Howard Hughes Medical Institute

Cytokines dimerize two receptor chains to activate Janus kinases and STAT transcription factors that regulate immune cells but have therapeutic liabilities. We engineered “Trikines” to compel cis formation of three-chain cytokine receptor complexes at the cell surface that induce bespoke STAT transcriptional signaling programs optimized for therapeutic efficacy. Designed Trikines co-activated pSTAT5 and pSTAT3 signatures distinct from any natural cytokines, by assembling trimeric combinations of Interleukin-2 (IL-2), Interleukin-10 (IL-10), and Interleukin-21 (IL-21) receptors. An IL-2-based-Trikine restrained terminal differentiation of T cells, promoted stemness, and enhanced durability of tumor control without toxicity. Unexpectedly, an IL-10-based Trikine induced immune infiltration into poorly immunogenic tumors, showing striking efficacy in small cell lung cancer and pancreatic cancer models. Trikines obviate the need for cell engineering to customize STAT signatures for immunotherapy.

Histones are small basic proteins that form the proteinaceous core of the nucleosome, the repeating building block of chromatin in all eukaryotes. Long thought to be exclusive to eukaryotes, histones are now increasingly appreciated for their roles in organizing genomes across all domains of life, namely in archaea, bacteria, and even viruses. We survey recent advances in our understanding of the imaginative uses of histones in disparate biological entities, ranging from nucleosome-like metastable particles in giant viruses to slinky-like hypernucleosomes in archaea to bacterial histones that bind DNA in decidedly unorthodox ways. Across these different contexts, we examine how DNA compaction and conformation emanate from evolutionarily conserved aspects of histone structure, including how the oligomeric states of histones dictate their capacity to contort DNA in different conformations. It appears that relatively small tweaks to the amino acid sequences of histones can result in structural and functional variations in DNA binding. As such, nucleosomes in eukaryotes sample only a narrow range of possible structures.

Fat body-specific FASN1 loss leads to fat-depleted but viable Drosophila that complete their developmental lifecycle by rewiring energy metabolism to store glycogen instead of fat FASN1-deficient larvae functionally rely on glycogen synthesis and glycolysis, but not lactate metabolism, and display blunted TCA metabolismMetabolic screening reveals a SREBP-dependent TG:glycogen metabolic switch in response to blockage of DNL fatty acid biosynthesisHistone acetyltransferases (HATs) Nej and Tip60, and acetyl-CoA synthase, are required for the TG:glycogen metabolic switch.

Tissues harbor memories of inflammation, which heighten sensitivity to diverse future assaults. Whether and how these adaptations are sustained through time and cell division remain poorly understood. We show that in mice, epidermal stem cells store lifelong, functional epigenetic records of psoriasis-like skin flares. Applying deep learning to investigate these chromatin dynamics, we unearth CpG dinucleotide density as a major driver of memory persistence. Although unnecessary for inflammation-induced transcription factors to open and establish memories, CpG-enriched sequences thereafter become essential, reinforcing accessibility across cellular generations by integrating DNA demethylation, methylation-sensitive transcription factors, sequence-intrinsic nucleosome disaffinity, and the nucleosome-destabilizing histone variant H2A.Z. Thus, once activated by inflammation-induced transcription factors, DNA sequences orchestrate persistent poise, imparting long-lasting memory to stress-sensitive genes and profoundly affecting tissue fitness upon recall.

Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by adenosine deaminases acting on RNA (ADARs), is a widespread modification in metazoans. Cumulative evidence has revealed the altered A-to-I editing profiles in cancers, but the underlying mechanism remains unclear. Here, we discover the well-known histone lysine methyltransferase enhancer of zeste homologue 2 (EZH2) as an unexplored ADAR interactor and editing regulator in prostate cancer (PCa). Through competing with interleukin enhancer binding factor 2 (ILF2) for ADAR1 binding, EZH2 reshapes the substrate selectivity of ADAR1 and thus exhibits a bidirectional role in editing regulation. Moreover, EZH2 depletion induces the translational repression of transportin-1 (TRN1), which further results in the accumulation of cytoplasmic ADAR1p110 isoform to protect many oncogenic transcripts from degradation. Consistently, depletion of ADAR1 dramatically enhances the sensitivity of cancer cells and tumors to EZH2 selective degraders. Collectively, our study sheds new light on a link between two layers of epigenetic regulations at histone modification and RNA editing levels, demonstrates a previously uncharacterized role of EZH2 in RNA editing and mRNA stability independently of its lysine methyltransferase activity, and reveals the significance of EZH2-ADAR1 cascade in governing RNA editing and mRNA stability, which may provide additional perspectives for the advancement of EZH2-targeting cancer therapies.

