CDK8 and CDK19 regulate intestinal differentiation and homeostasis via the chromatin remodeling complex SWI/SNF.

Initiation and maintenance of transcriptional states are critical for controlling normal tissue homeostasis and differentiation. The cyclin dependent kinases CDK8 and CDK19 (Mediator kinases) are regulatory components of Mediator, a highly conserved complex that orchestrates enhancer-mediated transcriptional output. While Mediator kinases have been implicated in the transcription of genes necessary for development and growth, its function in mammals has not been well defined. Using genetically defined models and pharmacological inhibitors, we showed that CDK8 and CDK19 function in a redundant manner to regulate intestinal lineage specification in humans and mice. The Mediator kinase module bound and phosphorylated key components of the chromatin remodeling complex switch/sucrose non-fermentable (SWI/SNF) in intestinal epithelial cells. Concomitantly, SWI/SNF and MED12-Mediator colocalized at distinct lineage-specifying enhancers in a CDK8/19-dependent manner. Thus, these studies reveal a transcriptional mechanism of intestinal cell specification, coordinated by the interaction between the chromatin remodeling complex SWI/SNF and Mediator kinase.

Pharmacodynamic Studies of Fluorescent Diamond Carriers of Doxorubicin in Liver Cancer Cells and Colorectal Cancer Organoids.

BACKGROUND: We recently reported on preferential deposition of bare fluorescent diamond particles FDP-NV-700/800nm (FDP-NV) in the liver following intravenous administration to rats. The pharmacokinetics of FDP-NV in that species indicated short residency in the circulation by rapid clearance by the liver. Retention of FDP-NV in the liver was not associated with any pathology. These observations suggested that cancer therapeutics, such as doxorubicin, linked to FDP-NV, could potentially serve for anti-cancer treatment while sparing toxicities of peripheral organs. PURPOSE: To generate proof-of-concept (POC) and detail mechanisms of action of doxorubicin-coated FDP-NV-700/800nm (FDP-DOX) as a prospective chemotherapeutic for metastatic liver cancer. METHODS: FDP-DOX was generated by adsorption chemistry. Experimental design included concentration and time-dependent efficacy studies as compared with naïve (baren) FDP-NV in in vitro liver cancer cells models. Uptake of FDP-NV and FDP-DOX by HepG-2, Hep-3B and hCRC organoids were demonstrated by flow-cytometry and fluorescent microscopy. FDP-DOX pharmacodynamic effects included metabolic as well as cell death biomarkers Annexin V, TUNEL and LDH leakage. DOX desorpted from FDP-DOX was assessed by confocal microscopy and chemical assay of cells fractions. RESULTS: FDP-DOX efficacy was dose- and time-dependent and manifested in both liver cancer cell lines and human CRC organoids. FDP-DOX was rapidly internalized into cancer cells/organoids leading to cancer growth inhibition and apoptosis. FDP-DOX disrupted cell membrane integrity as evident by LDH release and suppressing mitochondrial metabolic pathways (AlamarBlue assay). Access of free DOX to the nuclei was confirmed by direct UV-Visible fluorescent assay and confocal microscopy of DOX fluorescence. CONCLUSION: The rapid uptake and profound cancer inhibition observed using FDP-DOX in clinically relevant cancer models, highlight FDP-DOX promise for cancer chemotherapeutics. We also conclude that the in vitro data justify further investment in in vivo POC studies.

Genome-scale CRISPR-Cas9 screen of Wnt/ß-catenin signaling identifies therapeutic targets for colorectal cancer.

