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Transcription factors (TFs) bind specific sequences in promoter-proximal and -distal DNA elements to regulate gene transcription. RNA is transcribed from both of these DNA elements, and some DNA binding TFs bind RNA. Hence, RNA transcribed from regulatory elements may contribute to stable TF occupancy at these sites. We show that the ubiquitously expressed TF Yin-Yang 1 (YY1) binds to both gene regulatory elements and their associated RNA species across the entire genome. Reduced transcription of regulatory elements diminishes YY1 occupancy, whereas artificial tethering of RNA enhances YY1 occupancy at these elements. We propose that RNA makes a modest but important contribution to the maintenance of certain TFs at gene regulatory elements and suggest that transcription of regulatory elements produces a positive-feedback loop that contributes to the stability of gene expression programs.

The synthesis of pre-mRNA by RNA polymerase II (Pol II) involves the formation of a transcription initiation complex, and a transition to an elongation complex 1 – 4 . The large subunit of Pol II contains an intrinsically disordered C-terminal domain that is phosphorylated by cyclin-dependent kinases during the transition from initiation to elongation, thus influencing the interaction of the C-terminal domain with different components of the initiation or the RNA-splicing apparatus 5 , 6 . Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain 7 – 12 . Both the transcription-initiation machinery and the splicing machinery can form phase-separated condensates that contain large numbers of component molecules: hundreds of molecules of Pol II and mediator are concentrated in condensates at super-enhancers 7 , 8 , and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites 9 – 12 . Here we investigate whether the phosphorylation of the Pol II C-terminal domain regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing. We find that the hypophosphorylated C-terminal domain of Pol II is incorporated into mediator condensates and that phosphorylation by regulatory cyclin-dependent kinases reduces this incorporation. We also find that the hyperphosphorylated C-terminal domain is preferentially incorporated into condensates that are formed by splicing factors. These results suggest that phosphorylation of the Pol II C-terminal domain drives an exchange from condensates that are involved in transcription initiation to those that are involved in RNA processing, and implicates phosphorylation as a mechanism that regulates condensate preference. RNA polymerase II with a hypophosphorylated C-terminal domain preferentially incorporates into mediator condensates, and with a hyperphosphorylated C-terminal domain into splicing-factor condensates, revealing phosphorylation as a regulatory mechanism in condensate preference.

The nucleus contains diverse phase-separated condensates that compartmentalize and concentrate biomolecules with distinct physicochemical properties. Here, we investigated whether condensates concentrate small-molecule cancer therapeutics such that their pharmacodynamic properties are altered. We found that antineoplastic drugs become concentrated in specific protein condensates in vitro and that this occurs through physicochemical properties independent of the drug target. This behavior was also observed in tumor cells, where drug partitioning influenced drug activity. Altering the properties of the condensate was found to affect the concentration and activity of drugs. These results suggest that selective partitioning and concentration of small molecules within condensates contributes to drug pharmacodynamics and that further understanding of this phenomenon may facilitate advances in disease therapy.

One-carbon (1C) metabolism is a universal metabolic process that is required for purine synthesis and supports the high levels of proliferation in cancer cells. The transport of serine into mitochondria supplies most of the 1C units needed for biosynthesis. Kory et al. used a genetic screen to identify the long-sought-after mitochondrial serine transporter. Elucidating the key step of serine transport is important for our understanding of metabolism and has potential implications for cancer treatment. Science , this issue p. eaat9528 A mitochondrial serine transporter supports one-carbon metabolism, which is important for nucleotide synthesis in both normal and cancer cells. One-carbon metabolism generates the one-carbon units required to synthesize many critical metabolites, including nucleotides. The pathway has cytosolic and mitochondrial branches, and a key step is the entry, through an unknown mechanism, of serine into mitochondria, where it is converted into glycine and formate. In a CRISPR-based genetic screen in human cells for genes of the mitochondrial pathway, we found sideroflexin 1 (SFXN1), a multipass inner mitochondrial membrane protein of unclear function. Like cells missing mitochondrial components of one-carbon metabolism, those null for SFXN1 are defective in glycine and purine synthesis. Cells lacking SFXN1 and one of its four homologs, SFXN3, have more severe defects, including being auxotrophic for glycine. Purified SFXN1 transports serine in vitro. Thus, SFXN1 functions as a mitochondrial serine transporter in one-carbon metabolism.

