Lysine acetylation controls local protein conformation by influencing proline isomerization.

Gene transcription responds to stress and metabolic signals to optimize growth and survival. Histone H3 (H3) lysine 4 trimethylation (K4me3) facilitates state changes, but how levels are coordinated with the environment is unclear. Here, we show that isomerization of H3 at the alanine 15-proline 16 (A15-P16) peptide bond is influenced by lysine 14 (K14) and controls gene-specific K4me3 by balancing the actions of Jhd2, the K4me3 demethylase, and Spp1, a subunit of the Set1 K4 methyltransferase complex. Acetylation at K14 favors the A15-P16trans conformation and reduces K4me3. Environmental stress-induced genes are most sensitive to the changes at K14 influencing H3 tail conformation and K4me3. By contrast, ribosomal protein genes maintain K4me3, required for their repression during stress, independently of Spp1, K14, and P16. Thus, the plasticity in control of K4me3, via signaling to K14 and isomerization at P16, informs distinct gene regulatory mechanisms and processes involving K4me3.

Optimisation of a screening platform for determining IL-6 inflammatory signalling in the senescence-associated secretory phenotype (SASP).

Cellular senescence has been shown to be sufficient for the development of multiple age-related pathologies. Senescent cells adopt a secretory phenotype (the SASP) which comprises a large number of pro-inflammatory cytokines, chemokines and proteases. The SASP itself is thought to be causative in many pathologies of age-related diseases, and there is growing interest in developing seno-modifying agents that can suppress the SASP. However, in order to identify new agents, it is necessary to conduct moderate to high throughput screening with robust assays for the required outcome. Here, we describe optimisation and validation of a cell-based biosensor HEK cell line for measurement of IL-6 concentrations within the range secreted into conditioned medium by primary senescent fibroblasts, adapted for a 384 well plate format suitable for library screening applications. We further show that the assay can measure changes in IL-6 secretion dependent on cell population age, and that the assay is responsive to mTOR inhibition in the senescent cells, which reduces the SASP, including IL-6. Hence, we propose that this optimised biosensor, which we term HEK-SASP, may prove of value in studies requiring robust, renewable and relatively inexpensive assays for measuring SASP factors.

Correction to: Optimisation of a screening platform for determining IL-6 inflammatory signalling in the senescence-associated secretory phenotype (SASP).

In the original publication of the article, Fig. 2 was published incorrectly. The corrected Figure is given below. The original article has been corrected.

Sense and antisense transcription are associated with distinct chromatin architectures across genes.

Genes from yeast to mammals are frequently subject to non-coding transcription of their antisense strand; however the genome-wide role for antisense transcription remains elusive. As transcription influences chromatin structure, we took a genome-wide approach to assess which chromatin features are associated with nascent antisense transcription, and contrast these with features associated with nascent sense transcription. We describe a distinct chromatin architecture at the promoter and gene body specifically associated with antisense transcription, marked by reduced H2B ubiquitination, H3K36 and H3K79 trimethylation and increased levels of H3 acetylation, chromatin remodelling enzymes, histone chaperones and histone turnover. The difference in sense transcription between genes with high or low levels of antisense transcription is slight; thus the antisense transcription-associated chromatin state is not simply analogous to a repressed state. Using mutants in which the level of antisense transcription is reduced at GAL1, or altered genome-wide, we show that non-coding transcription is associated with high H3 acetylation and H3 levels across the gene, while reducing H3K36me3. Set1 is required for these antisense transcription-associated chromatin changes in the gene body. We propose that nascent antisense and sense transcription have fundamentally distinct relationships with chromatin, and that both should be considered canonical features of eukaryotic genes.

Dynamic regulation of ß1 subunit trafficking controls vascular contractility.

Ion channels composed of pore-forming and auxiliary subunits control physiological functions in virtually all cell types. A conventional view is that channels assemble with their auxiliary subunits before anterograde plasma membrane trafficking of the protein complex. Whether the multisubunit composition of surface channels is fixed following protein synthesis or flexible and open to acute and, potentially, rapid modulation to control activity and cellular excitability is unclear. Arterial smooth muscle cells (myocytes) express large-conductance Ca(2+)-activated potassium (BK) channel α and auxiliary ß1 subunits that are functionally significant modulators of arterial contractility. Here, we show that native BKα subunits are primarily (∼95%) plasma membrane-localized in human and rat arterial myocytes. In contrast, only a small fraction (∼10%) of total ß1 subunits are located at the cell surface. Immunofluorescence resonance energy transfer microscopy demonstrated that intracellular ß1 subunits are stored within Rab11A-postive recycling endosomes. Nitric oxide (NO), acting via cGMP-dependent protein kinase, and cAMP-dependent pathways stimulated rapid (≤1 min) anterograde trafficking of ß1 subunit-containing recycling endosomes, which increased surface ß1 almost threefold. These ß1 subunits associated with surface-resident BKα proteins, elevating channel Ca(2+) sensitivity and activity. Our data also show that rapid ß1 subunit anterograde trafficking is the primary mechanism by which NO activates myocyte BK channels and induces vasodilation. In summary, we show that rapid ß1 subunit surface trafficking controls functional BK channel activity in arterial myocytes and vascular contractility. Conceivably, regulated auxiliary subunit trafficking may control ion channel activity in a wide variety of cell types.

