Project description:Previously, we showed that peripheral administration of 2-ME (2-methoxyestradiol), a CYP1B1 (cytochrome P450 1B1)-catechol-O-methyltransferase (COMT) generated metabolite of E2 (17?-Estradiol), protects against angiotensin II-induced hypertension in female mice. The demonstration that central E2 inhibits angiotensin II-induced hypertension, together with the expression of CYP1B1 in the brain, led us to hypothesize that E2-CYP1B1 generated metabolite 2-ME in the brain mediates its protective action against angiotensin II-induced hypertension in female mice. To test this hypothesis, we examined the effect of intracerebroventricularly (ICV) administered E2 in ovariectomized (OVX)-wild-type (Cyp1b1+/+) and OVX-Cyp1b1-/- mice on the action of systemic angiotensin II. ICV-E2 attenuated the angiotensin II-induced increase in mean arterial blood pressure, impairment of baroreflex sensitivity, and sympathetic activity in OVX-Cyp1b1+/+ but not in ICV-injected short interfering (si)RNA-COMT or OVX-Cyp1b1-/- mice. ICV-2-ME attenuated the angiotensin II-induced increase in blood pressure in OVX-Cyp1b1-/- mice; this effect was inhibited by ICV-siRNA estrogen receptor-? (ER?) and G protein-coupled estrogen receptor 1 (GPER1). ICV-E2 in OVX-Cyp1b1+/+ but not in OVX-Cyp1b1-/- mice and 2-ME in the OVX-Cyp1b1-/- inhibited angiotensin II-induced increase in reactive oxygen species production in the subfornical organ and paraventricular nucleus, activation of microglia and astrocyte, and neuroinflammation in paraventricular nucleus. Furthermore, central CYP1B1 gene disruption in Cyp1b1+/+ mice by ICV-adenovirus-GFP (green fluorescence protein)-CYP1B1-short hairpin (sh)RNA elevated, while reconstitution by adenovirus-GFP-CYP1B1-DNA in the paraventricular nucleus but not in subfornical organ in Cyp1b1-/- mice attenuated the angiotensin II-induced increase in systolic blood pressure. These data suggest that E2-CYP1B1-COMT generated metabolite 2-ME, most likely in the paraventricular nucleus via estrogen receptor-? and GPER1, protects against angiotensin II-induced hypertension and neuroinflammation in female mice.

Project description:Throughout life, humans experience repeated exposure to viral antigens through infection and vaccination, resulting in the generation of diverse, and largely unique, antigen specific antibody repertoires. A paramount feature of antibodies that enables their critical contributions in counteracting recurrent and novel pathogens, and consequently fostering their utility as valuable targets for therapeutic and vaccine development, is the exquisite specificity displayed against their target antigens. Yet, there is still limited understanding of the determinants of antibody-antigen specificity, particularly as a function of antibody sequence. In recent years, experimental characterization of antibody repertoires has led to novel insights into fundamental properties of antibody sequences, but has been largely decoupled from at-scale antigen specificity analysis. Here, using the LIBRA-seq technology, we generated a large dataset mapping antibody sequence to antigen specificity for thousands of B cells, by screening the repertoires of a set of healthy individuals against twenty viral antigens representing diverse pathogens of biomedical significance. Analysis uncovered virus specific patterns in variable gene usage, gene pairing, somatic hypermutation, as well as the presence of convergent antiviral signatures across multiple individuals, including the presence of public antibody clonotypes. Notably, our results showed that, for B cell receptors originating from different individuals but leveraging an identical combination of heavy and light chain variable genes, there is a specific CDRH3/CDRL3 identity threshold that defines whether these B cells may share the same antigen specificity. This finding provides a quantifiable measure of the relationship between antibody sequence and antigen specificity and further defines experimentally grounded criteria for defining public antibody clonality. Understanding the fundamental rules of antibody-antigen interactions can lead to transformative new approaches for the development of antibody therapeutics and vaccines against current and emerging viruses.

Project description:To elucidate how genomic sequences build transcriptional control networks we need to understand the connection between DNA sequence and transcription factor binding and function. Binding predictions based solely on consensus predictions are limited because a single factor can use degenerate sequence motifs and related transcription factors often prefer identical sequences. The ETS family transcription factor, ETS1, exemplifies these challenges. Unexpected, redundant occupancy of ETS1 and other ETS proteins is observed at promoters of housekeeping genes in T cells due to common sequence preferences and the presence of strong consensus motifs. However, ETS1 exhibits a specific function in T cell activation, thus unique transcriptional targets are predicted. To uncover the sequence motifs that mediate specific functions of ETS1, chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) identified both promoter and enhancer binding events in Jurkat T cells. A comparison with DNase I sensitivity both validated the dataset and improved accuracy. Redundant occupancy of ETS1 with the ETS protein GABPA occurred primarily in promoters of housekeeping genes, whereas ETS1 specific occupancy occurred in the enhancers of T-cell specific genes. Two routes to ETS1 specificity were identified: an intrinsic preference of ETS1 for a variant of the ETS family consensus sequence and the presence of a composite sequence that can support cooperative binding with a RUNX transcription factor. Genome-wide occupancy of RUNX factors corroborated the importance of this partnership. Furthermore, genome-wide occupancy of co-activator CBP indicated tight co-localization with ETS1 at specific enhancers, but not redundant promoters. The distinct sequences associated with redundant versus specific ETS1 occupancy were predictive of promoter or enhancer location and the ontology of nearby genes. These findings demonstrate that diversity of binding motifs may enable variable transcription factor function at different genomic sites. Each ChIP sample was pooled from three independent immunoprecipitation experiments

Project description:To elucidate how genomic sequences build transcriptional control networks we need to understand the connection between DNA sequence and transcription factor binding and function. Binding predictions based solely on consensus predictions are limited because a single factor can use degenerate sequence motifs and related transcription factors often prefer identical sequences. The ETS family transcription factor, ETS1, exemplifies these challenges. Unexpected, redundant occupancy of ETS1 and other ETS proteins is observed at promoters of housekeeping genes in T cells due to common sequence preferences and the presence of strong consensus motifs. However, ETS1 exhibits a specific function in T cell activation, thus unique transcriptional targets are predicted. To uncover the sequence motifs that mediate specific functions of ETS1, chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) identified both promoter and enhancer binding events in Jurkat T cells. A comparison with DNase I sensitivity both validated the dataset and improved accuracy. Redundant occupancy of ETS1 with the ETS protein GABPA occurred primarily in promoters of housekeeping genes, whereas ETS1 specific occupancy occurred in the enhancers of T-cell specific genes. Two routes to ETS1 specificity were identified: an intrinsic preference of ETS1 for a variant of the ETS family consensus sequence and the presence of a composite sequence that can support cooperative binding with a RUNX transcription factor. Genome-wide occupancy of RUNX factors corroborated the importance of this partnership. Furthermore, genome-wide occupancy of co-activator CBP indicated tight co-localization with ETS1 at specific enhancers, but not redundant promoters. The distinct sequences associated with redundant versus specific ETS1 occupancy were predictive of promoter or enhancer location and the ontology of nearby genes. These findings demonstrate that diversity of binding motifs may enable variable transcription factor function at different genomic sites.

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Specificity determinants of CYP1B1 estradiol hydroxylation.

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Specificity determinants of CYP1B1 estradiol hydroxylation.

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