Males miss and females forgo: Auditory masking from vessel noise impairs foraging efficiency and success in killer whales.

Understanding how the environment mediates an organism's ability to meet basic survival requirements is a fundamental goal of ecology. Vessel noise is a global threat to marine ecosystems and is increasing in intensity and spatiotemporal extent due to growth in shipping coupled with physical changes to ocean soundscapes from ocean warming and acidification. Odontocetes rely on biosonar to forage, yet determining the consequences of vessel noise on foraging has been limited by the challenges of observing underwater foraging outcomes and measuring noise levels received by individuals. To address these challenges, we leveraged a unique acoustic and movement dataset from 25 animal-borne biologging tags temporarily attached to individuals from two populations of fish-eating killer whales (Orcinus orca) in highly transited coastal waters to (1) test for the effects of vessel noise on foraging behaviors-searching (slow-click echolocation), pursuit (buzzes), and capture and (2) investigate the mechanism of interference. For every 1 dB increase in maximum noise level, there was a 4% increase in the odds of searching for prey by both sexes, a 58% decrease in the odds of pursuit by females and a 12.5% decrease in the odds of prey capture by both sexes. Moreover, all but one deep (≥75 m) foraging attempt with noise ≥110 dB re 1 µPa (15-45 kHz band; n = 6 dives by n = 4 whales) resulted in failed prey capture. These responses are consistent with an auditory masking mechanism. Our findings demonstrate the effects of vessel noise across multiple phases of odontocete foraging, underscoring the importance of managing anthropogenic inputs into soundscapes to achieve conservation objectives for acoustically sensitive species. While the timescales for recovering depleted prey species may span decades, these findings suggest that complementary actions to reduce ocean noise in the short term offer a critical pathway for recovering odontocete foraging opportunities.

Combination of AID2 and BromoTag expands the utility of degron-based protein knockdowns.

Acute protein knockdown is a powerful approach to dissecting protein function in dynamic cellular processes. We previously reported an improved auxin-inducible degron system, AID2, but recently noted that its ability to induce degradation of some essential replication factors, such as ORC1 and CDC6, was not enough to induce lethality. Here, we present combinational degron technologies to control two proteins or enhance target depletion. For this purpose, we initially compare PROTAC-based degrons, dTAG and BromoTag, with AID2 to reveal their key features and then demonstrate control of cohesin and condensin with AID2 and BromoTag, respectively. We develop a double-degron system with AID2 and BromoTag to enhance target depletion and accelerate depletion kinetics and demonstrate that both ORC1 and CDC6 are pivotal for MCM loading. Finally, we show that co-depletion of ORC1 and CDC6 by the double-degron system completely suppresses DNA replication, and the cells enter mitosis with single-chromatid chromosomes, indicating that DNA replication is uncoupled from cell cycle control. Our combinational degron technologies will expand the application scope for functional analyses.

Deciphering lineage specification during early embryogenesis in mouse gastruloids using multilayered proteomics.

Gastrulation is a critical stage in embryonic development during which the germ layers are established. Advances in sequencing technologies led to the identification of gene regulatory programs that control the emergence of the germ layers and their derivatives. However, proteome-based studies of early mammalian development are scarce. To overcome this, we utilized gastruloids and a multilayered mass spectrometry-based proteomics approach to investigate the global dynamics of (phospho) protein expression during gastruloid differentiation. Our findings revealed many proteins with temporal expression and unique expression profiles for each germ layer, which we also validated using single-cell proteomics technology. Additionally, we profiled enhancer interaction landscapes using P300 proximity labeling, which revealed numerous gastruloid-specific transcription factors and chromatin remodelers. Subsequent degron-based perturbations combined with single-cell RNA sequencing (scRNA-seq) identified a critical role for ZEB2 in mouse and human somitogenesis. Overall, this study provides a rich resource for developmental and synthetic biology communities endeavoring to understand mammalian embryogenesis.