Abstract Brain resilience depends on collateral vessels whose geometries preserve blood flow when primary arteries are perturbed. How these protective vascular architectures are developmentally established remains unknown. Using longitudinal in vivo imaging in zebrafish, we show that mitochondrial state in embryonic angiogenic tip cells encodes the topology of basal and surface brain collateral networks. Mechanistically, microRNA-125a establishes the bioenergetic and redox balance of endothelial tip-cell mitochondria through conserved repression of the metabolic regulator PGC1a . Disruption of the microRNA-125a– PGC1a axis uncouples mitochondrial capacity from redox buffering in developing brain tip cells, redirecting their migration toward sparse, incompletely connected collateral network topologies and increasing adult cerebrovascular vulnerability. Consistent with this mechanism, humans with subclinical cerebrovascular injury exhibit reduced circulating microRNA-125a levels associated with incomplete basal collateral configurations. Together, these findings identify mitochondrial state as an instructive and conserved developmental program that encodes brain vascular reserve.

The mosquito Aedes aegypti is an important vector of viral pathogens and serves as a model for other vector species. Pathogens are transmitted when a mosquito bites a host animal, but the neural circuits that control seeking and biting behavior are not known. Here, we detail methods and protocols for the manipulation of neural activity in the mosquito using optogenetics, a key technique to determine the causal relationship between neural circuits and behavior. These methods include rearing mosquitoes for optogenetics and three assays that are designed to measure different steps in the sequence of arousal, attraction, proboscis probing, and engorgement on the blood of host animals. These behaviors occur at different spatial scales and in response to different sensory stimuli. Each behavioral assay is outfitted with red (625 nm) LEDs for optogenetic activation. To detect arousal in response to olfactory stimuli, flight and walking are measured in all three assays. To assay attraction or landing, mosquitoes are presented with a heated blood meal in a large arena. Proboscis probing and engorgement are assayed with video resolution that enables measurement of appendages and abdomen size. The protocol describes machine vision models to enable high-resolution temporal quantification of behavior as well as endpoint measurements of feeding. These methods can be used to test the function of any population of neurons in mosquito biting behavior and can be extended to additional behaviors.

While organellar genomes are typically a single circular molecule, some have undergone extreme evolutionary changes, such as fragmentation. Among plastid genomes, extensive fragmentation is rare but has been observed in the green alga Boodlea composita (Cladophorales), wherein single-stranded chromosomes adopt an unusual hairpin structure, stabilized by inverted repeats. This plastome is also highly reduced in coding content, containing only 21 protein-coding genes that are highly divergent in sequence, yet the plastome comprises a numerous and heterogeneous chromosome population. The lack of plastomes among close relatives has stymied our understanding of the evolutionary origins of Boodlea's unique plastome characteristics. Here, we report plastomes from three additional Cladophorales families. We show that elevated mutation rates and a massive reduction in coding content preceded fragmentation and that fragmentation resulted in a highly heterogeneous population with hundreds of minicircular chromosomes and a concomitant increase in GC content. Long inverted repeats are absent in minicircular chromosomes, suggesting the repetitive sequences that facilitate the formation of hairpin structures are unique to the Boodlea clade. Codon reassignments also occurred concurrently among fragmented and non-fragmented Cladophorales plastomes. Our findings document a clear stepwise progression of plastid topologies and sequence characteristics in Cladophorales. This assists in our understanding of the evolution of fragmented organellar genomes in general, as well as the limits to coding capacity reduction and sequence divergence able to be withstood by a functioning plastid.

Neural diversity is required for the brain to generate complex behaviors. During development, neural progenitors are exposed to different combinations of transient spatial cues for their identity specification. This identity is then interpreted by their progeny to activate terminal selector genes to become lineage-specific neurons. After spatial cues fade, it remains unclear how progenitors maintain their unique identity so that their progeny express the accurate, lineage-specific terminal selector genes. Using single-cell RNA sequencing in Drosophila, we identified a Forkhead domain transcription factor, Fd4, that is exclusively expressed in a single neural progenitor (neuroblast) and its new-born progeny. This neuroblast (NB), named NB7-1, forms at the intersection of the transient spatial cues Vnd (columnar expression) and En (row expression). We show that Fd4 expression overlaps spatial factor expression and terminal selector gene expression, thereby making Fd4 an excellent candidate for bridging transient spatial factors to lineage-specific terminal selector genes. We show that Fd4 is required for expression of terminal selector genes that maintain neuronal identity. Conversely, Fd4 misexpression generates ectopic NB7-1 progeny at the expense of Fd4-negative progenitor lineages. We conclude that Fd4 is continuously expressed in the NB7-1 and its new-born neuronal progeny where it activates terminal selector genes to produce lineage-specific neurons. We propose that Fd4 is a pioneering member of a class of 'lineage identity genes' that translate transient spatial cues into a long-term lineage identity.