SF1: ETV4 expression levels in endometrial cancer. SF2: Expression levels of ETV4 in candidate knockout clones. SF3: Short-term knockdown of ETV4 reduces ER genomic binding. SF4: Validation of rescue and overexpression of ETV4. SF5: Estrogen induced fold changes are higher in wildtype cells than ETV4 knockout cells and genes that lose estrogen induction are closer to ETV4 bound sites. SF6: Global chromatin accessibility is similar between wildtype and ETV4 knockout lines. SF7: Introduction of ETV4 in T-47D breast cancer cell line. SF8: GR localization does not change due to ETV4 loss. SF9: Growth phenotype observed in 2D growth of ETV4 knockout lines. SF10: ETV4 knockouts increase expression of cytokeratins, a marker of normal endometrium.

OBJECTIVES:To describe the risk factors for SARS-CoV-2 infection in UK healthcare workers (HCWs). METHODS:We conducted a prospective sero-epidemiological study of HCWs at a major UK teaching hospital using a SARS-CoV-2 immunoassay. Risk factors for seropositivity were analysed using multivariate logistic regression. RESULTS:410/5,698 (7·2%) staff tested positive for SARS-CoV-2 antibodies. Seroprevalence was higher in those working in designated COVID-19 areas compared with other areas (9·47% versus 6·16%) Healthcare assistants (aOR 2·06 [95%CI 1·14-3·71]; p=0·016) and domestic and portering staff (aOR 3·45 [95% CI 1·07-11·42]; p=0·039) had significantly higher seroprevalence than other staff groups after adjusting for age, sex, ethnicity and COVID-19 working location. Staff working in acute medicine and medical sub-specialities were also at higher risk (aOR 2·07 [95% CI 1·31-3·25]; p<0·002). Staff from Black, Asian and minority ethnic (BAME) backgrounds had an aOR of 1·65 (95% CI 1·32 - 2·07; p<0·001) compared to white staff; this increased risk was independent of COVID-19 area working. The only symptoms significantly associated with seropositivity in a multivariable model were loss of sense of taste or smell, fever, and myalgia; 31% of staff testing positive reported no prior symptoms. CONCLUSIONS:Risk of SARS-CoV-2 infection amongst HCWs is highly heterogeneous and influenced by COVID-19 working location, role, age and ethnicity. Increased risk amongst BAME staff cannot be accounted for solely by occupational factors.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) transmission is uncontrolled in many parts of the world, compounded in some areas by higher transmission potential of the B1.1.7 variant now seen in 50 countries. It is unclear whether responses to SARS-CoV-2 vaccines based on the prototypic strain will be impacted by mutations found in B.1.1.7. Here we assessed immune responses following vaccination with mRNA-based vaccine BNT162b2. We measured neutralising antibody responses following a single immunization using pseudoviruses expressing the wild-type Spike protein or the 8 amino acid mutations found in the B.1.1.7 spike protein. The vaccine sera exhibited a broad range of neutralising titres against the wild-type pseudoviruses that were modestly reduced against B.1.1.7 variant. This reduction was also evident in sera from some convalescent patients. Decreased B.1.1.7 neutralisation was also observed with monoclonal antibodies targeting the N-terminal domain (9 out of 10), the Receptor Binding Motif (RBM) (5 out of 31), but not in neutralising mAbs binding outside the RBM. Introduction of the E484K mutation in a B.1.1.7 background to reflect newly emerging viruses in the UK led to a more substantial loss of neutralising activity by vaccine-elicited antibodies and mAbs (19 out of 31) over that conferred by the B.1.1.7 mutations alone. E484K emergence on a B.1.1.7 background represents a threat to the vaccine BNT162b.