Widely tunable LP11 cladding-mode resonance in a twisted mechanically induced long-period fiber grating.

A record tunability of 35 nm for the LP(11) cladding-mode resonance in a twisted mechanically induced long-period fiber grating using standard single-mode communication fiber is demonstrated. By forming the LP(11) resonance far away from its cut-off wavelength and modifying the grooves of the grating in the form of smooth semicircular humps, a high twist sensitivity of 8.75 nm/(rad/cm) and a controlled tunability of 35 nm is achieved. The fiber with its lacquer coating is not broken even at a severe twist rate of 5.44 rad/cm. The present design can be used as a novel variable optical selective wavelength attenuator since the bandwidth, rejection efficiency, and center wavelength can be controlled by changing the grating length, pressure over the grating, and fiber twist, respectively. Using the results, a cost-effective tunable variable optical attenuator for selective channel-blanking applications is also demonstrated. A fine tunability of 1.5 nm is achieved for a twist rate change of 0.1 rad/cm.

The voltage-dependent L-type Ca2+ (CaV1.2) channel C-terminus fragment is a bi-modal vasodilator.

Voltage-dependent L-type Ca(2+) channels (CaV1.2) are the primary Ca(2+) entry pathway in vascular smooth muscle cells (myocytes). CaV1.2 channels control systemic blood pressure and organ blood flow and are pathologically altered in vascular diseases, which modifies vessel contractility. The CaV1.2 distal C-terminus is susceptible to proteolytic cleavage, which yields a truncated CaV1.2 subunit and a cleaved C-terminal fragment (CCt). Previous studies in cardiac myocytes and neurons have identified CCt as both a transcription factor and CaV1.2 channel inhibitor, with different signalling mechanisms proposed to underlie some of these effects. CCt existence and physiological functions in arterial myocytes are unclear, but important to study given the functional significance of CaV1.2 channels. Here, we show that CCt exists in myocytes of both rat and human resistance-size cerebral arteries, where it locates to both the nucleus and plasma membrane. Recombinant CCt expression in arterial myocytes inhibited CaV1.2 transcription and reduced CaV1.2 protein. CCt induced a depolarizing shift in the voltage dependence of both CaV1.2 current activation and inactivation, and reduced non-inactivating current in myocytes. Recombinant truncated CCt lacking a putative nuclear localization sequence (92CCt) did not locate to the nucleus and had no effect on arterial CaV1.2 transcription or protein. However, 92CCt shifted the voltage dependence of CaV1.2 activation and inactivation similarly to CCt. CCt and 92CCt both inhibited pressure- and depolarization-induced vasoconstriction, although CCt was a far more effective vasodilator. These data demonstrate that endogenous CCt exists and reduces both CaV1.2 channel expression and voltage sensitivity in arterial myocytes. Thus, CCt is a bi-modal vasodilator.

Preservation of microvascular barrier function requires CD31 receptor-induced metabolic reprogramming.

Endothelial barrier (EB) breaching is a frequent event during inflammation, and it is followed by the rapid recovery of microvascular integrity. The molecular mechanisms of EB recovery are poorly understood. Triggering of MHC molecules by migrating T-cells is a minimal signal capable of inducing endothelial contraction and transient microvascular leakage. Using this model, we show that EB recovery requires a CD31 receptor-induced, robust glycolytic response sustaining junction re-annealing. Mechanistically, this response involves src-homology phosphatase activation leading to Akt-mediated nuclear exclusion of FoxO1 and concomitant ß-catenin translocation to the nucleus, collectively leading to cMyc transcription. CD31 signals also sustain mitochondrial respiration, however this pathway does not contribute to junction remodeling. We further show that pathologic microvascular leakage in CD31-deficient mice can be corrected by enhancing the glycolytic flux via pharmacological Akt or AMPK activation, thus providing a molecular platform for the therapeutic control of EB response.

Hybrid Poly(hydroxy urethane)s: Folded-Sheet Morphology and Thermoreversible Adhesion.

