roles in smooth muscle cell phenotypic regulation are not known. We assessed the roles of p300 and CBP in human VSMC with knockdown, in inducible smooth muscle-specific knockout mice (inducible knockout [iKO]; p300iKO or CBPiKO), and in samples of human intimal hyperplasia. P300, CBP, and histone acetylation were differently regulated in VSMCs undergoing phenotypic switching and in vessel remodeling after vascular injury. Medial p300 expression and activity were repressed by injury, but CBP and histone acetylation were induced in neointima. Knockdown experiments revealed opposing effects of p300 and CBP in the VSMC phenotype: p300 promoted contractile protein expression and inhibited migration, but CBP inhibited contractile genes and enhanced migration. p300iKO mice exhibited severe intimal hyperplasia after arterial injury compared with controls, whereas CBPiKO mice were entirely protected. In normal aorta, p300iKO reduced, but CBPiKO enhanced, contractile protein expression and contractility compared with controls. Mechanistically, we found that these histone acetyl-transferases oppositely regulate histone acetylation, DNA hydroxymethylation, and PolII (RNA polymerase II) binding to promoters of differentiation-specific contractile genes. Our data indicate that p300 and TET2 function together, because p300 was required for TET2-dependent hydroxymethylation of contractile promoters, and TET2 was required for p300-dependent acetylation of these loci. TET2 coimmunoprecipitated with p300, and this interaction was enhanced by rapamycin but repressed by platelet-derived growth factor (PDGF) treatment, with p300 promoting TET2 protein stability. CBP did not associate with TET2, but instead facilitated recruitment of histone deacetylases (HDAC2, HDAC5) to contractile protein promoters. Furthermore, CBP inhibited TET2 mRNA levels. Immunostaining of cardiac allograft vasculopathy samples revealed that p300 expression is repressed but CBP is induced in human intimal hyperplasia. This work reveals that p300 and CBP serve nonredundant and opposing functions in VSMC phenotypic switching and coordinately regulate chromatin modifications through distinct functional interactions with TET2 or HDACs. Targeting specific histone acetyl-transferases may hold therapeutic promise for cardiovascular diseases.

Unveiling RCOR1 as a rheostat at transcriptionally permissive chromatin

RCOR1 is a known transcription repressor that recruits and positions LSD1 and HDAC1/2 on chromatin to erase histone methylation and acetylation. However, there is currently an incomplete understanding of RCOR1’s range of localization and function. Here, we probe RCOR1’s distribution on a genome-wide scale and unexpectedly find that RCOR1 is predominantly associated with transcriptionally active genes. Biochemical analysis reveals that RCOR1 associates with RNA Polymerase II (POL-II) during transcription and deacetylates its carboxy-terminal domain (CTD) at lysine 7. We provide evidence that this non-canonical RCOR1 activity is linked to dampening of POL-II productive elongation at actively transcribing genes. Thus, RCOR1 represses transcription in two ways—first, via a canonical mechanism by erasing transcriptionally permissive histone modifications through associating with HDACs and, second, via a non-canonical mechanism that deacetylates RNA POL-II’s CTD to inhibit productive elongation. We conclude that RCOR1 is a transcription rheostat.

In leukemia, knock-down of the death inducer-obliterator gene would inhibit the proliferation of endothelial cells by inhibiting the expression of CDK6 and CCND1.