Vaccines remain the cornerstone for containing the SARS-CoV-2 pandemic. mRNA vaccines provide protection in clinical trials using a two-dose approach, separated by a three to four week gap. UK policy in 2021 is to extend the dosing interval from three to twelve weeks and other countries are likely to follow suit given the demand for mRNA vaccines and ongoing uncontrolled transmission. There is a paucity of data in the elderly, even though these individuals are the first to receive vaccines due to risk of severe disease. Here we assessed real world immune responses following vaccination with mRNA-based vaccine BNT162b2. Median age was 81 years amongst 101 participants after the first dose of the BNT162b2 vaccine. Geometric mean neutralisation titres in participants over 80 years old after the first dose were lower than in younger individuals [83.4 (95% CI 52.0-133.7) vs 46.6 (95% CI 33.5-64.8) p 0.01]. A lower proportion of participants 80 years and older achieved adequate neutralisation titre of >1:20 for 50% neutralisation as compared to those under 80 (21% vs 51%, p 0.003). Binding IgG responses correlated with neutralisation. Sera from participants in both age groups showed significantly lower neutralisation potency against B.1.1.7 Spike pseudotyped viruses as compared to wild type. The adjusted ORs for inadequate neutralisation in the 80 years and above age group were 3.7 (95% CI 1.2-11.2) and 4.4 (95% CI 1.5-12.6) against wild type and B.1.1.7 pseudotyped viruses. We observed a trend towards lower somatic hypermutation in participants with suboptimal neutralisation, and elderly participants demonstrated clear reduction in class switched somatic hypermutation, driven by the IgA1/2 isotype. SARS-CoV-2 Spike specific T- cell IFN𝛾 and IL-2 responses were impaired in the older age group after 1 dose and although IFN𝛾 increased between vaccine doses, IL-2 responses did not significantly increase. There was a significantly higher risk of suboptimal neutralising antibody and T cell response following first dose vaccination with BNT162b2 in half of participants above the age of 80, persisting up to 12 weeks. These high risk populations warrant specific measures in order to mitigate against vaccine failure, particularly where SARS-CoV-2 variants of concern are circulating.

Two dose mRNA vaccination provides excellent protection against SARS-CoV-2. However, there are few data on vaccine efficacy in elderly individuals above the age of 801. Additionally, new variants of concern (VOC) with reduced sensitivity to neutralising antibodies have raised fears for vulnerable groups. Here we assessed humoral and cellular immune responses following vaccination with mRNA vaccine BNT162b22 in elderly participants prospectively recruited from the community and younger health care workers. Median age was 72 years and 51% were females amongst 140 participants. Neutralising antibody responses after the first vaccine dose diminished with increasing age, with a marked drop in participants over 80 years old. Sera from participants below and above 80 showed significantly lower neutralisation potency against B.1.1.7, B.1.351 and P.1. variants of concern as compared to wild type. Those over 80 were more likely to lack any neutralisation against VOC compared to younger participants following first dose. The adjusted odds ratio for inadequate neutralisation activity against the B.1.1.7, P.1 and B.1.351 variant in the older versus younger age group was 4.3 (95% CI 2.0-9.3, p<0.001), 6.7 (95% CI 1.7-26.3, p=0.008) and 1.7 (95% CI 0.5-5.7, p=0.41). Binding IgG and IgA antibodies were lower in the elderly, as was the frequency of SARS-CoV-2 Spike specific B-memory cells. We observed a trend towards lower somatic hypermutation in participants with suboptimal neutralisation, and elderly participants demonstrated clear reduction in class switched somatic hypermutation, driven by the IgA1/2 isotype. SARS-CoV-2 Spike specific T-cell IFNγ and IL-2 responses fell with increasing age, and both cytokines were secreted primarily by CD4 T cells. We conclude that the elderly are a high risk population that warrant specific measures in order to mitigate against vaccine failure, particularly where variants of concern are circulating.