Hybrid poly(hydroxy urethane)s (PHUs) are synthesized by copolymerizing aromatic/alicyclic cyclic carbonates with a polyether amine via addition polymerization. They result into polymers with an average molecular weight of 10 kDa and exhibit solubility in common organic solvents. The hybrid PHUs display T g up to 18 °C. PHUs are enriched with multiple H-bonded interactions and they are assessed using temperature-dependent 1H NMR and Fourier-transform infrared studies. PHUs possess folded-sheet morphology with nanogap between folds and nanowidth between chains. The secondary interactions bestow thermoreversible property to PHUs, and they display good adhesion to both polar (Al-Al) and nonpolar (HDPE-HDPE) substrates. Hybrid PHUs show improved optical transparency compared to homo PHUs. The PHUs are thermally stable up to 250 °C.

Cyclooxygenase-2 Selectively Controls Renal Blood Flow Through a Novel PPARß/δ-Dependent Vasodilator Pathway.

Cyclooxygenase-2 (COX-2) is an inducible enzyme expressed in inflammation and cancer targeted by nonsteroidal anti-inflammatory drugs. COX-2 is also expressed constitutively in discreet locations where its inhibition drives gastrointestinal and cardiovascular/renal side effects. Constitutive COX-2 expression in the kidney regulates renal function and blood flow; however, the global relevance of the kidney versus other tissues to COX-2-dependent blood flow regulation is not known. Here, we used a microsphere deposition technique and pharmacological COX-2 inhibition to map the contribution of COX-2 to regional blood flow in mice and compared this to COX-2 expression patterns using luciferase reporter mice. Across all tissues studied, COX-2 inhibition altered blood flow predominantly in the kidney, with some effects also seen in the spleen, adipose, and testes. Of these sites, only the kidney displayed appreciable local COX-2 expression. As the main site where COX-2 regulates blood flow, we next analyzed the pathways involved in kidney vascular responses using a novel technique of video imaging small arteries in living tissue slices. We found that the protective effect of COX-2 on renal vascular function was associated with prostacyclin signaling through PPARß/δ (peroxisome proliferator-activated receptor-ß/δ). These data demonstrate the kidney as the principle site in the body where local COX-2 controls blood flow and identifies a previously unreported PPARß/δ-mediated renal vasodilator pathway as the mechanism. These findings have direct relevance to the renal and cardiovascular side effects of drugs that inhibit COX-2, as well as the potential of the COX-2/prostacyclin/PPARß/δ axis as a therapeutic target in renal disease.

CRISPRi is not strand-specific at all loci and redefines the transcriptional landscape.

CRISPRi, an adapted CRISPR-Cas9 system, is proposed to act as a strand-specific roadblock to repress transcription in eukaryotic cells using guide RNAs (sgRNAs) to target catalytically inactive Cas9 (dCas9) and offers an alternative to genetic interventions for studying pervasive antisense transcription. Here, we successfully use click chemistry to construct DNA templates for sgRNA expression and show, rather than acting simply as a roadblock, sgRNA/dCas9 binding creates an environment that is permissive for transcription initiation/termination, thus generating novel sense and antisense transcripts. At HMS2 in Saccharomyces cerevisiae, sgRNA/dCas9 targeting to the non-template strand for antisense transcription results in antisense transcription termination, premature termination of a proportion of sense transcripts and initiation of a novel antisense transcript downstream of the sgRNA/dCas9-binding site. This redefinition of the transcriptional landscape by CRISPRi demonstrates that it is not strand-specific and highlights the controls and locus understanding required to properly interpret results from CRISPRi interventions.

An in vitro model to study the pathogenesis of the early endometriosis lesion.

OBJECTIVE: To determine if whole fragments of endometrium can adhere to peritoneum with intact mesothelium. DESIGN: Tissue culture and immunohistochemical study. SETTING: University Medical Center. PATIENTS: Reproductive-age women undergoing surgery for benign conditions. INTERVENTIONS: Whole explants of human peritoneum from the anterior abdominal wall and the posterior surface of the uterus were cultured with whole fragments of mechanically dispersed endometrium. MAIN OUTCOME MEASURES: Adhesion of endometrial fragments to the surface of the peritoneum was evaluated. Adherent fragments of endometrium were identified using the dissecting microscope and by performing serial sections of the peritoneum explants for light and confocal laser-scanning microscopy. Immunohistochemical staining of the mesothelium with antibodies to cytokeratin and vimentin was used to ensure an intact layer of mesothelium beneath the endometrial implants. Transmission electron microscopy was also used to evaluate the adhesion of endometrium to the mesothelium. RESULTS: Endometrium was identified attached to the surface of the peritoneum. After 18-24 hours of culture, the majority of implants did not have identifiable mesothelium beneath them, but most had intact mesothelium running up to the point of attachment. Approximately 10% of the endometrial implants had intact mesothelium at the site of attachment. After 1 hour of culture, both endometrial stromal and epithelial cells were attached to intact mesothelium in nearly all cases. Early transmesothelial invasion involves endometrial stromal cells. CONCLUSIONS: Endometrial stromal and epithelial cells can attach to the intact mesothelial surface of the peritoneum. Endometrial stromal cell invasion through the mesothelium occurs in less than 18-24 hours.