The capacity to generate functional hepatocytes from renewable human pluripotent stem cells (hPSCs) could address limited supplies of primary human hepatocytes. However, hepatocytes differentiated from hPSCs in vitro are functionally immature. To understand mechanisms regulating maturation of in vitro derived hepatocytes, we developed a 3D spheroid differentiation system and compared gene regulatory elements in uncultured human primary hepatocytes with those in hepatocytes that were differentiated in 2D or 3D conditions from human PSCs by RNA-seq, ATAC-seq, and H3K27Ac ChIP-seq. Three-dimensional differentiation improved enhancer activity and expression of transcription factor ONECUT1, but was insufficient to upregulate human-specific mature hepatocytes marker gene CYP3A4 or super-enhancer regulated transcription factor gene NFIC. Regulome comparisons showed reduced enrichment of thyroid receptor THRB motifs in accessible chromatin and in active enhancers without reduced transcription of THRB, suggesting the regulation at the level of THRB ligands in PSC-differentiated hepatocytes. Addition of thyroid hormone T3 to the PSC-differentiated hepatocytes increased CYP3A4 expression. T3 increased binding of THRB to the CYP3A4 proximal enhancer and restored the super-enhancer status and gene expression of NFIC and reduced expression of AFP. The resultant hPSC-hepatocytes showed gene expression, epigenetic status and super-enhancer landscape closer to primary hepatocytes and activated regulatory regions including non-coding SNPs associated with liver-related diseases. Transplanting the 3D PSC-hepatocytes into immunocompromised mice resulted in engraftment of human hepatocytes in the mouse liver parenchyma without disrupting normal liver histology at 6 months after transplantation. This work provides insights into the functions of nuclear receptor THRB and highlights the importance of the environmental factors-nuclear receptors axis in regulating maturation of human PSC-differentiated cell types. Competing Interest Statement R.J. is a cofounder of Fate, Fulcrum, and Omega Therapeutics and an advisor to Dewpoint and Camp4 Therapeutics. R.A.Y. is a founder and shareholder of Syros Pharmaceuticals, Camp4 Therapeutics, Omega Therapeutics, and Dewpoint Therapeutics. J.F.J. and M.B. are employees of Novo Nordisk A/S. T.L. is a shareholder of Syros Pharmaceuticals and a consultant to Camp4 Therapeutics. The remaining authors have no competing interests.