SARS-CoV-2 transmission is uncontrolled in many parts of the world, compounded in some areas by higher transmission potential of the B1.1.7 variant now seen in 50 countries. In addition, many countries are extending the interval between doses of vaccine is being extended in order to expand vaccine coverage. It is unclear whether real world neutralising antibody titres will follow those in clinical trials, particularly in elderly persons, and how this will be impacted by mutations in strains such as B1.1.7. Here we tested immune responses in patients 3 weeks following the first dose of the Pfizer BioNTech vaccine BNT162b2. We also tested neutralising antibody responses against pseudoviruses expressing wild type Spike proteins or expressing 3 key mutations present in B.1.1.7 (deletion 69/70, N501Y, A570D). IgG Spike antibody titres correlated well with neutralisation. We observed a range of neutralisation titres against wild type, with geometric mean titre (GMT) of 24. The vaccine sera exhibited a range of inhibitory dilutions giving 50% neutralisation (ID50) from <1:4 to 3449. However a lower proportion of participants over 80 years old achieved threshold neutralisation titre of >1:4 for 50% neutralisation as compared to those under 80 years old (8/15 versus 8/8 P=0.052) after the first dose. Neutralisation titres were not significantly impacted by the combination of three Spike mutations tested, but were reduced against the full set of Spike mutations present in the B.1.1.7 variant. The highest fold change was approxiamately 6 and the median fold change for the B.1.1.7 variant versus wild type was 3.85 (IQR 2.68-5.28). Further work is needed to establish the impact of these observations on real life vaccine efficacy.

SARS-CoV-2 transmission is uncontrolled in many parts of the world, compounded in some areas by higher transmission potential of the B1.1.7 variant now seen in 50 countries. It is unclear whether responses to SARS-CoV-2 vaccines based on the prototypic strain will be impacted by mutations found in B.1.1.7. Here we assessed immune responses following vaccination with mRNA-based vaccine BNT162b2. We measured neutralising antibody responses following a single immunization using pseudoviruses expressing the wild-type Spike protein or the 8 mutations found in the B.1.1.7 Spike protein. The vaccine sera exhibited a broad range of neutralizing titres against the wild-type pseudoviruses (<1:4 to 3449) that were reduced against B.1.1.7 variant by 3.85 fold (IQR 2.68-5.28). This reduction was also evident in sera from some convalescent patients. Decreased B.1.1.7 neutralization was also observed with monoclonal antibodies targeting the N-terminal domain (9 out of 10), the Receptor Binding Motif (RBM) (5 outof 29), but not in neutralizing mAbs binding outside the RBM. Introduction of the E484K mutation in a B.1.1.7 background led to a further loss of neutralizing activity by vaccine-elicited antibodies over that conferred by the B.1.1.7 mutations alone. Further work is needed to establish the impact of these observations on protective vaccine efficacy in the context of the evolving B.1.1.7 lineage that will likely acquire E484K.

AbstractEstrogen signaling through estrogen receptor alpha (ER) plays a major role in endometrial cancer risk and progression, however, the molecular mechanisms underlying ER's regulatory role in endometrial cancer are poorly understood. In breast cancer cells, ER genomic binding is enabled by FOXA1 and GATA3, but the transcription factors that control ER genomic binding in endometrial cancer cells remain unknown. We previously identified ETV4 as a candidate factor controlling ER genomic binding in endometrial cancer cells, and here we explore the functional importance of ETV4. Homozygous deletion of ETV4, using CRISPR/Cas9, led to greatly reduced ER binding at the majority of loci normally bound by ER. Consistent with the dramatic loss of ER binding, the gene expression response to estradiol was dampened for most genes. ETV4 contributes to estrogen signaling in two distinct ways. ETV4 loss affects chromatin accessibility at some ER bound loci and impairs ER nuclear translocation. The diminished estrogen signaling upon ETV4 deletion led to decreased growth, particularly in 3D culture, where hollow organoids were formed and in vivo in the context of estrogen-dependent growth. These results show that ETV4 plays an important role in estrogen signaling in endometrial cancer cells.Significance:Estrogen receptor alpha (ER) is a key oncogene in endometrial cancer. This study uncovers ETV4 as an important factor in controlling the activity of ER and the growth of endometrial cancer cells.