Aberrant activation of Wnt/ß-catenin pathway is a key driver of colorectal cancer (CRC) growth and of great therapeutic importance. In this study, we performed comprehensive CRISPR screens to interrogate the regulatory network of Wnt/ß-catenin signaling in CRC cells. We found marked discrepancies between the artificial TOP reporter activity and ß-catenin-mediated endogenous transcription and redundant roles of T cell factor/lymphoid enhancer factor transcription factors in transducing ß-catenin signaling. Compiled functional genomic screens and network analysis revealed unique epigenetic regulators of ß-catenin transcriptional output, including the histone lysine methyltransferase 2A oncoprotein (KMT2A/Mll1). Using an integrative epigenomic and transcriptional profiling approach, we show that KMT2A loss diminishes the binding of ß-catenin to consensus DNA motifs and the transcription of ß-catenin targets in CRC. These results suggest that KMT2A may be a promising target for CRCs and highlight the broader potential for exploiting epigenetic modulation as a therapeutic strategy for ß-catenin-driven malignancies.

Characterization of aromatase expression in the adult male and female mouse brain. I. Coexistence with oestrogen receptors α and ß, and androgen receptors.

Aromatase catalyses the last step of oestrogen synthesis. There is growing evidence that local oestrogens influence many brain regions to modulate brain development and behaviour. We examined, by immunohistochemistry, the expression of aromatase in the adult male and female mouse brain, using mice in which enhanced green fluorescent protein (EGFP) is transcribed following the physiological activation of the Cyp19A1 gene. EGFP-immunoreactive processes were distributed in many brain regions, including the bed nucleus of the stria terminalis, olfactory tubercle, medial amygdaloid nucleus and medial preoptic area, with the densest distributions of EGFP-positive cell bodies in the bed nucleus and medial amygdala. Differences between male and female mice were apparent, with the density of EGFP-positive cell bodies and fibres being lower in some brain regions of female mice, including the bed nucleus and medial amygdala. EGFP-positive cell bodies in the bed nucleus, lateral septum, medial amygdala and hypothalamus co-expressed oestrogen receptor (ER) α and ß, or the androgen receptor (AR), although single-labelled EGFP-positive cells were also identified. Additionally, single-labelled ERα-, ERß- or AR-positive cell bodies often appeared to be surrounded by EGFP-immunoreactive nerve fibres/terminals. The widespread distribution of EGFP-positive cell bodies and fibres suggests that aromatase signalling is common in the mouse brain, and that locally synthesised brain oestrogens could mediate biological effects by activating pre- and post-synaptic oestrogen α and ß receptors, and androgen receptors. The higher number of EGFP-positive cells in male mice may indicate that the autocrine and paracrine effects of oestrogens are more prominent in males than females.

Behavioural phenotype of APPC100.V717F transgenic mice over-expressing a mutant Abeta-bearing fragment is associated with reduced NMDA receptor density.

The aim of this study was to characterize APPC100.V717F transgenic (TgC100.V717F) mice which over-express a mutant C100 fragment of the amyloid precursor protein. The mice were compared to TgC100 wild type mice (TgC100.WT) and non-transgenic controls at 4-9 and 16-22 months of age. TgC100.V717F mice showed behavioural hyperactivity, particularly at a younger age, as shown by increased numbers of elevated plus maze arm entries and Y-maze arm entries, enhanced baseline locomotor activity in the open field, and enhanced amphetamine-induced hyperlocomotion. This hyperactivity was less pronounced in TgC100.WT which only displayed significant differences to non-transgenic controls at a younger age for the number of Y-maze arm entries and baseline locomotor activity in the open field. In addition, TgC100.V717F mice, but not TgC100.WT, demonstrated cognitive deficits, as shown by reduced spontaneous alternation in the Y-maze and markedly reduced retention in a passive avoidance test. At an older age, TgC100.V717F mice showed enhanced startle and increased immobility time in the forced swim test. In the TgC100.V717F mice, but not TgC100.WT, the behavioural changes were paralleled by a significant reduction in the expression of hippocampal NMDA receptor subunits types 1 and 2A. Concomitantly, we detected axonal disruption and apoptosis in the hippocampus of TgC100.V717F mice. In conclusion, these data demonstrate that the mutant C100 fragment is an effector of biochemical and both cognitive and non-cognitive behaviours. These transgenic mice may be a model for the psychotic features associated with early Alzheimer's disease.