BackgroundEndothelial cells (ECs) are a critical component of the hematopoietic niche, and the cross-talk between ECs and leukemia was reported recently. This study aimed to determine the genes involved in the proliferation inhibition of endothelial cells in leukemia.MethodsHuman umbilical vein endothelial cells (HUVEC) were cultured alone or co-cultured with K562 cell lines. GeneChip assays were performed to identify the differentially expressed genes. The Celigo, MTT assay, and flow cytometric analysis were used to determine the effect of RNAi DIDO on cell growth and apoptosis. The differently expressed genes were verified by qRT-PCR (quantitative real-time PCR) and western-blot.ResultsIn K562-HUVEC co-cultured cell lines, 323 down-regulated probes were identified and the extracellular signal-regulated kinase 5 (ERK5) signaling pathway was significantly inhibited. Among the down-regulated genes, the death inducer-obliterator gene (DIDO) is a part of the centrosome protein and may be involved in cell mitosis. As shown in the public data, leukemia patients with lower expression of DIDO showed a better overall survival (OS). The HUVEC cells were infected with shDIDO lentivirus, and reduced expression, inhibited proliferation, and increased apoptosis was observed in shDIDO cells. In addition, the expression of Cyclin-Dependent Kinase 6 (CDK6) and Cyclin D1 (CCND1) genes was inhibited in shDIDO cells. Finally, the public ChIP-seq data were used to analyze the regulators that bind with DIDO, and the H3K4me3 and PolII (RNA polymerase II) signals were found near the Exon1 and exon2 sites of DIDO.ConclusionThe knock-down of DIDO will inhibit the proliferation of endothelial cells in the leukemia environment. The expression of DIDO may be regulated by H3K4me3 and the inhibition of DIDO may lead to the down-regulation of CDK6 and CCND1. However, how DIDO interacts with CDK6 and CCND1 requires further study.

DNA-induced spatial entrapment of general transcription machinery can stabilize gene expression in a nondividing cell

An important characteristic of cell differentiation is its stability. Only rarely do cells or their stem cell progenitors change their differentiation pathway. If they do, it is often accompanied by a malfunction such as cancer. A mechanistic understanding of the stability of differentiated states would allow better prospects of alleviating the malfunctioning. However, such complete information is yet elusive. Earlier experiments performed in Xenopus oocytes to address this question suggest that a cell may maintain its gene expression by prolonged binding of cell type-specific transcription factors. Here, using DNA competition experiments, we show that the stability of gene expression in a nondividing cell could be caused by the local entrapment of part of the general transcription machinery in transcriptionally active regions. Strikingly, we found that transcriptionally active and silent forms of the same DNA template can stably coexist within the same nucleus. Both DNA templates are associated with the gene-specific transcription factor Ascl1, the core factor TBP2, and the polymerase II (Pol-II) ser5 C-terminal domain (CTD) phosphorylated form, while Pol-II ser2 CTD phosphorylation is restricted to the transcriptionally dominant template. We discover that the active and silent DNA forms are physically separated in the oocyte nucleus through partition into liquid-liquid phase-separated condensates. Altogether, our study proposes a mechanism of transcriptional regulation involving a spatial entrapment of general transcription machinery components to stabilize the active form of a gene in a nondividing cell.

Orphan Nuclear Receptor RORγ Modulates the Genome-Wide Binding of the Cholesterol Metabolic Genes during Mycotoxin-Induced Liver Injury.

Maintaining lipid homeostasis is crucial to liver function, the key organ that governs the whole-body energy metabolism. In contrast, lipid dysregulation has been implicated in mycotoxin-induced liver injury, by which the pathophysiological regulation and the molecular components involved remain elusive. Here we focused on the potential roles of orphan nuclear receptor (NR) RORγ in lipid programming, and aimed to explore its action on cholesterol regulation in the liver of mycotoxin-exposed piglets. We found that liver tissues were damaged in the mycotoxin-exposed piglets compared to the healthy controls, revealed by histological analysis, elevated seral ALT, AST and ALP levels, and increased caspase 3/7 activities. Consistent with the transcriptomic finding of down-regulated cholesterol metabolism, we demonstrated that both cholesterol contents and cholesterol biosynthesis/transformation gene expressions in the mycotoxin-exposed livers were reduced, including HMGCS1, FDPS, SQLE, EBP, FDFT1 and VLDLR. Furthermore, we reported that RORγ binds to the cholesterol metabolic genes in porcine hepatocytes using a genome-wide ChIP-seq analysis, whereas mycotoxin decreased the RORγ binding occupancies genome-wide, especially at the cholesterol metabolic pathway. In addition, we revealed the enrichment of co-factors p300 and SRC, the histone marks H3K27ac and H3K4me2, together with RNA Polymerase II (Pol-II) at the locus of HMGCS1 in hepatocytes, which were reduced by mycotoxin-exposure. Our results provide a deep insight into the cholesterol metabolism regulation during mycotoxin-induced liver injury, and propose NRs as therapeutic targets for anti-mycotoxin treatments.