The eukaryotic transcription factor ETS1 is regulated by an intrinsically disordered serine-rich region (SRR) that transiently associates with the adjacent ETS domain to inhibit DNA binding. In this study, we further elucidated the physicochemical basis for ETS1 autoinhibition by characterizing the interaction of its ETS domain with a series of synthetic peptides corresponding to the SRR. Binding is driven by the hydrophobic effect and enhanced electrostatically by phosphorylation of serines adjacent to aromatic residues in the amphipathic SRR. Structural characterization of the dynamic peptide/protein complex by NMR spectroscopy and X-ray crystallography revealed multiple modes of binding that lead to autoinhibition by synergistically blocking the DNA-binding interface of the ETS domain and stabilizing an appended helical inhibitory module against allosterically induced unfolding. Consistent with these conclusions, the SRR peptide does not interact with DNA-bound ETS1. In addition, we found that the ETS1 SRR phosphopeptide binds to distantly related PU.1 in vitro, indicating that autoinhibition exploits features of the ETS domain that are conserved across this family of transcription factors.

ETS transcription factors from the ERG and ETV1/4/5 subfamilies are overexpressed in the majority of prostate cancer patients and contribute to disease progression. Here, we have developed two in vitro assays for the interaction of ETS transcription factors with DNA that are amenable to high-throughput screening. Using ETS1 as a model, we applied these assays to screen 110 compounds derived from a high-throughput virtual screen. We found that the use of lower-affinity DNA binding sequences, similar to those that ERG and ETV1 bind to in prostate cells, allowed for higher inhibition from many of these test compounds. Further pilot experiments demonstrated that the in vitro assays are robust for ERG, ETV1, and ETV5, three of the ETS transcription factors that are overexpressed in prostate cancer.

Many different transcription factors (TFs) regulate gene expression in a combinatorial fashion, often by binding in close proximity to each other on composite cis-regulatory DNA elements. Here, we investigated how ETS TFs bind with the AP1 TFs JUN-FOS at composite DNA-binding sites. DNA-binding ability with JUN-FOS correlated with the phenotype of ETS proteins in prostate cancer. We found that the oncogenic ETS-related gene (ERG) and ETS variant (ETV) 1/4/5 subfamilies co-occupy ETS-AP1 sites with JUN-FOS in vitro, whereas JUN-FOS robustly inhibited DNA binding by the tumor suppressors ETS homologous factor (EHF) and SAM pointed domain-containing ETS TF (SPDEF). EHF bound ETS-AP1 DNA with tighter affinity than ERG in the absence of JUN-FOS, possibly enabling EHF to compete with ERG and JUN-FOS for binding to ETS-AP1 sites. Genome-wide mapping of EHF- and ERG-binding sites in prostate epithelial cells revealed that EHF is preferentially excluded from closely spaced ETS-AP1 DNA sequences. Structural modeling and mutational analyses indicated that adjacent positively charged surfaces from EHF and JUN-FOS use electrostatic repulsion to disfavor simultaneous DNA binding. Conservation of positive residues on the JUN-FOS interface identified E74-like ETS TF 1 (ELF1) as an additional ETS TF exhibiting anticooperative DNA binding with JUN-FOS, and we found that ELF1 is frequently down-regulated in prostate cancer. In summary, divergent electrostatic features of ETSTFs at their JUN-FOS interface enable distinct binding events at ETS-AP1 DNA sites, which may drive specific targeting of ETS TFs to facilitate distinct transcriptional programs.

Autoinhibition enables spatial and temporal regulation of cellular processes by coupling protein activity to surrounding conditions, often via protein partnerships or signaling pathways. We report the molecular basis of DNA-binding autoinhibition of ETS transcription factors ETV1, ETV4 and ETV5, which are often overexpressed in prostate cancer. Inhibitory elements that cooperate to repress DNA binding were identified in regions N- and C-terminal of the ETS domain. Crystal structures of these three factors revealed an α-helix in the C-terminal inhibitory domain that packs against the ETS domain and perturbs the conformation of its DNA-recognition helix. Nuclear magnetic resonance spectroscopy demonstrated that the N-terminal inhibitory domain (NID) is intrinsically disordered, yet utilizes transient intramolecular interactions with the DNA-recognition helix of the ETS domain to mediate autoinhibition. Acetylation of selected lysines within the NID activates DNA binding. This investigation revealed a distinctive mechanism for DNA-binding autoinhibition in the ETV1/4/5 subfamily involving a network of intramolecular interactions not present in other ETS factors. These distinguishing inhibitory elements provide a platform through which cellular triggers, such as protein-protein interactions or post-translational modifications, may specifically regulate the function of these oncogenic proteins.