Fetal testis dysgenesis and compromised Leydig cell function in Tgfbr3 (beta glycan) knockout mice.

Betaglycan (Tgfbr3) is a coreceptor for transforming growth factor-beta (TGFB) superfamily ligands. In the current study, a defect in seminiferous cord formation was detected in 12.5-13.5 days postcoitum (dpc) beta glycan null murine testis. Immunohistochemistry with antibodies against cell-specific markers revealed defects in somatic cell populations. To confirm these data, quantitative real-time PCR was performed to determine changes in the expression levels of genes involved in fetal testis cell differentiation and function. The expression levels of the Leydig cell markers Insl3, Cyp17a1, Cyp11a1, Star, and Hsd3b1 were reduced in knockout testis compared to wild-type testis, beginning at 12.5 dpc. Whole mount in situ hybridization confirmed that Cyp11a1 expression was reduced in the null testis, but its distribution pattern was unchanged. Apoptosis was not affected by the loss of beta glycan, but proliferation within the interstitium was reduced at 14.5 dpc. However, morphometric analysis showed no changes in Leydig cell counts between the wild-type and the knockout testes at 14.5 dpc, indicating that fetal Leydig function, rather than number, was affected by the loss of beta glycan. The expression levels of Sertoli cell markers Dhh, Sox9, and Amh were also reduced in the knockout testis at 14.5 dpc. However, the expression of fetal germ cell markers Pou5f1 and DDX4 were not changed across the genotypes at any age examined. Our data show that the presence of beta glycan is required for normal cord formation, normal fetal Leydig cell development, and the establishment of fetal testis endocrine function, thus implicating TGFB superfamily members as regulators of early fetal testis structure and function.

Estrogen deficiency results in apoptosis in the frontal cortex of adult female aromatase knockout mice.

The aromatase knockout (ArKO) mouse is completely estrogen deficient. We previously detected apoptosis in the hypothalamus of 1 year-old male ArKO mice. This study shows that 12 week-old female ArKO mice display spontaneous apoptosis of pyramidal neurons in the frontal cortex while wild-type (WT) littermates show no signs of apoptosis. Concomitantly, bcl-2 related anti-apoptotic genes are down-regulated whereas the pro-apoptotic gene TRADD is up-regulated in the female ArKO frontal cortex. This phenotype can be rescued by 3-week replacement of 17beta-estradiol. Furthermore, the apoptosis phenotype is exacerbated in 12-15 month-old female ArKO mice, which have 30% less neurons in the frontal cortex and lower brain weights than WT counterparts. These data show that estrogens are essential for the survival of female cortical neurons even in the absence of pathological conditions or external assaults. Our observations also demonstrate the sexually dimorphic susceptibility of neurons to estrogen deficiency.

Differential expression of TGFBR3 (betaglycan) in mouse ovary and testis during gonadogenesis.

TGFBR3 is an accessory receptor that binds to and modulates the activities of both transforming growth factor-beta (TGFbeta) and inhibin, two members of the TGFbeta superfamily of growth factors that regulate many aspects of reproductive biology. Tgfbr3 is known to be expressed in adult testis and ovary, but little is known about this receptor during gonadogenesis. Herein, we describe Tgfbr3 expression in the male and female fetal and neonatal murine gonad. Real-time PCR analysis revealed that Tgfbr3 mRNA was expressed at higher levels in the developing testis compared to ovary. TGFBR3 was expressed within the fetal testis interstitium, predominantly by Leydig cells, but expression shifted inside the seminiferous cords at birth. In contrast, TGFBR3 was detected in both the somatic and germ cell lineages in the fetal and neonatal ovary. This differential expression pattern suggests divergent roles for this TGFBR3 in developing testis and ovary.

Betanodavirus B2 is an RNA interference antagonist that facilitates intracellular viral RNA accumulation.

