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Gene activator proteins comprise distinct DNA-binding and transcriptional activation domains (ADs). Because few ADs have been described, we tested domains tiling all yeast transcription factors for activation in vivo and identified 150 ADs. By mRNA display, we showed that 73% of ADs bound the Med15 subunit of Mediator, and that binding strength was correlated with activation. AD-Mediator interaction in vitro was unaffected by a large excess of free activator protein, pointing to a dynamic mechanism of interaction. Structural modeling showed that ADs interact with Med15 without shape complementarity (‘fuzzy’ binding). ADs shared no sequence motifs, but mutagenesis revealed biochemical and structural constraints. Finally, a neural network trained on AD sequences accurately predicted ADs in human proteins and in other yeast proteins, including chromosomal proteins and chromatin remodeling complexes. These findings solve the longstanding enigma of AD structure and function and provide a rationale for their role in biology. Cells adapt and respond to changes by regulating the activity of their genes. To turn genes on or off, they use a family of proteins called transcription factors. Transcription factors influence specific but overlapping groups of genes, so that each gene is controlled by several transcription factors that act together like a dimmer switch to regulate gene activity. The presence of transcription factors attracts proteins such as the Mediator complex, which activates genes by gathering the protein machines that read the genes. The more transcription factors are found near a specific gene, the more strongly they attract Mediator and the more active the gene is. A specific region on the transcription factor called the activation domain is necessary for this process. The biochemical sequences of these domains vary greatly between species, yet activation domains from, for example, yeast and human proteins are often interchangeable. To understand why this is the case, Sanborn et al. analyzed the genome of baker’s yeast and identified 150 activation domains, each very different in sequence. Three-quarters of them bound to a subunit of the Mediator complex called Med15. Sanborn et al. then developed a machine learning algorithm to predict activation domains in both yeast and humans. This algorithm also showed that negatively charged and greasy regions on the activation domains were essential to be activated by the Mediator complex. Further analyses revealed that activation domains used different poses to bind multiple sites on Med15, a behavior known as ‘fuzzy’ binding. This creates a high overall affinity even though the binding strength at each individual site is low, enabling the protein complexes to remain dynamic. These weak interactions together permit fine control over the activity of several genes, allowing cells to respond quickly and precisely to many changes. The computer algorithm used here provides a new way to identify activation domains across species and could improve our understanding of how living things grow, adapt and evolve. It could also give new insights into mechanisms of disease, particularly cancer, where transcription factors are often faulty.

Gene expression is controlled by dynamic localization of thousands of regulatory proteins to precise genomic regions. Understanding this cell type-specific process has been a longstanding goal yet remains challenging because DNA–protein mapping methods generally study one protein at a time. Here, to address this, we developed chromatin immunoprecipitation done in parallel (ChIP-DIP) to generate genome-wide maps of hundreds of diverse regulatory proteins in a single experiment. ChIP-DIP produces highly accurate maps within large pools (>160 proteins) for all classes of DNA-associated proteins, including modified histones, chromatin regulators and transcription factors and across multiple conditions simultaneously. First, we used ChIP-DIP to measure temporal chromatin dynamics in primary dendritic cells following LPS stimulation. Next, we explored quantitative combinations of histone modifications that define distinct classes of regulatory elements and characterized their functional activity in human and mouse cell lines. Overall, ChIP-DIP generates context-specific protein localization maps at consortium scale within any molecular biology laboratory and experimental system. ChIP-DIP (ChIP done in parallel) is a highly multiplex assay for protein–DNA binding, scalable to hundreds of proteins including modified histones, chromatin regulators and transcription factors, offering a refined view of the cis -regulatory code.