Harmony of Protein Tags and Chimeric Molecules Empowers Targeted Protein Ubiquitination and Beyond.

Post-translational modifications (PTMs) are crucial mechanisms that underlie the intricacies of biological systems and disease mechanisms. This review focuses on the latest advancements in the design of heterobifunctional small molecules that hijack PTM machineries for target-specific modifications in living systems. A key innovation in this field is the development of proteolysis-targeting chimeras (PROTACs), which promote the ubiquitination of target proteins for proteasomal degradation. The past decade has seen several adaptations of the PROTAC concept to facilitate targeted (de)phosphorylation and acetylation. Protein fusion tags have been particularly vital in these proof-of-concept studies, aiding in the investigation of the functional roles of post-translationally modified proteins linked to diseases. This overview delves into protein-tagging strategies that enable the targeted modulation of ubiquitination, phosphorylation, and acetylation, emphasizing the synergies and challenges of integrating heterobifunctional molecules with protein tags in PTM research. Despite significant progress, many PTMs remain to be explored, and protein tag-assisted PTM-inducing chimeras will continue to play an important role in understanding the fundamental roles of protein PTMs and in exploring the therapeutic potential of manipulating protein modifications, particularly for targets not yet addressed by existing drugs.

A degron-based approach to manipulate Eomes functions in the context of the developing mouse embryo.

The T-box transcription factor Eomesodermin (Eomes), also known as Tbr2, plays essential roles in the early mouse embryo. Loss-of-function mutant embryos arrest at implantation due to Eomes requirements in the trophectoderm cell lineage. Slightly later, expression in the visceral endoderm promotes anterior visceral endoderm formation and anterior-posterior axis specification. Early induction in the epiblast beginning at day 6 is necessary for nascent mesoderm to undergo epithelial to mesenchymal transition (EMT). Eomes acts in a temporally and spatially restricted manner to sequentially specify the yolk sac haemogenic endothelium, cardiac mesoderm, definitive endoderm, and axial mesoderm progenitors during gastrulation. Little is known about the underlying molecular mechanisms governing Eomes actions during the formation of these distinct progenitor cell populations. Here, we introduced a degron-tag and mCherry reporter sequence into the Eomes locus. Our experiments analyzing homozygously tagged embryonic stem cells and embryos demonstrate that the degron-tagged Eomes protein is fully functional. dTAG (degradation fusion tag) treatment in vitro results in rapid protein degradation and recapitulates the Eomes-null phenotype. However in utero administration of dTAG resulted in variable and lineage-specific degradation, likely reflecting diverse cell type-specific Eomes expression dynamics. Finally, we demonstrate that Eomes protein rapidly recovers following dTAG wash-out in vitro. The ability to temporally manipulate Eomes protein expression in combination with cell marking by the mCherry-reporter offers a powerful tool for dissecting Eomes-dependent functional roles in these diverse cell types in the early embryo.

The Insulin Receptor and Insulin like Growth Factor Receptor 5' UTRs Support Translation Initiation Independently of EIF4G1.

IRES mediated translation initiation requires a different repertoire of factors than canonical cap-dependent translation. Treatments that inhibit the canonical translation factor EIF4G1 have little or no effect on the ability of the Insr and Igf1r cellular IRESes to promote translation. Transcripts for two cellular receptors contain RNA elements that facilitate translation initiation without intact EIF4G1. Cellular IRES mechanisms may resemble viral type III IRESes allowing them to promote translate with a limited number of initiation factors allowing them to work under stress conditions when canonical translation is repressed.

Identification of RNA-binding proteins' direct effects on gene expression via the degradation tag system.

