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2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly-adenosine diphosphate (ADP)-ribose polymerase (TIPARP/PARP7), an aryl hydrocarbon receptor (AHR) target gene and mono-ADP-ribosyltransferase, acts as part of a negative feedback loop to repress AHR signaling. This process is prevented by a single H532A mutation in TIPARP that destroys its catalytic activity. We hypothesized that the loss of TIPARP catalytic activity would increase sensitivity to TCDD-induced toxicity in vivo. To test this, we created a catalytically deficient mouse line (TiparpH532A) by introducing a single H532A mutation in TIPARP. Treatment of mouse embryonic fibroblasts or hepatocytes isolated from TiparpH532A mice confirmed the increased TCDD-induced expression of the AHR target genes Cyp1a1, Cyp1b1, and Tiparp. TiparpH532A mice given a single injection of 10 µg/kg TCDD, a nonlethal dose in Tiparp+/+ mice, did not survive beyond day 10. All Tiparp+/+ mice survived the 30-day treatment. TCDD-treated TiparpH532A mice displayed increased expression of AHR target genes, increased steatohepatitis and hepatotoxicity. Hepatic RNA-sequencing revealed 7-fold more differentially expressed genes in TiparpH532A mice than in Tiparp+/+ mice (4542 vs 647 genes) 6 days after TCDD treatment. Differentially expressed genes included genes involved in xenobiotic metabolism, lipid homeostasis and inflammation. Taken together, these data further support TIPARP as a critical negative regulator of AHR activity and show that loss of its catalytic activity is sufficient to increase sensitivity to TCDD-induced steatohepatitis and lethality. Since TIPARP inhibition has recently emerged as a potential anticancer therapy, the impact on AHR signaling, TCDD and polycyclic aromatic hydrocarbon toxicity will need to be carefully considered under conditions of therapeutic TIPARP inhibition.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly-ADP-ribose polymerase (TIPARP) is an enzyme that adds a single ADP-ribose moiety to itself or other proteins. Tiparp is highly expressed in the brain; however, its function in this organ is unknown. Here, we used Tiparp–/– mice to determine Tiparp’s role in the development of the prefrontal cortex., 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly-ADP-ribose polymerase (TIPARP) is an enzyme that adds a single ADP-ribose moiety to itself or other proteins. Tiparp is highly expressed in the brain; however, its function in this organ is unknown. Here, we used Tiparp–/– mice to determine Tiparp’s role in the development of the prefrontal cortex. Loss of Tiparp resulted in an aberrant organization of the mouse cortex, where the upper layers presented increased cell density in the knock-out mice compared with wild type. Tiparp loss predominantly affected the correct distribution and number of GABAergic neurons. Furthermore, neural progenitor cell proliferation was significantly reduced. Neural stem cells (NSCs) derived from Tiparp–/– mice showed a slower rate of migration. Cytoskeletal components, such as α-tubulin are key regulators of neuronal differentiation and cortical development. α-tubulin mono-ADP ribosylation (MAR) levels were reduced in Tiparp–/– cells, suggesting that Tiparp plays a role in the MAR of α-tubulin. Despite the mild phenotype presented by Tiparp–/– mice, our findings reveal an important function for Tiparp and MAR in the correct development of the cortex. Unravelling Tiparp’s role in the cortex, could pave the way to a better understanding of a wide spectrum of neurological diseases which are known to have increased expression of TIPARP.

TCDD-inducible poly-ADP-ribose polymerase (TIPARP; also known as PARP7 and ARTD14) is a mono-ADP-ribosyltransferase that has emerged as an important regulator of innate immunity, stem cell pluripotency, and transcription factor regulation. Characterizing TIPARP's catalytic activity and identifying its target proteins are critical to understanding its cellular function. Here we describe methods that we use to characterize TIPARP catalytic activity and its mono-ADP-ribosylation of its target proteins.