APOLO lncRNA, a self-calibrating switch of root development

APOLO lncRNA, a self-calibrating switch of root development

Structures of mammalian RNA polymerase II pre-initiation complexes.

The initiation of transcription is a focal point for the regulation of gene activity during mammalian cell differentiation and development. To initiate transcription, RNA polymerase II (PolII) assembles with general transcription factors into a pre-initiation complex (PIC) that opens promoter DNA. Previous work provided the molecular architecture of the yeast1-9 and human10,11 PIC and a topological model for DNA opening by the general transcription factor TFIIH12-14. Here we report the high-resolution cryo-electron microscopy structure of PIC comprising human general factors and Sus scrofa domesticus PolII, which is 99.9% identical to human PolII. We determine the structures of PIC with closed and opened promoter DNA at 2.5-2.8Å resolution, and resolve the structure of TFIIH at 2.9-4.0Å resolution. We capture the TFIIH translocase XPB in the pre- and post-translocation states, and show that XPB induces and propagates a DNA twist to initiate the opening of DNA approximately 30 base pairs downstream of the TATA box. We also provide evidence that DNA opening occurs in two steps and leads to the detachment of TFIIH from the core PIC, which may stop DNA twisting and enable RNA chain initiation.

Structure of the human Mediator-RNA polymerase II pre-initiation complex.

Mediator is a conserved coactivator complex that enables the regulated initiation of transcription at eukaryotic genes1-3. Mediator is recruited by transcriptional activators and binds the pre-initiation complex (PIC) to stimulate the phosphorylation of RNA polymeraseII (PolII) and promoter escape1-6. Here we prepare a recombinant version of human Mediator, reconstitute a 50-subunit Mediator-PIC complex and determine the structure of the complex by cryo-electron microscopy. The head module of Mediator contacts the stalk of PolII and the general transcription factors TFIIB and TFIIE, resembling the Mediator-PIC interactions observed in the corresponding complex in yeast7-9. The metazoan subunits MED27-MED30 associate with exposed regions in MED14 and MED17 to form the proximal part of the Mediator tail module that binds activators. Mediator positions the flexibly linked cyclin-dependent kinase (CDK)-activating kinase of the general transcription factor TFIIH near the linker to the C-terminal repeat domain of PolII. The Mediator shoulder domain holds the CDK-activating kinase subunit CDK7, whereas the hook domain contacts a CDK7 element that flanks the kinase active site. The shoulder and hook domains reside in the Mediator head and middle modules, respectively, which can move relative to each other and may induce an active conformation of the CDK7 kinase to allosterically stimulate phosphorylationof the C-terminal domain.

TOX4 Promotes Promoter Proximal Pause of RNA Polymerase Ii by Facilitating the Recruitment of DSIF and NELF

Protein phosphatase 1 complex (PP1C), consisting of one of the phosphatases, PP1α, β and γ, and three regulatory subunits, PNUTS, TOX4 and WDR82, plays critical roles in gene regulation and is mutated in over 20% of uterine corpus endometrial carcinoma cases. TOX4 is the least understood PP1C subunit. Here we show that chromatin occupancy pattern of TOX4 resembles that of RNA polymerase II (Pol II), and its loss increases the phosphorylation of the C-terminal domain and decreases the global occupancy of Pol II. Mechanistically, it promotes promoter-proximal pause of Pol II by facilitating the recruitment of DSIF and NELF likely in a dephosphorylation dependent manner. Moreover, quantitative proteomic analyses revealed that TOX4 mainly facilitates protein dephosphorylation by PP1 phosphatases. Furthermore, Tox4 knockout in mice impairs T cell development by dysregulating genes critical for survival, proliferation and fate determination. These results lay the foundation for further understanding roles of TOX4 in transcription, development and diseases.

Oscillating and stable genome topologies underlie hepatic physiological rhythms during the circadian cycle.