Inhibition of CD44 N- and O-linked glycosylation decreases endometrial cell lines attachment to peritoneal mesothelial cells.

The attachment of endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs) to peritoneal mesothelial cells (PMCs) with and without inhibition of N- and O-linked glycosylation, the viability of EECs and ESCs, and the expression of CD44 surface density were evaluated. Inhibition of CD44 N- and O-linked glycosylation by using tunicamycin and/or B-GalNAc statistically significantly inhibited endometrial cell attachment to peritoneal mesothelial cells, suggesting a role in establishment of early endometriotic lesions.

Nucleosome remodeling and transcriptional repression are distinct functions of Isw1 in Saccharomyces cerevisiae.

The SANT domain is a nucleosome recognition module found in transcriptional regulatory proteins, including chromatin-modifying enzymes. It shows high functional degeneracy between species, varying in sequence and copy number. Here, we investigate functions in vivo associated with two SANT motifs, SANT and SLIDE, in the Saccharomyces cerevisiae Isw1 chromatin-remodeling ATPase. We show that differences in the primary structures of the SANT and SLIDE domains in yeast and Drosophila melanogaster reflect their different functions. In yeast, the SLIDE domain is required for histone interactions, while this is a function of the SANT domain in flies. In yeast, both motifs are required for optimal association with chromatin and for formation of the Isw1b complex (Isw1, Ioc2, and Ioc4). Moreover, nucleosome remodeling at the MET16 locus is defective in strains lacking the SANT or SLIDE domain. In contrast, the SANT domain is dispensable for the interaction between Isw1 and Ioc3 in the Isw1a complex. We show that, although defective in nucleosome remodeling, Isw1 lacking the SANT domain is able to repress transcription initiation at the MET16 promoter. Thus, chromatin remodeling and transcriptional repression are distinct activities of Isw1.

Endometrial stromal sarcoma mimicking a myoma.

Uterine malignancies are not uncommonly misdiagnosed for the more ubiquitous leiomyoma. A case of endometrial stromal sarcoma with ultrasound and color Doppler imaging is described.

Modeling the early endometriotic lesion: mesothelium-endometrial cell co-culture increases endometrial invasion and alters mesothelial and endometrial gene transcription.

OBJECTIVE: To determine the role of peritoneal mesothelial cells (PMCs) in the process of endometrial invasion into the peritoneum and to evaluate gene expression after endometrial-PMC co-culture. DESIGN: In vitro study. SETTING: University laboratory. PATIENT(S): Reproductive-age women without endometriosis. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The rate of endometrial invasion through modeled peritoneum in the presence and absence of PMCs was evaluated. The influence of endometrial-PMC attachment on the expression of target genes, implicated in the pathogenesis of endometriosis, was examined by using reverse transcription polymerase chain reaction. RESULT(S): Endometrial stromal cell (ESC) invasion through invasion chambers coated with Matrigel (MTGL) and with growth factor-reduced Matrigel (GFR-MTGL) was increased 10-fold when a PMC monolayer was present. Endometrial epithelioid cell (EM42) invasion increased greater than threefold through the MTGL and GFR-MTGL-coated membranes when a PMC monolayer was present. Endometrial stromal cell, EM42, and PMC transcription of extracellular signal-related kinase, colony stimulating factor-1, c-fms, and c-Met was increased after endometrial-PMC attachment. Similar changes were not seen when endometrial cells were exposed to PMC-conditioned media and when PMCs were exposed to endometrial cell conditioned media. CONCLUSION(S): Peritoneal mesothelial cells increased invasion of ESCs and EM42s through modeled peritoneum. Endometrial-PMC co-culture led to alterations in gene transcription by endometrial cells and PMCs. This study suggests that PMCs contribute to the process of endometrial invasion into the peritoneum.

Dynamic lysine methylation on histone H3 defines the regulatory phase of gene transcription.

Covalent modifications to histones are key epigenetic marks that control gene transcription. Multiple lysine residues on histone H3 are methylated (me), but their functions are unclear. Here, we demonstrate two phases of combinatorial and dynamic H3 methylation during induction of transcription at MET16 in yeast. K4me3 with K36me2/3 define a postinitiation regulatory phase and precede the appearance of K4me2 with K79me2 at the onset of transcript elongation. The Isw1 ATPase delays the release of initiated RNA polymerase II (RNAPII) into elongation to facilitate chromatin modifications. The Spp1 subunit of complex associated with Set1 (COMPASS) and Set2, determining K4me3 and K36me2/3, respectively, are required for transient NuA4-dependent H4K8ac. This releases RNAPII from Isw1 control and promotes controlled transcription elongation and termination. We propose that newly initiated RNAPII is under epigenetic control.