Limits...
The TrxG Complex Mediates Cytokine Induced De Novo Enhancer Formation in Islets.

Tennant BR, Hurley P, Dhillon J, Gill A, Whiting C, Hoffman BG - PLoS ONE (2015)

Bottom Line: Roughly 10% of the de novo and increased regions were enriched for the repressive histone modification histone 3 lysine 27 trimethylation (H3K27me3) in untreated cells, and these were frequently associated with chemokine genes.We show that GSK-J4 is particularly effective in blunting IFNγ, Il-1β, and TNFα-induced chemokine gene expression in β-cells; however, it induced significant islet-cell apoptosis and β-cell dysfunction.Taken together these data suggest that the TrxG complex is potentially a viable target for preventing cytokine induced chemokine gene expression in β-cells.

View Article: PubMed Central - PubMed

Affiliation: Child and Family Research Institute, British Columbia Children's Hospital and Sunny Hill Health Centre, 950 W28th Avenue, Vancouver, British Columbia, Canada.

ABSTRACT
To better understand how β-cells respond to proinflammatory cytokines we mapped the locations of histone 3 lysine 4 monomethylation (H3K4me1), a post-translational histone modification enriched at active and poised cis-regulatory regions, in IFNγ, Il-1β, and TNFα treated pancreatic islets. We identified 96,721 putative cis-regulatory loci, of which 3,590 were generated de novo, 3,204 had increased H3K4me1, and 5,354 had decreased H3K4me1 in IFNγ, Il-1β, and TNFα exposed islets. Roughly 10% of the de novo and increased regions were enriched for the repressive histone modification histone 3 lysine 27 trimethylation (H3K27me3) in untreated cells, and these were frequently associated with chemokine genes. We show that IFNγ, Il-1β, and TNFα exposure overcomes this repression and induces chemokine gene activation in as little as three hours, and that this expression persists for days in absence of continued IFNγ, Il-1β, and TNFα exposure. We implicate trithorax group (TrxG) complexes as likely players in the conversion of these repressed loci to an active state. To block the activity of these complexes, we suppressed Wdr5, a core component of the TrxG complexes, and used the H3K27me3 demethylase inhibitor GSK-J4. We show that GSK-J4 is particularly effective in blunting IFNγ, Il-1β, and TNFα-induced chemokine gene expression in β-cells; however, it induced significant islet-cell apoptosis and β-cell dysfunction. Wdr5 suppression also reduced IFNγ, Il-1β, and TNFα induced chemokine gene expression in β-cells without affecting islet-cell survival or β-cell function after 48hrs, but did begin to increase islet-cell apoptosis and β-cell dysfunction after four days of treatment. Taken together these data suggest that the TrxG complex is potentially a viable target for preventing cytokine induced chemokine gene expression in β-cells.

No MeSH data available.


Related in: MedlinePlus

The small molecule GSK-J4, an inhibitor of Jmjd3 and Utx, significantly blunts IFNγ, Il-1β, and TNFα-induced chemokine gene expression in islets.(A) A dose-response curve of Ccl2 expression in islets exposed a titration of concentrations of GSK-J4 as determined by qPCR. (B) Expression of selected chemokine and cytokine genes in islets exposed to 20μM GSK-J4 or a vehicle control (DMSO) and simultaneously untreated or treated with IFNγ, Il-1β, and TNFα (cytokine) as determined by qPCR. (C) Expression of selected genes involved in regulating β-cell function in islets exposed to 20μM GSK-J4 or a vehicle control (DMSO) and simultaneously untreated or treated with IFNγ, Il-1β, and TNFα (cytokine) as determined by qPCR. (D) Glucose-induced insulin secretion assays of islets exposed to 20μM GSK-J4 or a vehicle control (DMSO) and simultaneously untreated or treated with IFNγ, Il-1β, and TNFα (cytokine) as determined by qPCR. (E) Propidium iodide (PI) incorporation in islets exposed to 20μM GSK-J4 or a vehicle control (DMSO) and simultaneously untreated or treated with IFNγ, Il-1β, and TNFα (cytokine) as determined by qPCR. (F) Representative images of islets treated with DMSO or GSK for three and six hours respectively. PI positive cells are marked in red.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4623983&req=5