The circadian clock drives extensive temporal gene expression programs controlling daily changes in behavior and physiology. In mouse liver, transcription factors dynamics, chromatin modifications, and RNA Polymerase II (PolII) activity oscillate throughout the 24-hour (24h) day, regulating the rhythmic synthesis of thousands of transcripts. Also, 24h rhythms in gene promoter-enhancer chromatin looping accompany rhythmic mRNA synthesis. However, how chromatin organization impinges on temporal transcription and liver physiology remains unclear. Here, we applied time-resolved chromosome conformation capture (4C-seq) in livers of WT and arrhythmic Bmal1 knockout mice. In WT, we observed 24h oscillations in promoter-enhancer loops at multiple loci including the core-clock genes Period1, Period2 and Bmal1. In addition, we detected rhythmic PolII activity, chromatin modifications and transcription involving stable chromatin loops at clock-output gene promoters representing key liver function such as glucose metabolism and detoxification. Intriguingly, these contacts persisted in clock-impaired mice in which both PolII activity and chromatin marks no longer oscillated. Finally, we observed chromatin interaction hubs connecting neighbouring genes showing coherent transcription regulation across genotypes. Thus, both clock-controlled and clock-independent chromatin topology underlie rhythmic regulation of liver physiology.

Motif-driven interactions between RNA and PRC2 are rheostats that regulate transcription elongation.

Although Polycomb repressive complex 2 (PRC2) is now recognized as an RNA-binding complex, the full range of binding motifs and why PRC2-RNA complexes often associate with active genes have not been elucidated. Here we identify high-affinity RNA motifs whose mutations weaken PRC2 binding and attenuate its repressive function in mouse embryonic stem cells. Interactions occur at promoter-proximal regions and frequently coincide with pausing of RNA Polymerase II (POL-II). Surprisingly, while PRC2-associated nascent transcripts are highly expressed, ablating PRC2 further upregulates expression via loss of pausing and enhanced transcription elongation. Thus, PRC2-nascent RNA complexes operate as rheostats to fine-tune transcription by regulating transitions between pausing and elongation, explaining why PRC2-RNA complexes frequently occur within active genes. Nascent RNA also targets PRC2 in cis and downregulates neighboring genes. We propose a unifying model in which RNA specifically recruits PRC2 to repress genes through POL-II pausing and, more classically, H3K27-trimethylation.

Time-restricted feeding downregulates cholesterol biosynthesis program via ROR\u03b3-mediated chromatin modification in porcine liver organoids

BackgroundTime-restricted feeding (TRF) is a dieting strategy based on nutrients availability and diurnal rhythm, shown to improve lipid metabolism efficiency. We have demonstrated previously that retinoic acid-related (RAR) orphan receptor (ROR) γ is the primary transcription factor controlling cholesterol (CHO) biosynthesis program of animals. However, the functional role of RORγ in liver physiology of pigs in response to TRF has not been determined, largely due to the lack of functional models and molecular tools. In the present study, we established porcine liver organoids and subjected them to restricted nutrients supply for 10-h during the light portion of the day.ResultsOur results showed that TRF regimen did not alter hepatocyte physiology, including unchanged cell viability, caspase 3/7 enzyme activity and the gene signature of cell proliferation in porcine liver organoids, compared to the control group (P > 0.05). Furthermore, we found that TRF downregulated the hepatic CHO biosynthesis program at both mRNA and protein levels, along with the reduced cellular CHO content in porcine liver organoids (P < 0.05). Using unbiased bioinformatic analysis of a previous ChIP-seq data and ChIP-qPCR validation, we revealed RORγ as the predominant transcription factor that responded to TRF, amongst the 12 targeted nuclear receptors (NRs) (P < 0.05). This was likely through RORγ direct binding to the MVK gene (encoding mevalonate kinase). Finally, we showed that RORγ agonists and overexpression enhanced the enrichment of co-factor p300, histone marks H3K27ac and H3K4me1/2, as well as RNA polymerase II (Pol-II) at the locus of MVK, in TRF-porcine liver organoids, compared to TRF-vector control (P < 0.05).ConclusionsOur findings demonstrate that TRF triggers the RORγ-mediated chromatin remodeling at the locus of CHO biosynthesis genes in porcine liver organoids and further improves lipid metabolism.