Development of functional resident macrophages in human pluripotent stem cell-derived colonic organoids and human fetal colon.

Most organs have tissue-resident immune cells. Human organoids lack these immune cells, which limits their utility in modeling many normal and disease processes. Here, we describe that pluripotent stem cell-derived human colonic organoids (HCOs) co-develop a diverse population of immune cells, including hemogenic endothelium (HE)-like cells and erythromyeloid progenitors that undergo stereotypical steps in differentiation, resulting in the generation of functional macrophages. HCO macrophages acquired a transcriptional signature resembling human fetal small and large intestine tissue-resident macrophages. HCO macrophages modulate cytokine secretion in response to pro- and anti-inflammatory signals and were able to phagocytose and mount a robust response to pathogenic bacteria. When transplanted into mice, HCO macrophages were maintained within the colonic organoid tissue, established a close association with the colonic epithelium, and were not displaced by the host bone-marrow-derived macrophages. These studies suggest that HE in HCOs gives rise to multipotent hematopoietic progenitors and functional tissue-resident macrophages.

Tissue Responses to Shiga Toxin in Human Intestinal Organoids.

BACKGROUND & AIMS: Shiga toxin (Stx)-producing Escherichia coli (eg, O157:H7) infection produces bloody diarrhea, while Stx inhibits protein synthesis and causes the life-threatening systemic complication of hemolytic uremic syndrome. The murine intestinal tract is resistant to O157:H7 and Stx, and human cells in culture fail to model the complex tissue responses to intestinal injury. We used genetically identical, human stem cell-derived intestinal tissues of varying complexity to study Stx toxicity in vitro and in vivo. METHODS: In vitro susceptibility to apical or basolateral exposure to Stx was assessed using human intestinal organoids (HIOs) derived from embryonic stem cells, or enteroids derived from multipotent intestinal stem cells. HIOs contain a lumen, with a single layer of differentiated epithelium surrounded by mesenchymal cells. Enteroids only contain epithelium. In vivo susceptibility was assessed using HIOs, with or without an enteric nervous system, transplanted into mice. RESULTS: Stx induced necrosis and apoptotic death in both epithelial and mesenchymal cells. Responses that require protein synthesis (cellular proliferation and wound repair) also were observed. Epithelial barrier function was maintained even after epithelial cell death was seen, and apical to basolateral translocation of Stx was seen. Tissue cross-talk, in which mesenchymal cell damage caused epithelial cell damage, was observed. Stx induced mesenchymal expression of the epithelial marker E-cadherin, the initial step in mesenchymal-epithelial transition. In vivo responses of HIO transplants injected with Stx mirrored those seen in vitro. CONCLUSIONS: Intestinal tissue responses to protein synthesis inhibition by Stx are complex. Organoid models allow for an unprecedented examination of human tissue responses to a deadly toxin.

Mot1, Ino80C, and NC2 Function Coordinately to Regulate Pervasive Transcription in Yeast and Mammals.

Pervasive transcription initiates from cryptic promoters and is observed in eukaryotes ranging from yeast to mammals. The Set2-Rpd3 regulatory system prevents cryptic promoter function within expressed genes. However, conserved systems that control pervasive transcription within intergenic regions have not been well established. Here we show that Mot1, Ino80 chromatin remodeling complex (Ino80C), and NC2 co-localize on chromatin and coordinately suppress pervasive transcription in S. cerevisiae and murine embryonic stem cells (mESCs). In yeast, all three proteins bind subtelomeric heterochromatin through a Sir3-stimulated mechanism and to euchromatin via a TBP-stimulated mechanism. In mESCs, the proteins bind to active and poised TBP-bound promoters along with promoters of polycomb-silenced genes apparently lacking TBP. Depletion of Mot1, Ino80C, or NC2 by anchor away in yeast or RNAi in mESCs leads to near-identical transcriptome phenotypes, with new subtelomeric transcription in yeast, and greatly increased pervasive transcription in both yeast and mESCs.

Shiga Toxin Mediated Neurologic Changes in Murine Model of Disease.