Gene activator proteins comprise distinct DNA-binding and transcriptional activation domains (ADs). Because few ADs have been described, we tested domains tiling all yeast transcription factors for activation in vivo and identified 150 ADs. By mRNA display, we showed that 73% of ADs bound the Med15 subunit of Mediator, and that binding strength was correlated with activation. AD-Mediator interaction in vitro was unaffected by a large excess of free activator protein, pointing to a dynamic mechanism of interaction. Structural modeling showed that ADs interact with Med15 without shape complementarity (‘fuzzy’ binding). ADs shared no sequence motifs, but mutagenesis revealed biochemical and structural constraints. Finally, a neural network trained on AD sequences accurately predicted ADs in human proteins and in other yeast proteins, including chromosomal proteins and chromatin remodeling complexes. These findings solve the longstanding enigma of AD structure and function and provide a rationale for their role in biology.

in vivo and identified 150 ADs. By mRNA display, we showed that 73% of ADs bound the Med15 subunit of Mediator, and that binding strength was correlated with activation. AD-Mediator interaction in vitro was unaffected by a large excess of free activator protein, pointing to a dynamic mechanism of interaction. Structural modeling showed that ADs interact with Med15 without shape complementarity (“fuzzy” binding). ADs shared no sequence motifs, but mutagenesis revealed biochemical and structural constraints. Finally, a neural network trained on AD sequences accurately predicted ADs in human proteins and in other yeast proteins, including chromosomal proteins and chromatin remodeling complexes. These findings solve the longstanding enigma of AD structure and function and provide a rationale for their role in biology. Competing Interest Statement The authors have declared no competing interest. Footnotes * ↵+ Lead Contact

Methylation patterns of circulating cell-free DNA (cfDNA) contain rich information about recent cell death events in the body. Here, we present an approach for unbiased determination of the tissue origins of cfDNA, using a reference methylation atlas of 25 human tissues and cell types. The method is validated using in silico simulations as well as in vitro mixes of DNA from different tissue sources at known proportions. We show that plasma cfDNA of healthy donors originates from white blood cells (55%), erythrocyte progenitors (30%), vascular endothelial cells (10%) and hepatocytes (1%). Deconvolution of cfDNA from patients reveals tissue contributions that agree with clinical findings in sepsis, islet transplantation, cancer of the colon, lung, breast and prostate, and cancer of unknown primary. We propose a procedure which can be easily adapted to study the cellular contributors to cfDNA in many settings, opening a broad window into healthy and pathologic human tissue dynamics. The methylation status of circulating cell-free DNA (cfDNA) can be informative about recent cell death events. Here the authors present an approach to determine the tissue origins of cfDNA, using a reference methylation atlas of 25 human tissues and cell types, and find that cfDNA from patients reveals tissue contributions that agree with clinical findings.

El Niño–Southern Oscillation (ENSO) is a naturally occurring mode of tropical Pacific variability, with global impacts on society and natural ecosystems. While it has long been known that El Niño events display a diverse range of amplitudes, triggers, spatial patterns, and life cycles, the realization that ENSO’s impacts can be highly sensitive to this event-to-event diversity is driving a renewed interest in the subject. This paper surveys our current state of knowledge of ENSO diversity, identifies key gaps in understanding, and outlines some promising future research directions.

Base editors use DNA-modifying enzymes targeted with a catalytically impaired CRISPR protein to precisely install point mutations. Here, we develop phage-assisted continuous evolution of base editors (BE–PACE) to improve their editing efficiency and target sequence compatibility. We used BE–PACE to evolve cytosine base editors (CBEs) that overcome target sequence context constraints of canonical CBEs. One evolved CBE, evoAPOBEC1-BE4max, is up to 26-fold more efficient at editing cytosine in the GC context, a disfavored context for wild-type APOBEC1 deaminase, while maintaining efficient editing in all other sequence contexts tested. Another evolved deaminase, evoFERNY, is 29% smaller than APOBEC1 and edits efficiently in all tested sequence contexts. We also evolved a CBE based on CDA1 deaminase with much higher editing efficiency at difficult target sites. Finally, we used data from evolved CBEs to illuminate the relationship between deaminase activity, base editing efficiency, editing window width and byproduct formation. These findings establish a system for rapid evolution of base editors and inform their use and improvement. Improved base editors are generated by continuous evolution.