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of dioxin (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin; TCDD), which includes thymic atrophy, steatohepatitis, and a lethal wasting syndrome in laboratory rodents. Although the mechanisms of dioxin toxicity remain unknown, AHR signaling in hepatocytes is necessary for dioxin-induced liver toxicity. We previously reported that loss of TCDD-inducible poly(adenosine diphosphate [ADP]-ribose) polymerase (TIPARP/PARP7/ARTD14), an AHR target gene and mono-ADP-ribosyltransferase, increases the sensitivity of mice to dioxin-induced toxicities. To test the hypothesis that TIPARP is a negative regulator of AHR signaling in hepatocytes, we generated Tiparp(fl/fl) mice in which exon 3 of Tiparp is flanked by loxP sites, followed by Cre-lox technology to create hepatocyte-specific (Tiparp(fl/fl)Cre(Alb)) and whole-body (Tiparp(fl/fl)Cre(CMV); Tiparp(Ex3−/−)) Tiparp null mice. Tiparp(fl/fl)Cre(Alb) and Tiparp(Ex3−/−) mice given a single injection of 10 μg/kg dioxin did not survive beyond days 7 and 9, respectively, while all Tiparp(+/+) mice survived the 30-day treatment. Dioxin-exposed Tiparp(fl/fl)Cre(Alb) and Tiparp(Ex3−/−) mice had increased steatohepatitis and hepatotoxicity as indicated by greater staining of neutral lipids and serum alanine aminotransferase activity than similarly treated wild-type mice. Tiparp(fl/fl)Cre(Alb) and Tiparp(Ex3−/−) mice exhibited augmented AHR signaling, denoted by increased dioxin-induced gene expression. Metabolomic studies revealed alterations in lipid and amino acid metabolism in liver extracts from Tiparp(fl/fl)Cre(Alb) mice compared with wild-type mice. Taken together, these data illustrate that TIPARP is an important negative regulator of AHR activity, and that its specific loss in hepatocytes is sufficient to increase sensitivity to dioxin-induced steatohepatitis and lethality.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor and member of the basic helix-loop-helix-PAS family. AHR is activated by numerous dietary and endogenous compounds that contribute to its regulation of genes in diverse signaling pathways including xenobiotic metabolism, vascular development, immune responses and cell cycle control. However, it is most widely studied for its role in mediating 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity. The AHR target gene and mono-ADP-ribosyltransferase, TCDD-inducible poly-ADP-ribose polymerase (TIPARP), was recently shown to be part of a novel negative feedback loop regulating AHR activity through mono-ADP-ribosylation. However, the molecular characterization of how AHR regulates TIPARP remains elusive. Here we show that activated AHR is recruited to the TIPARP promoter, through its binding to two genomic regions that each contain multiple AHR response elements (AHREs), AHR regulates the expression of both TIPARP but also TIPARP-AS1, a long non-coding RNA (lncRNA) which lies upstream of TIPARP exon 1 and is expressed in the opposite orientation. Reporter gene and deletion studies showed that the distal AHRE cluster predominantly regulated TIPARP expression while the proximal cluster regulated TIPARP-AS1. Moreover, time course and promoter activity assays suggest that TIPARP and TIPARP-AS1 work in concert to regulate AHR signaling. Collectively, these data show an added level of complexity in the AHR signaling cascade which involves lncRNAs, whose functions remain poorly understood.

Differentiation of human endometrial stromal cells (HESC) into decidual cells represents a highly coordinated process essential for embryo implantation. We show that decidualizing HESC down-regulate the histone methyltransferase enhancer of Zeste homolog 2 (EZH2), resulting in declining levels of trimethylation of histone 3 on lysine 27 (H3K27me3) at the proximal promoters of key decidual marker genes PRL and IGFBP1. Loss of H3K27me3 was associated with a reciprocal enrichment in acetylation of the same lysine residue, indicating active remodeling from repressive to transcriptionally permissive chromatin. Chromatin immunoprecipitation coupled with DNA microarray analysis demonstrated that decidualization triggers genome-wide changes in H3K27me3 distribution that only partly overlap those observed upon EZH2 knockdown in undifferentiated HESC. Gene ontology revealed that gain of the repressive H3K27me3 mark in response to decidualization and upon EZH2 knockdown in undifferentiated cells was enriched at the promoter regions of genes involved in transcriptional regulation and growth/cell proliferation, respectively. However, loss of the H3K27me3 mark (indicating increased chromatin accessibility) in decidualizing cells and upon EZH2 knockdown occurred at selective loci enriched for genes functionally implicated in responses to stimulus. In agreement, EZH2 knockdown in undifferentiated HESC was sufficient to augment the induction of decidual marker genes in response to cyclic AMP and progesterone signaling. Thus, loss of EZH2-dependent methyltransferase activity in the endometrium is integral to the process of chromatin remodeling that enables the transition from a proliferative to a decidual phenotype in response to differentiation cues.