Herpesvirus saimiri (HVS) is an oncogenic γ-herpesvirus that causes fatal T-cell lymphomas and leukemias in primates and transforms human primary T cells. The most abundant viral transcripts in latently-infected marmoset T cells are seven small U-rich noncoding (nc) RNAs called HSURs. Although they were discovered over 25 years ago, little is known about their functions. In 2010, the Steitz lab found that HSUR 1 base pairs with a highly-conserved host microRNA, miR-27, leading to its degradation in a sequence-specific and binding-dependent manner. However, the biological function of miR-27 in T cells was unknown, confounding the reason why miR-27 degradation is important for HVS. To understand the role of miR-27 in HVS-infected T cells, I first used high-throughput sequencing of RNA after crosslinking immunoprecipitation (HITS-CLIP) to identify messenger RNA (mRNA) targets of miR-27. Genes in the T-Cell Receptor (TOR) signaling pathway, including the major adaptor protein: growth factor receptor-bound protein 2 (GRB2), are enriched among miR-27 targets. Accordingly, I found that transfection of miR-27 into human T cells attenuates TCR-induced activation of mitogen-activated protein kinases (MAPKs) and induction of a cell-surface marker, CD69. MiR-27 also robustly downregulates semaphorin 7A (SEMA7A) and interferon-γ (lFN-γ), key modulators and effectors of T-cell activation. In HVS-infected cells, HSUR 1 upregulates expression of these key T-cell activation proteins by mediating miR-27 degradation. My results uncovered the molecular mechanism of HSUR 1's contribution to the constitutive activation and proliferation of HVS-infected T cells, and thus the persistent infection and propagation of HVS. Furthermore, I made the surprising discovery that two distantly-related oncogenic γ-herpesviruses, Alcelaphine herpesvirus 1 (AIHV-1) and Ovine herpesvirus 2 (OvHV-2), which also cause lethal T-cell lymphoproliferative disorders (known as malignant catarrhal fever, or MCF) in certain ruminants, do not carry a ncRNA to degrade miR-27 but instead encode viral homologs of miR-27 target genes, including SEMA7A, in the syntenic region. My research also suggests that similar alternative approaches for enhancing host-cell gene expression operate in cytomegalovirus (CMV) infection. I also worked on developing new biochemical methods for discovering targets of miRNAs. Because synthetic 3'-biotin-tagged microRNAs have often been used to select interacting mRNA and ncRNA targets, I examined the extent of association of 3'-end biotinylated miR-27 with Argonaute proteins in transfected human cells using a co-immunoprecipitation assay followed by Northern blot analysis. I found that biotinylated miR-27 is not efficiently associated with Ago compared to unmodified miR-27. These results suggest that 3'-end biotin-modified miRNAs are questionable monitors of miRNA function in cells.

Existing natural language processing (NLP) methods to convert free-text clinical notes into structured data often require problem-specific annotations and model training. This study aims to evaluate ChatGPT’s capacity to extract information from free-text medical notes efficiently and comprehensively. We developed a large language model (LLM)-based workflow, utilizing systems engineering methodology and spiral “prompt engineering” process, leveraging OpenAI’s API for batch querying ChatGPT. We evaluated the effectiveness of this method using a dataset of more than 1000 lung cancer pathology reports and a dataset of 191 pediatric osteosarcoma pathology reports, comparing the ChatGPT-3.5 (gpt-3.5-turbo-16k) outputs with expert-curated structured data. ChatGPT-3.5 demonstrated the ability to extract pathological classifications with an overall accuracy of 89%, in lung cancer dataset, outperforming the performance of two traditional NLP methods. The performance is influenced by the design of the instructive prompt. Our case analysis shows that most misclassifications were due to the lack of highly specialized pathology terminology, and erroneous interpretation of TNM staging rules. Reproducibility shows the relatively stable performance of ChatGPT-3.5 over time. In pediatric osteosarcoma dataset, ChatGPT-3.5 accurately classified both grades and margin status with accuracy of 98.6% and 100% respectively. Our study shows the feasibility of using ChatGPT to process large volumes of clinical notes for structured information extraction without requiring extensive task-specific human annotation and model training. The results underscore the potential role of LLMs in transforming unstructured healthcare data into structured formats, thereby supporting research and aiding clinical decision-making.