RNA-binding proteins (RBPs) are critical regulators of gene expression. An RBP typically binds to multiple mRNAs and modulates their expression. Although loss-of-function experiments on an RBP can infer how it regulates a specific target mRNA, the results are confounded by potential secondary effects due to the attenuation of all other interactions of the target RBP. For example, regarding the interaction between Trim71, an evolutionarily conserved RBP, and Ago2 mRNA, although Trim71 binds to Ago2 mRNA and overexpression of Trim71 represses Ago2 mRNA translation, it is puzzling that AGO2 protein levels are not altered in the Trim71 knockdown/knockout cells. To address this, we adapted the dTAG (degradation tag) system for determining the direct effects of the endogenous Trim71. Specifically, we knocked in the dTAG to the Trim71 locus, enabling inducible rapid Trim71 protein degradation. We observed that following the induction of Trim71 degradation, Ago2 protein levels first increased, confirming the Trim71-mediated repression, and then returned to the original levels after 24 h post-induction, revealing that the secondary effects from the Trim71 knockdown/knockout counteracted its direct effects on Ago2 mRNA. These results highlight a caveat in interpreting the results from loss-of-function studies on RBPs and provide a method to determine the primary effect(s) of RBPs on their target mRNAs.

Divergent foraging strategies between populations of sympatric matrilineal killer whales.

In cooperative species, human-induced rapid environmental change may threaten cost-benefit tradeoffs of group behavioral strategies that evolved in past environments. Capacity for behavioral flexibility can increase population viability in novel environments. Whether the partitioning of individual responsibilities within social groups is fixed or flexible across populations is poorly understood, despite its relevance for predicting responses to global change at the population and species levels and designing successful conservation programs. We leveraged bio-logging data from two populations of fish-eating killer whales (Orcinus orca) to quantify patterns of fine-scale foraging movements and their relationships with demography. We reveal striking interpopulation differences in patterns of individual foraging behavior. Females from the endangered Southern Resident (SRKW) population captured less prey and spent less time pursuing prey than SRKW males or Northern Resident (NRKW) females, whereas NRKW females captured more prey than NRKW males. The presence of a calf (≤3 years) reduced the number of prey captured by adult females from both populations, but disproportionately so for SRKW. SRKW adult males with a living mother captured more prey than those whose mother had died, whereas the opposite was true for NRKW adult males. Across populations, males foraged in deeper areas than females, and SRKW captured prey deeper than NRKW. These population-level differences in patterns of individual foraging behavior challenge the existing paradigm that females are the disproportionate foragers in gregarious resident killer whales, and demonstrate considerable variation in the foraging strategies across populations of an apex marine predator experiencing different environmental stressors.

High-efficiency knock-in of degradable tags (dTAG) at endogenous loci in cell lines.

The dTAG system is a versatile strategy for tunable control of protein abundance and facilitates the time-resolved assessment of disease-associated protein function. A "co-opted" fusion-based degron peptide, the "dTAG" facilitates the study of endogenous protein function when knocked-in at the endogenous genetic loci of proteins of interest. We combine CRISPR/Cas9 mediated induction of double-strand breaks (DSB) with the delivery of a single-stranded DNA HDR-donor-template via crude preparations of recombinant adeno-associated virus (rAAV). Our approach to knock-in of large (1-2kb) DNA fragments via crude-rAAV mediated HDR donor delivery is rapid and inexpensive. It facilitates genetic modification of a variety of human as well as mouse cell lines at high efficiency and precision.

EBF1 is continuously required for stabilizing local chromatin accessibility in pro-B cells.

The establishment of de novo chromatin accessibility in lymphoid progenitors requires the "pioneering" function of transcription factor (TF) early B cell factor 1 (EBF1), which binds to naïve chromatin and induces accessibility by recruiting the BRG1 chromatin remodeler subunit. However, it remains unclear whether the function of EBF1 is continuously required for stabilizing local chromatin accessibility. To this end, we replaced EBF1 by EBF1-FKBPF36V in pro-B cells, allowing the rapid degradation by adding the degradation TAG13 (dTAG13) dimerizer. EBF1 degradation results in a loss of genome-wide EBF1 occupancy and EBF1-targeted BRG1 binding. Chromatin accessibility was rapidly diminished at EBF1-binding sites with a preference for sites whose occupancy requires the pioneering activity of the C-terminal domain of EBF1. Diminished chromatin accessibility correlated with altered gene expression. Thus, continuous activity of EBF1 is required for the stable maintenance of the transcriptional and epigenetic state of pro-B cells.