© 2015 AlphaMed Press.Menstruation drives cyclic activation of endometrial progenitor cells, tissue regeneration, and maturation of stromal cells, which differentiate into specialized decidual cells prior to and during pregnancy. Aberrant responsiveness of human endometrial stromal cells (HESCs) to deciduogenic cues is strongly associated with recurrent pregnancy loss (RPL), suggesting a defect in cellular maturation. MeDIP-seq analysis of HESCs did not reveal gross perturbations in CpG methylation in RPL cultures, although quantitative differences were observed in or near genes that are frequently deregulated in vivo. However, RPL was associated with a marked reduction in methylation of defined CA-rich motifs located throughout the genome but enriched near telomeres. Non-CpG methylation is a hallmark of cellular multipotency. Congruently, we demonstrate that RPL is associated with a deficiency in endometrial clonogenic cell populations. Loss of epigenetic stemness features also correlated with intragenic CpG hypomethylation and reduced expression of HMGB2, coding high mobility group protein 2. We show that knockdown of this sequence-independent chromatin protein in HESCs promotes senescence and impairs decidualization, exemplified by blunted time-dependent secretome changes. Our findings indicate that stem cell deficiency and accelerated stromal senescence limit the differentiation capacity of the endometrium and predispose for pregnancy failure. Stem Cells 2016;34:346-356 Recurrent pregnancy loss is caused by endometrial stem cell deficiency, triggering heightened tissue senescence and impaired decidualization.

Cyclic differentiation of human endometrial stromal cells (HESCs) into decidual cells is a highly coordinated process essential for embryo implantation and pregnancy. This differentiation process is closely recapitulated in culture upon exposure of purified HESCs to cyclic AMP and progesterone signaling. Mining of gene expression data revealed that HESCs express 147 genes coding for epigenetic effectors, 33 (22%) of which are significantly regulated (P < 0.05) upon decidualization. Among these are genes encoding for histone-modifying proteins and their cofactors, histone-binding proteins, histone variants, CpG-binding proteins and DNA methyltransferases (DNMTs). Interestingly, more than two-thirds of differentially expressed chromatin-modifying genes are down-regulated upon the transition from a proliferative to a differentiated HESC phenotype. Despite the strong regulation of DNMTs, colorimetric and long interspersed nuclear element 1 methylation assays did not show global changes in DNA methylation levels upon differentiation of HESCs. Taken together, the coordinated regulation of diverse effector molecules suggests that complex epigenetic modification at specific loci underpins the acquisition of a decidual endometrial phenotype.

Bacterial flagella play key roles in surface attachment and host-bacterial interactions as well as driving motility. Here, we have investigated the ability of Caulobacter crescentus to assemble its flagellar filament from six flagellins: FljJ, FljK, FljL, FljM, FljN, and FljO. Flagellin gene deletion combinations exhibited a range of phenotypes from no motility or impaired motility to full motility. Characterization of the mutant collection showed the following: (i) that there is no strict requirement for any one of the six flagellins to assemble a filament; (ii) that there is a correlation between slower swimming speeds and shorter filament lengths in Δ fljK Δ fljM mutants; (iii) that the flagellins FljM to FljO are less stable than FljJ to FljL; and (iv) that the flagellins FljK, FljL, FljM, FljN, and FljO alone are able to assemble a filament.