pone.0141470.g008: The small molecule GSK-J4, an inhibitor of Jmjd3 and Utx, significantly blunts IFNγ, Il-1β, and TNFα-induced chemokine gene expression in islets.(A) A dose-response curve of Ccl2 expression in islets exposed a titration of concentrations of GSK-J4 as determined by qPCR. (B) Expression of selected chemokine and cytokine genes in islets exposed to 20μM GSK-J4 or a vehicle control (DMSO) and simultaneously untreated or treated with IFNγ, Il-1β, and TNFα (cytokine) as determined by qPCR. (C) Expression of selected genes involved in regulating β-cell function in islets exposed to 20μM GSK-J4 or a vehicle control (DMSO) and simultaneously untreated or treated with IFNγ, Il-1β, and TNFα (cytokine) as determined by qPCR. (D) Glucose-induced insulin secretion assays of islets exposed to 20μM GSK-J4 or a vehicle control (DMSO) and simultaneously untreated or treated with IFNγ, Il-1β, and TNFα (cytokine) as determined by qPCR. (E) Propidium iodide (PI) incorporation in islets exposed to 20μM GSK-J4 or a vehicle control (DMSO) and simultaneously untreated or treated with IFNγ, Il-1β, and TNFα (cytokine) as determined by qPCR. (F) Representative images of islets treated with DMSO or GSK for three and six hours respectively. PI positive cells are marked in red.

Mentions: Our data above indicated that targeting the TrxG complex to prevent IFNγ, Il-1β, and TNFα-induced chromatin state changes may be a promising approach to prevent cytokine induced chemokine and cytokine gene expression in β-cells. To seek a more effective approach we next used the ethyl ester pro-drug GSK-J4 that is converted to GSK-J1, a selective inhibitor of Jmjd3 and Utx [58,59], within the cells. We hypothesized this approach might be effective as we had determined that many of the cis-regulatory loci adjacent to the cytokine induced chemokine and cytokine genes are normally in a H3K27me3 enriched, repressive or bivalent state, and thus require H3K27me3 removal for activation. We first performed a dose-response curve and found that 20μM GSK-J4 resulted in greater than 90% suppression of IFNγ, Il-1β, and TNFα-induced Ccl2 gene expression in islets (Fig 8A). Using this dose on islets treated with IFNγ, Il-1β, and TNFα for three hours resulted in highly significant reductions in the induction of Ccl2 (15.6 fold, p<0.001), Ccl20 (8.0 fold, p<0.01), Cxcl10 (22.3 fold, p<0.001), Cxcl11 (37.9 fold, p<0.001), Cxcl9 (4.8 fold, p<0.05), Il-15 (9.1 fold, p<0.01), and in iNos (33.8 fold, p<0.001) (Fig 8B). Further, exposure of islets to this dose of GSK-J4 for three hours, in the presence or absence of IFNγ, Il-1β, and TNFα had no significant effect on the expression of genes involved in β-cell function (Fig 8C). However, it did significantly decrease glucose-stimulated insulin secretion (Fig 8D). Also, although GSK-J4 did not increase islet cell apoptosis after three hours (Fig 8E and 8F), after six hours of exposure to GSK-J4 almost all the islet-cells were PI positive suggesting they had undergone apoptosis (Fig 8F). Lower concentrations of GSK-J4 (down to 2μM) also resulted in significant cell death after six hours (S2 Fig) indicating the use of lower doses of GSK-J4 for longer treatment periods is not a viable approach to reduce IFNγ, Il-1β, and TNFα-induced chemokine gene expression in islets. Despite this, these results further confirm that targeting the TrxG complex may be a useful approach to prevent cytokine induced chemokine and cytokine gene expression in β-cells; however, alternative mechanisms need to be sought.