Control of ribosomal RNA synthesis by hematopoietic transcription factors.

Ribosomal RNAs (rRNAs) are the most abundant cellular RNAs, and their synthesis from rDNA repeats by RNA polymerase I accounts for the bulk of all transcription. Despite substantial variation in rRNA transcription rates across cell types, little is known about cell-type-specific factors that bind rDNA and regulate rRNA transcription to meet tissue-specific needs. Using hematopoiesis as a model system, we mapped about 2,200 ChIP-seq datasets for 250 transcription factors (TFs) and chromatin proteins to human and mouse rDNA and identified robust binding of multiple TF families to canonical TF motifs on rDNA. Using a 47S-FISH-Flow assay developed for nascent rRNA quantification, we demonstrated that targeted degradation of C/EBP alpha (CEBPA), a critical hematopoietic TF with conserved rDNA binding, caused rapid reduction in rRNA transcription due to reduced RNA Pol I occupancy. Our work identifies numerous potential rRNA regulators and provides a template for dissection of TF roles in rRNA transcription.

Target protein localization and its impact on PROTAC-mediated degradation.

Proteolysis-targeting chimeras (PROTACs) bring a protein of interest (POI) into spatial proximity of an E3 ubiquitin ligase, promoting POI ubiquitylation and proteasomal degradation. PROTACs rely on endogenous cellular machinery to mediate POI degradation, therefore the subcellular location of the POI and access to the E3 ligase being recruited potentially impacts PROTAC efficacy. To interrogate whether the subcellular context of the POI influences PROTAC-mediated degradation, we expressed either Halo or FKBP12F36V (dTAG) constructs consisting of varying localization signals and tested the efficacy of their degradation by von Hippel-Lindau (VHL)- or cereblon (CRBN)-recruiting PROTACs targeting either Halo or dTAG. POIs were localized to the nucleus, cytoplasm, outer mitochondrial membrane, endoplasmic reticulum, Golgi, peroxisome or lysosome. Differentially localized Halo or FKBP12F36V proteins displayed varying levels of degradation using the same respective PROTACs, suggesting therefore that the subcellular context of the POI can influence the efficacy of PROTAC-mediated POI degradation.

RNA Pol II pausing facilitates phased pluripotency transitions by buffering transcription.

Promoter-proximal RNA Pol II pausing is a critical step in transcriptional control. Pol II pausing has been predominantly studied in tissue culture systems. While Pol II pausing has been shown to be required for mammalian development, the phenotypic and mechanistic details of this requirement are unknown. Here, we found that loss of Pol II pausing stalls pluripotent state transitions within the epiblast of the early mouse embryo. Using Nelfb -/- mice and a NELFB degron mouse pluripotent stem cell model, we show that embryonic stem cells (ESCs) representing the naïve state of pluripotency successfully initiate a transition program but fail to balance levels of induced and repressed genes and enhancers in the absence of NELF. We found an increase in chromatin-associated NELF during transition from the naïve to later pluripotent states. Overall, our work defines the acute and long-term molecular consequences of NELF loss and reveals a role for Pol II pausing in the pluripotency continuum as a modulator of cell state transitions.

Temporal resolution of gene derepression and proteome changes upon PROTAC-mediated degradation of BCL11A protein in erythroid cells.