Massachusetts has one of the highest cumulative incidence rates of coronavirus disease 2019 (COVID-19) cases in the US. Understanding which specific demographic, economic, and occupational factors have contributed to disparities in COVID-19 incidence rates across the state is critical to informing public health strategies. We performed a cross-sectional study of 351 Massachusetts cities and towns from January 1 to May 6, 2020, and found that a 10-percentage-point increase in the Black non-Latino population was associated with an increase of 312.3 COVID-19 cases per 100,000 population, whereas a 10-percentage-point increase in the Latino population was associated with an increase of 258.2 cases per 100,000. Independent predictors of higher COVID-19 rates included the proportion of foreign-born noncitizens living in a community, mean household size, and share of food service workers. After adjustment for these variables, the association between the Latino population and COVID-19 rates was attenuated. In contrast, the association between the Black population and COVID-19 rates persisted but may be explained by other systemic inequities. Public health and policy efforts that improve care for foreign-born noncitizens, address crowded housing, and protect food service workers may help mitigate the spread of COVID-19 among minority communities.

Treatment with aspirin and a P2Y12 inhibitor is commonly used in patients with cardiovascular disorders. The overall effect of such treatment on all-cause mortality is unknown. In the Dual Antiplatelet Therapy (DAPT) Study, continuation of dual antiplatelet therapy beyond 12 months after coronary stenting was associated with an unexpected increase in non-cardiovascular death. In view of the potential public health importance of these findings, we aimed to assess the effect of extended duration dual antiplatelet therapy on mortality by doing a meta-analysis of all randomised, controlled trials of treatment duration in various cardiovascular disorders. We searched Medline, Embase, and Cochrane Central Register of Controlled Trials (CENTRAL) to identify randomised controlled trials assessing the effect of extended duration versus no or short duration dual antiplatelet therapy, published before Oct 1, 2014. We did a meta-analysis to pool results with a hierarchical Bayesian random-effects model. The primary outcomes were hazard ratios comparing rates of all-cause, cardiovascular, and non-cardiovascular death. Including the DAPT Study, we identified 14 eligible trials that randomly assigned 69 644 participants to different durations of dual antiplatelet therapy. Compared with aspirin alone or short duration dual antiplatelet therapy (≤6 months), continued treatment was not associated with a difference in all-cause mortality (hazard ratio [HR] 1·04, 95% credible interval [CrI] 0·96–1·18; p=0·17). Similarly, cardiovascular (1·01, 0·93–1·12; p=0·81) and non-cardiovascular mortality (1·04, 0·90–1·26; p=0·66) were no different with extended duration versus short duration dual antiplatelet therapy or aspirin alone. Extended duration dual antiplatelet therapy was not associated with a difference in the risk of all-cause, cardiovascular, or non-cardiovascular death compared with aspirin alone or short duration dual antiplatelet therapy. None.

Glutathione (GSH) is a small-molecule thiol that is abundant in all eukaryotes and has key roles in oxidative metabolism 1 . Mitochondria, as the major site of oxidative reactions, must maintain sufficient levels of GSH to perform protective and biosynthetic functions 2 . GSH is synthesized exclusively in the cytosol, yet the molecular machinery involved in mitochondrial GSH import remains unknown. Here, using organellar proteomics and metabolomics approaches, we identify SLC25A39, a mitochondrial membrane carrier of unknown function, as a regulator of GSH transport into mitochondria. Loss of SLC25A39 reduces mitochondrial GSH import and abundance without affecting cellular GSH levels. Cells lacking both SLC25A39 and its paralogue SLC25A40 exhibit defects in the activity and stability of proteins containing iron–sulfur clusters. We find that mitochondrial GSH import is necessary for cell proliferation in vitro and red blood cell development in mice. Heterologous expression of an engineered bifunctional bacterial GSH biosynthetic enzyme (GshF) in mitochondria enables mitochondrial GSH production and ameliorates the metabolic and proliferative defects caused by its depletion. Finally, GSH availability negatively regulates SLC25A39 protein abundance, coupling redox homeostasis to mitochondrial GSH import in mammalian cells. Our work identifies SLC25A39 as an essential and regulated component of the mitochondrial GSH-import machinery. SLC25A39 and its paralogue SLC25A40 have redundant roles in the import of glutathione into mitochondria of mammalian cells.