The TrxG Complex Mediates Cytokine Induced De Novo Enhancer Formation in Islets.

Tennant BR, Hurley P, Dhillon J, Gill A, Whiting C, Hoffman BG - PLoS ONE (2015)

The small molecule GSK-J4, an inhibitor of Jmjd3 and Utx, significantly blunts IFNγ, Il-1β, and TNFα-induced chemokine gene expression in islets.(A) A dose-response curve of Ccl2 expression in islets exposed a titration of concentrations of GSK-J4 as determined by qPCR. (B) Expression of selected chemokine and cytokine genes in islets exposed to 20μM GSK-J4 or a vehicle control (DMSO) and simultaneously untreated or treated with IFNγ, Il-1β, and TNFα (cytokine) as determined by qPCR. (C) Expression of selected genes involved in regulating β-cell function in islets exposed to 20μM GSK-J4 or a vehicle control (DMSO) and simultaneously untreated or treated with IFNγ, Il-1β, and TNFα (cytokine) as determined by qPCR. (D) Glucose-induced insulin secretion assays of islets exposed to 20μM GSK-J4 or a vehicle control (DMSO) and simultaneously untreated or treated with IFNγ, Il-1β, and TNFα (cytokine) as determined by qPCR. (E) Propidium iodide (PI) incorporation in islets exposed to 20μM GSK-J4 or a vehicle control (DMSO) and simultaneously untreated or treated with IFNγ, Il-1β, and TNFα (cytokine) as determined by qPCR. (F) Representative images of islets treated with DMSO or GSK for three and six hours respectively. PI positive cells are marked in red.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4623983&req=5

pone.0141470.g008: The small molecule GSK-J4, an inhibitor of Jmjd3 and Utx, significantly blunts IFNγ, Il-1β, and TNFα-induced chemokine gene expression in islets.(A) A dose-response curve of Ccl2 expression in islets exposed a titration of concentrations of GSK-J4 as determined by qPCR. (B) Expression of selected chemokine and cytokine genes in islets exposed to 20μM GSK-J4 or a vehicle control (DMSO) and simultaneously untreated or treated with IFNγ, Il-1β, and TNFα (cytokine) as determined by qPCR. (C) Expression of selected genes involved in regulating β-cell function in islets exposed to 20μM GSK-J4 or a vehicle control (DMSO) and simultaneously untreated or treated with IFNγ, Il-1β, and TNFα (cytokine) as determined by qPCR. (D) Glucose-induced insulin secretion assays of islets exposed to 20μM GSK-J4 or a vehicle control (DMSO) and simultaneously untreated or treated with IFNγ, Il-1β, and TNFα (cytokine) as determined by qPCR. (E) Propidium iodide (PI) incorporation in islets exposed to 20μM GSK-J4 or a vehicle control (DMSO) and simultaneously untreated or treated with IFNγ, Il-1β, and TNFα (cytokine) as determined by qPCR. (F) Representative images of islets treated with DMSO or GSK for three and six hours respectively. PI positive cells are marked in red.
Mentions: Our data above indicated that targeting the TrxG complex to prevent IFNγ, Il-1β, and TNFα-induced chromatin state changes may be a promising approach to prevent cytokine induced chemokine and cytokine gene expression in β-cells. To seek a more effective approach we next used the ethyl ester pro-drug GSK-J4 that is converted to GSK-J1, a selective inhibitor of Jmjd3 and Utx [58,59], within the cells. We hypothesized this approach might be effective as we had determined that many of the cis-regulatory loci adjacent to the cytokine induced chemokine and cytokine genes are normally in a H3K27me3 enriched, repressive or bivalent state, and thus require H3K27me3 removal for activation. We first performed a dose-response curve and found that 20μM GSK-J4 resulted in greater than 90% suppression of IFNγ, Il-1β, and TNFα-induced Ccl2 gene expression in islets (Fig 8A). Using this dose on islets treated with IFNγ, Il-1β, and TNFα for three hours resulted in highly significant reductions in the induction of Ccl2 (15.6 fold, p<0.001), Ccl20 (8.0 fold, p<0.01), Cxcl10 (22.3 fold, p<0.001), Cxcl11 (37.9 fold, p<0.001), Cxcl9 (4.8 fold, p<0.05), Il-15 (9.1 fold, p<0.01), and in iNos (33.8 fold, p<0.001) (Fig 8B). Further, exposure of islets to this dose of GSK-J4 for three hours, in the presence or absence of IFNγ, Il-1β, and TNFα had no significant effect on the expression of genes involved in β-cell function (Fig 8C). However, it did significantly decrease glucose-stimulated insulin secretion (Fig 8D). Also, although GSK-J4 did not increase islet cell apoptosis after three hours (Fig 8E and 8F), after six hours of exposure to GSK-J4 almost all the islet-cells were PI positive suggesting they had undergone apoptosis (Fig 8F). Lower concentrations of GSK-J4 (down to 2μM) also resulted in significant cell death after six hours (S2 Fig) indicating the use of lower doses of GSK-J4 for longer treatment periods is not a viable approach to reduce IFNγ, Il-1β, and TNFα-induced chemokine gene expression in islets. Despite this, these results further confirm that targeting the TrxG complex may be a useful approach to prevent cytokine induced chemokine and cytokine gene expression in β-cells; however, alternative mechanisms need to be sought.