Reactivation of fetal hemoglobin expression by the downregulation of BCL11A is a promising treatment for ß-hemoglobinopathies. A detailed understanding of BCL11A-mediated repression of γ-globin gene (HBG1/2) transcription is lacking, as studies to date used perturbations by shRNA or CRISPR-Cas9 gene editing. We leveraged the dTAG PROTAC degradation platform to acutely deplete BCL11A protein in erythroid cells and examined consequences by nascent transcriptomics, proteomics, chromatin accessibility, and histone profiling. Among 31 genes repressed by BCL11A, HBG1/2 and HBZ show the most abundant and progressive changes in transcription and chromatin accessibility upon BCL11A loss. Transcriptional changes at HBG1/2 were detected in <2 h. Robust HBG1/2 reactivation upon acute BCL11A depletion occurred without the loss of promoter 5-methylcytosine (5mC). Using targeted protein degradation, we establish a hierarchy of gene reactivation at BCL11A targets, in which nascent transcription is followed by increased chromatin accessibility, and both are uncoupled from promoter DNA methylation at the HBG1/2 loci.

Rapid and efficient degradation of endogenous proteins in vivo identifies stage-specific roles of RNA Pol II pausing in mammalian development.

Targeted protein degradation methods offer a unique avenue to assess a protein's function in a variety of model systems. Recently, these approaches have been applied to mammalian cell culture models, enabling unprecedented temporal control of protein function. However, the efficacy of these systems at the tissue and organismal levels in vivo is not well established. Here, we tested the functionality of the degradation tag (dTAG) degron system in mammalian development. We generated a homozygous knock-in mouse with a FKBP12F36V tag fused to negative elongation factor b (Nelfb) locus, a ubiquitously expressed regulator of transcription. In our validation of targeted endogenous protein degradation across mammalian development and adulthood, we demonstrate that irrespective of the route of administration the dTAG system is non-toxic, rapid, and efficient in embryos from the zygote-to-mid-gestation stages. Additionally, acute depletion of NELFB revealed a specific role in zygote-to-2-cell development and zygotic genome activation (ZGA).

Two distinct mechanisms of RNA polymerase II elongation stimulation in vivo.

Transcription by RNA polymerase II (RNA Pol II) relies on the elongation factors PAF1 complex (PAF), RTF1, and SPT6. Here, we use rapid factor depletion and multi-omics analysis to investigate how these elongation factors influence RNA Pol II elongation activity in human cells. Whereas depletion of PAF subunits PAF1 and CTR9 has little effect on cellular RNA synthesis, depletion of RTF1 or SPT6 strongly compromises RNA Pol II activity, albeit in fundamentally different ways. RTF1 depletion decreases RNA Pol II velocity, whereas SPT6 depletion impairs RNA Pol II progression through nucleosomes. These results show that distinct elongation factors stimulate either RNA Pol II velocity or RNA Pol II progression through chromatin in vivo. Further analysis provides evidence for two distinct barriers to early elongation: the promoter-proximal pause site and the +1 nucleosome. It emerges that the first barrier enables loading of elongation factors that are required to overcome the second and subsequent barriers to transcription.

Design and site-directed immobilization of single-chain Fv antibody to polystyrene latex beads via material-binding peptides and application to latex turbidimetric assay.

In this study, immobilization of single-chain Fv (scFv) antibodies on the surfaces of polystyrene (PS) latex beads via material-binding peptides was investigated for sensitive immuno-turbidimetric assay of C-reactive protein (CRP). Anti-CRP scFvs fused with polystyrene-binding peptide (PS-tag) and poly(methylmethacrylate)-binding peptide (PMMA-tag) were over-expressed in Escherichia coli cells and recovered in the active form following refolding. The beads with PMMA-tag-fused scFv (scFv-PM) were successfully suspended with sufficient dispersion at pH 8.0. Three types of alternative scFv-PMs with a penta-asparatic acid tag (D5-tag) introduced at different positions were then designed. All of the D5-tagged scFv-PMs were successfully immobilized on the surfaces of beads with no significant change in the diameter of the latex beads at pH levels ranging from 6.0 to 8.0. According to the results of turbidimetric assay for the detection of CRP, 13 ng/ml of CRP was detectable using beads with D5-tagged scFv-PMs at 400 ng/cm3, and no turbidity change was observed in the absence of antigen. When the density of scFv-PM was 250 ng/cm2, which was 63% of the maximum density, the beads were dispersed well and reactive with the antigen at a concentration range comparable to those with D5-tagged scFv-PMs. These results indicate that controlling charge density on the surface of beads after site-directed immobilization is definitely important in order to maintain high levels of dispersion and reactivity. Thus, the usefulness of the scFv-PM as well as D5-tagged scFv-PMs developed in the present study should be significant when used as ligand antibodies in the preparation of immuno-latex beads.