Seizures and neurologic involvement have been reported in patients infected with Shiga toxin (Stx) producing E. coli, and hemolytic uremic syndrome (HUS) with neurologic involvement is associated with more severe outcome. We investigated the extent of renal and neurologic damage in mice following injection of the highly potent form of Stx, Stx2a, and less potent Stx1. As observed in previous studies, Stx2a brought about moderate to acute tubular necrosis of proximal and distal tubules in the kidneys. Brain sections stained with hematoxylin and eosin (H&E) appeared normal, although some red blood cell congestion was observed. Microglial cell responses to neural injury include up-regulation of surface-marker expression (e.g., Iba1) and stereotypical morphological changes. Mice injected with Stx2a showed increased Iba1 staining, mild morphological changes associated with microglial activation (thickening of processes), and increased microglial staining per unit area. Microglial changes were observed in the cortex, hippocampus, and amygdala regions, but not the nucleus. Magnetic resonance imaging (MRI) of Stx2a-treated mice revealed no hyper-intensities in the brain, although magnetic resonance spectroscopy (MRS) revealed significantly decreased levels of phosphocreatine in the thalamus. Less dramatic changes were observed following Stx1 challenge. Neither immortalized microvascular endothelial cells from the cerebral cortex of mice (bEnd.3) nor primary human brain microvascular endothelial cells were found to be susceptible to Stx1 or Stx2a. The lack of susceptibility to Stx for both cell types correlated with an absence of receptor expression. These studies indicate Stx causes subtle, but identifiable changes in the mouse brain.

EP400 Deposits H3.3 into Promoters and Enhancers during Gene Activation.

Gene activation in metazoans is accompanied by the presence of histone variants H2AZ and H3.3 within promoters and enhancers. It is not known, however, what protein deposits H3.3 into chromatin or whether variant chromatin plays a direct role in gene activation. Here we show that chromatin containing acetylated H2AZ and H3.3 stimulates transcription in vitro. Analysis of the Pol II pre-initiation complex on immobilized chromatin templates revealed that the E1A binding protein p400 (EP400) was bound preferentially to and required for transcription stimulation by acetylated double-variant chromatin. EP400 also stimulated H2AZ/H3.3 deposition into promoters and enhancers and influenced transcription in vivo at a step downstream of the Mediator complex. EP400 efficiently exchanged recombinant histones H2A and H3.1 with H2AZ and H3.3, respectively, in a chromatin- and ATP-stimulated manner in vitro. Our data reveal that EP400 deposits H3.3 into chromatin alongside H2AZ and contributes to gene regulation after PIC assembly.

Molecular basis of recognition of quadruplexes human telomere and c-myc promoter by the putative anticancer agent sanguinarine.

BACKGROUND: Interaction of putative anticancer agent sanguinarine with two quadruplex forming sequences, human telomeric DNA (H24) and NHE III1 upstream of the P1 promoter of c-myc (Pu27), has been studied to understand the structural basis of the recognition. METHODS: Absorption, fluorescence and circular dichroism spectroscopy have been employed to characterize the association. Energetics of the interaction was studied by isothermal titration and differential scanning calorimetry. TRAP assay was done to assess the inhibitory potential of sanguinarine. RESULTS: Absorption and fluorescence studies show that sanguinarine has high binding affinity of ~10(5)M(-1) for both sequences. Binding stoichiometry is 2:1 for H24 and 3:1 for Pu27. Results suggest stacking interaction between planar sanguinarine moiety and G-quartets. Circular dichroism spectra show that sanguinarine does not cause structural perturbation in the all-parallel Pu27 but causes a structural transition from mixed hybrid to basket form at higher sanguinarine concentration in case of H24. The interaction is characterized by total enthalpy-entropy compensation and high heat capacity values. Differential scanning calorimetry studies suggest that sanguinarine binding increases the melting temperature and also the total enthalpy of transition of both quadruplexes. TRAP results show that sanguinarine effectively blocks telomerase activity in a concentration dependent manner in cell extracts from MDAMB-231 breast cancer cell lines. CONCLUSION: These results suggest that there is a difference in the structural modes of association of sanguinarine to the quadruplexes. GENERAL SIGNIFICANCE: It helps to understand the role of quadruplex structures as a target of small molecule inhibitors of telomerase.

Human telomere d[(TTAGGG)4] undergoes a conformational transition to the Na+-form upon binding with sanguinarine in presence of K+.