Transmission of the novel avian influenza A H7N9 virus seems to be predominantly between poultry and people. In the major Chinese cities of Shanghai, Hangzhou, Huzhou, and Nanjing—where most human cases of infection have occurred—live poultry markets (LPMs) were closed in April, 2013, soon after the initial outbreak, as a precautionary public health measure. Our objective was to quantify the effect of LPM closure in these cities on poultry-to-person transmission of avian influenza A H7N9 virus. We obtained information about every laboratory-confirmed human case of avian influenza A H7N9 virus infection reported in the four cities by June 7, 2013, from a database built by the Chinese Center for Disease Control and Prevention. We used data for age, sex, location, residence type (rural or urban area), and dates of illness onset. We obtained information about LPMs from official sources. We constructed a statistical model to explain the patterns in incidence of cases reported in each city on the basis of the assumption of a constant force of infection before LPM closure, and a different constant force of infection after closure. We fitted the model with Markov chain Monte Carlo methods. 85 human cases of avian influenza A H7N9 virus infection were reported in Shanghai, Hangzhou, Huzhou, and Nanjing by June 7, 2013, of which 60 were included in our main analysis. Closure of LPMs reduced the mean daily number of infections by 99% (95% credibility interval 93–100%) in Shanghai, by 99% (92–100%) in Hangzhou, by 97% (68–100%) in Huzhou, and by 97% (81–100%) in Nanjing. Because LPMs were the predominant source of exposure to avian influenza A H7N9 virus for confirmed cases in these cities, we estimated that the mean incubation period was 3·3 days (1·4–5·7). LPM closures were effective in the control of human risk of avian influenza A H7N9 virus infection in the spring of 2013. In the short term, LPM closure should be rapidly implemented in areas where the virus is identified in live poultry or people. In the long term, evidence-based discussions and deliberations about the role of market rest days and central slaughtering of all live poultry should be renewed. Ministry of Science and Technology, China; Research Fund for the Control of Infectious Disease; Hong Kong University Grants Committee; China–US Collaborative Program on Emerging and Re-emerging Infectious Diseases; Harvard Center for Communicable Disease Dynamics; and the US National Institutes of Health.

The unprecedented early spring frost that appears as a cold stress adversely affects growth and productivity in tea (Camellia sinensis L.); therefore, it is indispensable to develop approaches to improve the cold tolerance of tea. Here, we investigated the effect of pretreatment with exogenous melatonin on the net photosynthetic rate, the maximum photochemical efficiency of PSII, chlorophyll content, lipid peroxidation, reactive oxygen species (ROS) accumulation, antioxidant potential, and redox homeostasis in leaves of tea plants following cold stress. Our results revealed that cold treatment induced oxidative stress by increasing ROS accumulation, which in turn affected the photosynthetic process in tea leaves. However, treatment with melatonin mitigated cold-induced reductions in photosynthetic capacity by reducing oxidative stress through enhanced antioxidant potential and redox homeostasis. This study provides strong evidence that melatonin could alleviate cold-induced adverse effects in tea plants.

Adhesion G-protein-coupled receptors (aGPCRs) are important for organogenesis, neurodevelopment, reproduction and other processes 1 – 6 . Many aGPCRs are activated by a conserved internal (tethered) agonist sequence known as the Stachel sequence 7 – 12 . Here, we report the cryogenic electron microscopy (cryo-EM) structures of two aGPCRs in complex with G s : GPR133 and GPR114. The structures indicate that the Stachel sequences of both receptors assume an α-helical–bulge–β-sheet structure and insert into a binding site formed by the transmembrane domain (TMD). A hydrophobic interaction motif (HIM) within the Stachel sequence mediates most of the intramolecular interactions with the TMD. Combined with the cryo-EM structures, biochemical characterization of the HIM motif provides insight into the cross-reactivity and selectivity of the Stachel sequences. Two interconnected mechanisms, the sensing of Stachel sequences by the conserved ‘toggle switch’ W 6.53 and the constitution of a hydrogen-bond network formed by Q 7.49 /Y 7.49 and the P 6.47 /V 6.47 φφG 6.50 motif (φ indicates a hydrophobic residue), are important in Stachel sequence-mediated receptor activation and G s coupling. Notably, this network stabilizes kink formation in TM helices 6 and 7 (TM6 and TM7, respectively). A common G s -binding interface is observed between the two aGPCRs, and GPR114 has an extended TM7 that forms unique interactions with G s . Our structures reveal the detailed mechanisms of aGPCR activation by Stachel sequences and their G s coupling. Adhesion GPCRs involved in cell and matrix interactions signal through a distinct self-cleavage, self-activation mechanism.