Bottom Line: Roughly 10% of the de novo and increased regions were enriched for the repressive histone modification histone 3 lysine 27 trimethylation (H3K27me3) in untreated cells, and these were frequently associated with chemokine genes.We show that GSK-J4 is particularly effective in blunting IFNγ, Il-1β, and TNFα-induced chemokine gene expression in β-cells; however, it induced significant islet-cell apoptosis and β-cell dysfunction.Taken together these data suggest that the TrxG complex is potentially a viable target for preventing cytokine induced chemokine gene expression in β-cells.

View Article: PubMed Central - PubMed

Affiliation: Child and Family Research Institute, British Columbia Children's Hospital and Sunny Hill Health Centre, 950 W28th Avenue, Vancouver, British Columbia, Canada.

ABSTRACT
To better understand how β-cells respond to proinflammatory cytokines we mapped the locations of histone 3 lysine 4 monomethylation (H3K4me1), a post-translational histone modification enriched at active and poised cis-regulatory regions, in IFNγ, Il-1β, and TNFα treated pancreatic islets. We identified 96,721 putative cis-regulatory loci, of which 3,590 were generated de novo, 3,204 had increased H3K4me1, and 5,354 had decreased H3K4me1 in IFNγ, Il-1β, and TNFα exposed islets. Roughly 10% of the de novo and increased regions were enriched for the repressive histone modification histone 3 lysine 27 trimethylation (H3K27me3) in untreated cells, and these were frequently associated with chemokine genes. We show that IFNγ, Il-1β, and TNFα exposure overcomes this repression and induces chemokine gene activation in as little as three hours, and that this expression persists for days in absence of continued IFNγ, Il-1β, and TNFα exposure. We implicate trithorax group (TrxG) complexes as likely players in the conversion of these repressed loci to an active state. To block the activity of these complexes, we suppressed Wdr5, a core component of the TrxG complexes, and used the H3K27me3 demethylase inhibitor GSK-J4. We show that GSK-J4 is particularly effective in blunting IFNγ, Il-1β, and TNFα-induced chemokine gene expression in β-cells; however, it induced significant islet-cell apoptosis and β-cell dysfunction. Wdr5 suppression also reduced IFNγ, Il-1β, and TNFα induced chemokine gene expression in β-cells without affecting islet-cell survival or β-cell function after 48hrs, but did begin to increase islet-cell apoptosis and β-cell dysfunction after four days of treatment. Taken together these data suggest that the TrxG complex is potentially a viable target for preventing cytokine induced chemokine gene expression in β-cells.

No MeSH data available.


Related in: MedlinePlus