Interplay of condensation and chromatin binding underlies BRD4 targeting.

Nuclear compartments form via biomolecular phase separation, mediated through multivalent properties of biomolecules concentrated within condensates. Certain compartments are associated with specific chromatin regions, including transcriptional initiation condensates, which are composed of transcription factors and transcriptional machinery, and form at acetylated regions including enhancer and promoter loci. While protein self-interactions, especially within low-complexity and intrinsically disordered regions, are known to mediate condensation, the role of substrate-binding interactions in regulating the formation and function of biomolecular condensates is underexplored. Here, utilizing live-cell experiments in parallel with coarse-grained simulations, we investigate how chromatin interaction of the transcriptional activator BRD4 modulates its condensate formation. We find that both kinetic and thermodynamic properties of BRD4 condensation are affected by chromatin binding: nucleation rate is sensitive to BRD4-chromatin interactions, providing an explanation for the selective formation of BRD4 condensates at acetylated chromatin regions, and thermodynamically, multivalent acetylated chromatin sites provide a platform for BRD4 clustering below the concentration required for off-chromatin condensation. This provides a molecular and physical explanation of the relationship between nuclear condensates and epigenetically modified chromatin that results in their mutual spatiotemporal regulation, suggesting that epigenetic modulation is an important mechanism by which the cell targets transcriptional condensates to specific chromatin loci.

An FGF timer for zygotic genome activation.

Zygotic genome activation has been extensively studied in a variety of systems including flies, frogs, and mammals. However, there is comparatively little known about the precise timing of gene induction during the earliest phases of embryogenesis. Here we used high-resolution in situ detection methods, along with genetic and experimental manipulations, to study the timing of zygotic activation in the simple model chordate Ciona with minute-scale temporal precision. We found that two Prdm1 homologs in Ciona are the earliest genes that respond to FGF signaling. We present evidence for a FGF timing mechanism that is driven by ERK-mediated derepression of the ERF repressor. Depletion of ERF results in ectopic activation of FGF target genes throughout the embryo. A highlight of this timer is the sharp transition in FGF responsiveness between the eight- and 16-cell stages of development. We propose that this timer is an innovation of chordates that is also used by vertebrates.

WNK kinases sense molecular crowding and rescue cell volume via phase separation.

When challenged by hypertonicity, dehydrated cells must recover their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless condensates, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to restore cell volume. WNK1 condensate formation is driven by its intrinsically disordered C terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated in vivo by molecular crowding rather than changes in cell size. This allows kinase activity despite an inhibitory ionic milieu and permits cell volume recovery through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.

ERK signaling dissolves ERF repression condensates in living embryos.

Phase separation underlies the organization of the nucleus, including the biogenesis of nucleoli and the packaging of heterochromatin. Here we explore the regulation of transcription factor condensates involved in gene repression by ERK signaling in gastrulating embryos of a simple proto-vertebrate (Ciona). ERK signaling induces nuclear export of the transcriptional repressor Ets-2 repressive factor (ERF), which has been linked to various human developmental disorders. Using high-resolution imaging, we show that ERF is localized within discrete nuclear condensates that dissolve upon ERK activation. Interestingly, we observe dynamic pulses of assembly and dissociation during interphase, providing visualization of a nuclear phase separation process regulated by cell signaling. We discuss the implications of these observations for producing sharp on/off switches in gene activity and suppressing noise in cell-cell signaling events.

Hepatic demethylation of methoxy-bromodiphenyl ethers and conjugation of the resulting hydroxy-bromodiphenyl ethers in a marine fish, the red snapper, Lutjanus campechanus, and a freshwater fish, the channel catfish, Ictalurus punctatus.