The recruitment of transcriptional cofactors by sequence-specific transcription factors challenges the basis of high affinity and selective interactions. Extending previous studies that the N-terminal activation domain (AD) of ETV5 interacts with Mediator subunit 25 (MED25), we establish that similar, aromatic-rich motifs located both in the AD and in the DNA-binding domain (DBD) of the related ETS factor ETV4 interact with MED25. These ETV4 regions bind MED25 independently, display distinct kinetics, and combine to contribute to a high-affinity interaction of full-length ETV4 with MED25. High-affinity interactions with MED25 are specific for the ETV1/4/5 subfamily as other ETS factors display weaker binding. The AD binds to a single site on MED25 and the DBD interacts with three MED25 sites, allowing for simultaneous binding of both domains in full-length ETV4. MED25 also stimulates the in vitro DNA binding activity of ETV4 by relieving autoinhibition. ETV1/4/5 factors are often overexpressed in prostate cancer and genome-wide studies in a prostate cancer cell line indicate that ETV4 and MED25 occupy enhancers that are enriched for ETS-binding sequences and are both functionally important for the transcription of genes regulated by these enhancers. AP1-motifs, which bind JUN and FOS transcription factor families, were observed in MED25-occupied regions and JUN/FOS also contact MED25; FOS strongly binds to the same MED25 site as ETV4 AD and JUN interacts with the other two MED25 sites. In summary, we describe features of the multivalent ETV4- and AP1-MED25 interactions, thereby implicating these factors in the recruitment of MED25 to transcriptional control elements.

The ETS transcriptional repressor ETV6 (or TEL) is autoinhibited by an α-helix that sterically blocks its DNA-binding ETS domain. The inhibitory helix is marginally stable and unfolds when ETV6 binds to either specific or non-specific DNA. Using NMR spectroscopy, we show that folding of the inhibitory helix requires a buried charge-dipole interaction with helix H1 of the ETS domain. This interaction also contributes directly to autoinhibition by precluding a highly conserved dipole-enhanced hydrogen bond between the phosphodiester backbone of bound DNA and the N terminus of helix H1. To probe further the thermodynamic basis of autoinhibition, ETV6 variants were generated with amino acid substitutions introduced along the solvent exposed surface of the inhibitory helix. These changes were designed to increase the intrinsic helical propensity of the inhibitory helix without perturbing its packing interactions with the ETS domain. NMR-monitored amide hydrogen exchange measurements confirmed that the stability of the folded inhibitory helix increases progressively with added helix-promoting substitutions. This also results in progressively reinforced autoinhibition and decreased DNA-binding affinity. Surprisingly, locking the inhibitory helix onto the ETS domain by a disulfide bridge severely impairs, but does not abolish DNA binding. Weak interactions still occur via an interface displaced from the canonical ETS domain DNA-binding surface. Collectively, these studies establish a direct thermodynamic linkage between inhibitory helix stability and ETV6 autoinhibition, and demonstrate that helix unfolding does not strictly precede DNA binding. Modulating inhibitory helix stability provides a potential route for the in vivo regulation of ETV6 activity.

PAS domain containing protein kinase (Pask) is an evolutionarily conserved protein kinase implicated in energy homeostasis and metabolic regulation across eukaryotic species. We now describe an unexpected role of Pask in promoting the differentiation of myogenic progenitor cells, embryonic stem cells and adipogenic progenitor cells. This function of Pask is dependent upon its ability to phosphorylate Wdr5, a member of several protein complexes including those that catalyze histone H3 Lysine 4 trimethylation (H3K4me3) during transcriptional activation. Our findings suggest that, during myoblast differentiation, Pask stimulates the conversion of repressive H3K4me1 to activating H3K4me3 marks on the promoter of the differentiation gene myogenin (Myog) via Wdr5 phosphorylation. This enhances accessibility of the MyoD transcription factor and enables transcriptional activation of the Myog promoter to initiate muscle differentiation. Thus, as an upstream kinase of Wdr5, Pask integrates signaling cues with the transcriptional network to regulate the differentiation of progenitor cells.