Targeted Protein Degradation Tools: Overview and Future Perspectives.

Targeted protein inactivation (TPI) is an elegant approach to investigate protein function and its role in the cellular landscape, overcoming limitations of genetic perturbation strategies. These systems act in a reversible manner and reduce off-target effects exceeding the limitations of CRISPR/Cas9 and RNA interference, respectively. Several TPI have been developed and wisely improved, including compartment delocalization tools and protein degradation systems. However, unlike chemical tools such as PROTACs (PROteolysis TArgeting Chimeras), which work in a wild-type genomic background, TPI technologies require adding an aminoacidic signal sequence (tag) to the protein of interest (POI). On the other hand, the design and optimization of PROTACs are very laborious and time-consuming. In this review, we focus on anchor-away, deGradFP, auxin-inducible degron (AID) and dTAG technologies and discuss their recent applications and advances. Finally, we propose nano-grad, a novel nanobody-based protein degradation tool, which specifically proteolyzes endogenous tag-free target protein.

The long-range echo scene of the sperm whale biosonar.

Sperm whales use their gigantic nose to produce the most powerful sounds in the animal kingdom, presumably to echolocate deep-sea prey at long ranges and possibly to debilitate prey. To test these hypotheses, we deployed sound recording tags (DTAG-4) on the tip of the nose of three sperm whales. One of these recordings yielded over 6000 echo streams from organisms detected up to 144 m ahead of the whale, supporting a long-range prey detection function of the sperm whale biosonar. The whale navigated this complex acoustic scene by maintaining a stable, long-range acoustic gaze suggesting continual resource evaluation. Less than 10% of the echoic organisms recorded by the tag were targeted for capture and only 18% of the buzzes were emitted within the 50 m depth interval of maximum organism encounter rate, demonstrating echo-guided prey selection. Buzzes were initiated more than 20 m from the prey, showing that sperm whales do not debilitate their prey with sound, but trade echo levels for reduced forward masking and rapid updates on prey location in keeping with the lower manoeuvrability of these large predators. We conclude that the powerful biosonar of sperm whales enables long-range echolocation and selection of prey, but not acoustic debilitation.

When the noise goes on: received sound energy predicts sperm whale responses to both intermittent and continuous navy sonar.

Anthropogenic noise sources range from intermittent to continuous, with seismic and navy sonar technology moving towards near-continuous transmissions. Continuous active sonar (CAS) may be used at a lower amplitude than traditional pulsed active sonar (PAS), but potentially with greater cumulative sound energy. We conducted at-sea experiments to contrast the effects of navy PAS versus CAS on sperm whale behaviour using animal-attached sound- and movement-recording tags (n=16 individuals) in Norway. Changes in foraging effort and proxies for foraging success and cost during sonar and control exposures were assessed while accounting for baseline variation [individual effects, time of day, bathymetry and blackfish (pilot/killer whale) presence] in generalized additive mixed models (GAMMs). We found no reduction in time spent foraging during exposures to medium-level PAS (MPAS) transmitted at the same peak amplitude as CAS. In contrast, we found similar reductions in foraging during CAS (d.f.=1, F=8.0, P=0.005) and higher amplitude PAS (d.f.=1, F=20.8, P<0.001) when received at similar energy levels integrated over signal duration. These results provide clear support for sound energy over amplitude as the response driver. We discuss the importance of exposure context and the need to measure cumulative sound energy to account for intermittent versus more continuous sources in noise impact assessments.