Guanine-rich telomeric sequences fold into G-quadruplex conformation and are known to bind a variety of ligands including potential drug candidates. By means of CD spectroscopy and fluorescence lifetime measurements we demonstrate that putative anticancer therapeutic sanguinarine (SGR) exhibits two distinct interactions with human telomere d[(TTAGGG)(4)] (H24) in presence of K(+). Up to about 1:2 M ratio of H24:SGR (10 µM H24), two molecules of SGR bind H24. Above this molar ratio, SGR induces a conformational transition in H24 from the K(+)-form to the Na(+)-form. The demonstration of SGR-induced conformational transition in a G-quadruplex formed by a human telomeric sequence could provide new insights into interaction of drugs with quadruplex DNA structure.

Mechanism of interaction of small transcription inhibitors with DNA in the context of chromatin and telomere.

Small molecules from natural and synthetic sources have long been employed as human drugs. The transcription inhibitory potential of one class of these molecules has paved their use as anticancer drugs. The principal mode of action of these molecules is via reversible interaction with genomic DNA, double and multiple stranded. In this article we have revisited the mechanism of the interaction in the context of chromatin and telomere. The established modes of association of these molecules with double helical DNA provide a preliminary mechanism of their transcription inhibitory potential, but the scenario assumes a different dimension when the genomic DNA is associated with proteins in the transcription apparatus of both prokaryotic and eukaryotic organisms. We have discussed this altered scenario as a prelude to understand the chemical biology of their action in the cell. For the telomeric quadruplex DNA, we have reviewed the mechanism of their association with the quadruplex and resultant cellular consequence.

Identification of a novel inhibitor of coactivator-associated arginine methyltransferase 1 (CARM1)-mediated methylation of histone H3 Arg-17.

Methylation of the arginine residues of histones by methyltransferases has important consequences for chromatin structure and gene regulation; however, the molecular mechanism(s) of methyltransferase regulation is still unclear, as is the biological significance of methylation at particular arginine residues. Here, we report a novel specific inhibitor of coactivator-associated arginine methyltransferase 1 (CARM1; also known as PRMT4) that selectively inhibits methylation at arginine 17 of histone H3 (H3R17). Remarkably, this plant-derived inhibitor, called TBBD (ellagic acid), binds to the substrate (histone) preferentially at the signature motif, "KAPRK," where the proline residue (Pro-16) plays a critical role for interaction and subsequent enzyme inhibition. In a promoter-specific context, inhibition of H3R17 methylation represses expression of p21, a p53-responsive gene, thus implicating a possible role for H3 Arg-17 methylation in tumor suppressor function. These data establish TBBD as a novel specific inhibitor of arginine methylation and demonstrate substrate sequence-directed inhibition of enzyme activity by a small molecule and its physiological consequence.

Inhibition of lysine acetyltransferase KAT3B/p300 activity by a naturally occurring hydroxynaphthoquinone, plumbagin.

Lysine acetyltransferases (KATs), p300 (KAT3B), and its close homologue CREB-binding protein (KAT3A) are probably the most widely studied KATs with well documented roles in various cellular processes. Hence, the dysfunction of p300 may result in the dysregulation of gene expression leading to the manifestation of many disorders. The acetyltransferase activity of p300/CREB-binding protein is therefore considered as a target for new generation therapeutics. We describe here a natural compound, plumbagin (RTK1), isolated from Plumbago rosea root extract, that inhibits histone acetyltransferase activity potently in vivo. Interestingly, RTK1 specifically inhibits the p300-mediated acetylation of p53 but not the acetylation by another acetyltransferase, p300/CREB-binding protein -associated factor, PCAF, in vivo. RTK1 inhibits p300 histone acetyltransferase activity in a noncompetitive manner. Docking studies and site-directed mutagenesis of the p300 histone acetyltransferase domain suggest that a single hydroxyl group of RTK1 makes a hydrogen bond with the lysine 1358 residue of this domain. In agreement with this, we found that indeed the hydroxyl group-substituted plumbagin derivatives lost the acetyltransferase inhibitory activity. This study describes for the first time the chemical entity (hydroxyl group) required for the inhibition of acetyltransferase activity.

Sanguinarine interacts with chromatin, modulates epigenetic modifications, and transcription in the context of chromatin.