The affinity of the Ets-1 transcription factor for DNA is autoinhibited by an intrinsically disordered serine-rich region (SRR) and a helical inhibitory module (IM) appended to its winged helix-turn-helix ETS domain. Using NMR spectroscopy, we investigated how Ets-1 recognizes specific versus nonspecific DNA, with a focus on the roles of protein dynamics and autoinhibition in these processes. Upon binding either DNA, the two marginally stable N-terminal helices of the IM predominantly unfold, but still sample partially ordered conformations. Also, on the basis of amide chemical shift perturbation mapping, Ets-1 associates with both specific and nonspecific DNA through the same canonical ETS domain interface. These interactions are structurally independent of the SRR, and thus autoinhibition does not impart DNA-binding specificity. However, relative to the pronounced NMR spectroscopic changes in Ets-1 resulting from specific DNA binding, the spectra of the nonspecific DNA complexes showed conformational exchange broadening and lacked several diagnostic amide and indole signals attributable to hydrogen bonding interactions seen in reported X-ray crystallographic structures of this transcription factor with its cognate DNA sequences. Such differences are highlighted by the chemical shift and relaxation properties of several interfacial lysine and arginine side chains. Collectively, these data support a general model in which Ets-1 interacts with nonspecific DNA via dynamic electrostatic interactions, whereas hydrogen bonding drives the formation of well-ordered complexes with specific DNA.

Histone H3 lysine 4 (H3K4) methylation is a dynamic modification. In budding yeast, H3K4 methylation is catalyzed by the Set1-COMPASS methyltransferase complex and is removed by Jhd2, a JMJC domain family demethylase. The catalytic JmjC and JmjN domains of Jhd2 have the ability to remove all three degrees (mono-, di-, and tri-) of H3K4 methylation. Jhd2 also contains a plant homeodomain (PHD) finger required for its chromatin association and H3K4 demethylase functions. The Jhd2 PHD finger associates with chromatin independent of H3K4 methylation and the H3 N-terminal tail. Therefore, how Jhd2 associates with chromatin to perform H3K4 demethylation has remained unknown. We report a novel interaction between the Jhd2 PHD finger and histone H2A. Two residues in H2A (Phe-26 and Glu-57) serve as a binding site for Jhd2 in vitro and mediate its chromatin association and H3K4 demethylase functions in vivo. Using RNA sequencing, we have identified the functional target genes for Jhd2 and the H2A Phe-26 and Glu-57 residues. We demonstrate that H2A Phe-26 and Glu-57 residues control chromatin association and H3K4 demethylase functions of Jhd2 during positive or negative regulation of transcription at target genes. Importantly, we show that H2B Lys-123 ubiquitination blocks Jhd2 from accessing its binding site on chromatin, and thereby, we have uncovered a second mechanism by which H2B ubiquitination contributes to the trans-histone regulation of H3K4 methylation. Overall, our study provides novel insights into the chromatin binding dynamics and H3K4 demethylase functions of Jhd2.

Significance Eukaryotic proteins often contain intrinsically disordered regions that lack well-defined conformations, yet still play key roles in numerous biological processes. The molecular mechanisms underlying the functions of such unstructured regions are often poorly understood. In the case of the E26 transformation-specific transcription factor, DNA binding by the ETS domain is autoinhibited through transient interactions with an adjacent disordered serine-rich region (SRR). Phosphorylation of the SRR in response to cellular signals increases these interactions and thereby reinforces autoinhibition. In this study, we demonstrate that phosphorylation-enhanced autoinhibition requires the presence of phenylalanine or tyrosine residues neighboring the SRR phosphoacceptor serines. This highlights a previously unrecognized role of aromatic residues and their synergy with phosphoserines in an intrinsically disordered regulatory sequence.