Methoxylated bromodiphenyl ethers (MeO-BDEs), marine natural products, can be demethylated by cytochrome P450 to produce hydroxylated bromodiphenyl ethers (OH-BDEs), potentially toxic metabolites that are also formed by hydroxylation of BDE flame retardants. The OH-BDEs may be detoxified by glucuronidation and sulfonation. This study examined the demethylation of 6-MeO-BDE47, 2'-MeO-BDE68 and 4'-MeO-BDE68, in hepatic microsomes from the red snapper, Lutjanus campechanus, a marine fish likely to be exposed naturally to MeO-BDEs, and the channel catfish, Ictalurus punctatus, a freshwater fish in which pathways of xenobiotic biotransformation have been studied. We further studied the glucuronidation and sulfonation of the resulting OH-BDEs as well as of 6-OH-2'-MeO-BDE68 in hepatic microsomes and cytosol fractions of these fish. The three studied biotransformation pathways were active in both species, with high individual variability. The range of activities overlapped in the two species. Demethylation of MeO-BDEs, studied in the concentration range 10-500 µM, followed Michaelis-Menten kinetics in both fish species, however enzyme efficiencies were low, ranging from 0.024 to 0.334 µL min.mg protein. Conjugation of the studied OH-BDEs followed Michaelis-Menten kinetics in the concentration ranges 1-50 µM (glucuronidation) or 2.5-100 µM (sulfonation). These OH-BDEs were readily glucuronidated and sulfonated in the fish livers of both species, with enzyme efficiencies one to three orders of magnitude higher than for demethylation of the precursor MeO-BDEs. The relatively low efficiencies of demethylation of the MeO-BDEs, compared with higher efficiencies for OH-BDE conjugation, suggests that MeO-BDEs are more likely than OH-BDEs to bioaccumulate in tissues of exposed fish.

Age-Related Changes in miRNA Expression Influence GSTZ1 and Other Drug Metabolizing Enzymes.

Previous work has shown that hepatic levels of human glutathione transferase zeta 1 (GSTZ1) protein, involved in tyrosine catabolism and responsible for metabolism of the investigational drug dichloroacetate, increase in cytosol after birth before reaching a plateau around age 7. However, the mechanism regulating this change of expression is still unknown, and previous studies showed that GSTZ1 mRNA levels did not correlate with GSTZ1 protein expression. In this study, we addressed the hypothesis that microRNAs (miRNAs) could regulate expression of GSTZ1. We obtained liver samples from donors aged less than 1 year or older than 13 years and isolated total RNA for use in a microarray to identify miRNAs that were downregulated in the livers of adults compared with children. From a total of 2578 human miRNAs tested, 63 miRNAs were more than 2-fold down-regulated in adults, of which miR-376c-3p was predicted to bind to the 3' untranslated region of GSTZ1 mRNA. There was an inverse correlation of miR-376c-3p and GSTZ1 protein expression in the liver samples. Using cell culture, we confirmed that miR-376c-3p could downregulate GSTZ1 protein expression. Our findings suggest that miR-376c-3p prevents production of GSTZ1 through inhibition of translation. These experiments further our understanding of GSTZ1 regulation. Furthermore, our array results provide a database resource for future studies on mechanisms regulating human hepatic developmental expression. SIGNIFICANCE STATEMENT: Hepatic glutathione transferase zeta 1 (GSTZ1) is responsible for metabolism of the tyrosine catabolite maleylacetoacetate as well as the investigational drug dichloroacetate. Through examination of microRNA (miRNA) expression in liver from infants and adults and studies in cells, we showed that expression of GSTZ1 is controlled by miRNA. This finding has application to the dosing regimen of the drug dichloroacetate. The miRNA expression profiles are provided and will prove useful for future studies of drug-metabolizing enzymes in infants and adults.

Effects of extreme potassium stress on blood pressure and renal tubular sodium transport.

We characterized mouse blood pressure and ion transport in the setting of commonly used rodent diets that drive K+ intake to the extremes of deficiency and excess. Male 129S2/Sv mice were fed either K+-deficient, control, high-K+ basic, or high-KCl diets for 10 days. Mice maintained on a K+-deficient diet exhibited no change in blood pressure, whereas K+-loaded mice developed an ~10-mmHg blood pressure increase. Following challenge with NaCl, K+-deficient mice developed a salt-sensitive 8 mmHg increase in blood pressure, whereas blood pressure was unchanged in mice fed high-K+ diets. Notably, 10 days of K+ depletion induced diabetes insipidus and upregulation of phosphorylated NaCl cotransporter, proximal Na+ transporters, and pendrin, likely contributing to the K+-deficient NaCl sensitivity. While the anionic content with high-K+ diets had distinct effects on transporter expression along the nephron, both K+ basic and KCl diets had a similar increase in blood pressure. The blood pressure elevation on high-K+ diets correlated with increased Na+-K+-2Cl- cotransporter and γ-epithelial Na+ channel expression and increased urinary response to furosemide and amiloride. We conclude that the dietary K+ maneuvers used here did not recapitulate the inverse effects of K+ on blood pressure observed in human epidemiological studies. This may be due to the extreme degree of K+ stress, the low-Na+-to-K+ ratio, the duration of treatment, and the development of other coinciding events, such as diabetes insipidus. These factors must be taken into consideration when studying the physiological effects of dietary K+ loading and depletion.