DNA-binding anticancer agents cause alteration in chromatin structure and dynamics. We report the dynamic interaction of the DNA intercalator and potential anticancer plant alkaloid, sanguinarine (SGR), with chromatin. Association of SGR with different levels of chromatin structure was enthalpy driven with micromolar dissociation constant. Apart from DNA, it binds with comparable affinity with core histones and induces chromatin aggregation. The dual binding property of SGR leads to inhibition of core histone modifications. Although it potently inhibits H3K9 methylation by G9a in vitro, H3K4 and H3R17 methylation are more profoundly inhibited in cells. SGR inhibits histone acetylation both in vitro and in vivo. It does not affect the in vitro transcription from DNA template but significantly represses acetylation-dependent chromatin transcription. SGR-mediated repression of epigenetic marks and the alteration of chromatin geography (nucleography) also result in the modulation of global gene expression. These data, conclusively, show an anticancer DNA binding intercalator as a modulator of chromatin modifications and transcription in the chromatin context.

Mechanism of p300 specific histone acetyltransferase inhibition by small molecules.

Dysfunction of histone acetyltransferases (HATs) leads to several diseases including cancer, diabetes, and asthma. Therefore, small molecule inhibitors and activators of HATs are being considered as new generation therapeutics. Here, we report the molecular mechanisms of p300 HAT inhibition by specific and nonspecific HAT inhibitors: garcinol, isogarcinol, and 1 (LTK14). The p300 specific HAT inhibitor 1 behaves as a noncompetitive inhibitor for both acetyl-CoA and histone, unlike nonspecific HAT inhibitors garcinol and isogarcinol. The isothermal calorimetric data suggest that there is a high affinity enthalpy driven single binding site for 1 on p300HAT domain in contrast to two binding sites for garcinol and isogarcinol. Furthermore, the precise nature of molecular interactions was determined by using fluorescence, docking, and mutational studies. On the basis of these observations, we have proposed the mechanisms of specific versus nonspecific HAT inhibition by these small molecule compounds, which may be useful to design therapeutically favorable HAT inhibitors.

Chromatin as a target for the DNA-binding anticancer drugs.

Chemotherapy has been a major approach to treat cancer. Both constituents of chromatin, chromosomal DNA and the associated chromosomal histone proteins are the molecular targets of the anticancer drugs. Small DNA binding ligands, which inhibit enzymatic processes with DNA substrate, are well known in cancer chemotherapy. These drugs inhibit the polymerase and topoisomerase activity. With the advent in the knowledge of chromatin chemistry and biology, attempts have shifted from studies of the structural basis of the association of these drugs or small ligands (with the potential of drugs) with DNA to their association with chromatin and nucleosome. These drugs often inhibit the expression of specific genes leading to a series of biochemical events. An overview will be given about the latest understanding of the molecular basis of their action. We shall restrict to those drugs, synthetic or natural, whose prime cellular targets are so far known to be chromosomal DNA.

Marinomonas polaris sp. nov., a psychrohalotolerant strain isolated from coastal sea water off the subantarctic Kerguelen islands.

Two aerobic, psychrohalotolerant, motile bacterial isolates, CK13T and CK16, isolated from sea-water samples collected off the subantarctic Kerguelen island, were characterized by using a polyphasic taxonomic approach. On the basis of 16S rRNA gene sequence data, the strains were 99.6% similar and exhibited 93-97% similarity with the seven recognized species of Marinomonas. The most closely related species were Marinomonas pontica and Marinomonas primoryensis, with 97 and 96 % similarity at the 16S rRNA gene sequence level, respectively. DNA-DNA hybridization values between strain CK13T and M. pontica and M. primoryensis were only 58 and 40%, respectively. The major fatty acids present in strain CK13T were iso-C(16:0), C(16:0), C(16:1)omega7c and C(18:1)omega7c. The DNA G+C content of strain CK13T was 41.2 mol%. Phosphatidylethanolamine and phosphatidylglycerol were identified as the predominant phospholipids. All the above characteristics support the affiliation of strain CK13T to the genus Marinomonas. Phylogenetic analysis and phenotypic and genotypic distinctiveness confirmed that strains CK13T and CK16 are members of a novel species of the genus Marinomonas, for which the name Marinomonas polaris sp. nov. is proposed. The type strain is CK13T (=MTCC 6645T=DSM 16579T=JCM 12522T).