Betanodaviruses are small positive-sense bipartite RNA viruses that infect a wide variety of fish species and are notorious for causing lethal outbreaks in juvenile fish hatcheries worldwide. The function of a small nonstructural protein, B2, encoded by the subgenomic RNA3 of betanodaviruses, has remained obscure. Greasy grouper nervous necrosis virus, a betanodavirus model, was used to develop a facile DNA-based reverse genetics system that recapitulated the virus infection cycle, and we used this system to show that B2 is a small nonstructural protein that is essential for high level accumulation of viral RNA1 after RNA transfection of fish, mammalian, and avian cells. The defect in RNA1 accumulation in a B2 mutant was partially complemented by supplying B2 RNA in trans. Confocal analysis of the cellular distribution of B2 indicated that B2 is able to enter the nucleus and accumulates there during the late stages of GGNNV infection. Using human HeLa cells as a cellular RNA interference model, we found that B2 could efficiently antagonize RNA interference, which is a property shared by the distantly related alphanodavirus B2 proteins. This function provides appears to provide an explanation, at least in part, for why B2 mutant RNA1 is severely impaired in its intracellular accumulation.

RING-H2 protein WSSV249 from white spot syndrome virus sequesters a shrimp ubiquitin-conjugating enzyme, PvUbc, for viral pathogenesis.

Modification of proteins by ubiquitin is essential for numerous cellular processes. The RING-H2 finger motif has been implicated in ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Four proteins, WSSV199, WSSV222, WSSV249, and WSSV403, from white spot syndrome virus (WSSV) contain the RING-H2 motif. Here we report that WSSV249 physically interacts with a shrimp ubiquitin-conjugating enzyme, PvUbc, and mediates ubiquitination through its RING-H2 motif in the presence of E1 and PvUbc. Mutations of the putative zinc coordination residues in the RING-H2 domain of WSSV249, however, ablate ubiquitination efficiency. In addition, the RING-H2 domain of WSSV249 is capable of ubiquitination with UbcH1, UbcH2, UbcH5a, UbcH5b, UbcH5c, UbcH6, and UbcH10, respectively, exhibiting a low degree of E2 specificity. Significantly, the expression of WSSV249 and PvUbc increased during infection, as revealed by real-time PCR. Furthermore, in situ hybridization showed that WSSV249 and PvUbc display similar expression patterns in infected shrimps, and immunofluorescence and immunohistochemistry assays showed an increase of PvUbc in infected shrimp cells. These results suggest that the RING-H2 protein WSSV249 from WSSV may function as an E3 ligase via sequestration of PvUbc for viral pathogenesis in shrimp.

The male seahorse synthesizes and secretes a novel C-type lectin into the brood pouch during early pregnancy.

The male seahorse incubates its young in a manner resembling that of a mammalian pregnancy. After the female deposits her eggs into the male's brood pouch they are fertilized and the embryos develop and grow for several weeks until they are able to withstand the external environmental conditions independently, at which point they are irreversibly released. Although the precise function of the brood pouch is not clear, it is probably related to providing a suitable protective and osmotic environment for the young. The aim of this project was to construct and characterize a cDNA library made from the tissue lining the pouch, in order to help understand the molecular mechanisms regulating its development and function. The library profile indicates expression of genes encoding proteins involved in metabolism and transport, as well as structural proteins, gene regulatory proteins, and other proteins whose function is unknown. However, a large portion of the library contained genes encoding C-type lectins (CTLs), of which three full-length proteins were identified and found to contain a signal peptide and a single C-lectin domain, possessing all the conserved structural elements. We have produced recombinant protein for one of these and raised antisera; we have shown, using Western analysis and 2D electrophoresis, that this protein is secreted in significant quantities into the pouch fluid specifically during early pregnancy. Preliminary functional studies indicate that this CTL causes erythrocyte agglutination and may help to repress bacterial growth.