Limits...
Ikaros mediates the DNA methylation-independent silencing of MCJ/DNAJC15 gene expression in macrophages.

Navasa N, Martin-Ruiz I, Atondo E, Sutherland JD, Angel Pascual-Itoiz M, Carreras-González A, Izadi H, Tomás-Cortázar J, Ayaz F, Martin-Martin N, Torres IM, Barrio R, Carracedo A, Olivera ER, Rincón M, Anguita J - Sci Rep (2015)

Bottom Line: IFNγ, a protective cytokine against cardiac inflammation during Lyme borreliosis, represses MCJ transcription in macrophages.The transcriptional regulator, Ikaros, binds to the MCJ promoter in a Casein kinase II-dependent manner, and mediates the repression of MCJ expression.These results identify the MCJ gene as a transcriptional target of IFNγ and provide evidence of the dynamic adaptation of normal tissues to changes in the environment as a way to adapt metabolically to new conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary and Animal Sciences. University of Massachusetts Amherst. Amherst, MA 01003.

ABSTRACT
MCJ (DNAJC15) is a mitochondrial protein that regulates the mitochondrial metabolic status of macrophages and their response to inflammatory stimuli. CpG island methylation in cancer cells constitutes the only mechanism identified for the regulation of MCJ gene expression. However, whether DNA methylation or transcriptional regulation mechanisms are involved in the physiological control of this gene expression in non-tumor cells remains unknown. We now demonstrate a mechanism of regulation of MCJ expression that is independent of DNA methylation. IFNγ, a protective cytokine against cardiac inflammation during Lyme borreliosis, represses MCJ transcription in macrophages. The transcriptional regulator, Ikaros, binds to the MCJ promoter in a Casein kinase II-dependent manner, and mediates the repression of MCJ expression. These results identify the MCJ gene as a transcriptional target of IFNγ and provide evidence of the dynamic adaptation of normal tissues to changes in the environment as a way to adapt metabolically to new conditions.

No MeSH data available.


Related in: MedlinePlus

IFNγ induces the binding of Ikaros to the MCJ promoter through the activation of CK2.(A) Chromatin immunoprecipitation using an antibody specific for Ikaros. BMMs were stimulated with 100 ng/mL of IFNγ for 16 h or left unstimulated. Chromatin was processed and immunoprecipitated as described in Experimental Procedures. The values presented correspond to one experiment of 2 with similar results. (B) Luciferase activity driven by the 1 kb MCJ proximal promoter (MCJ-luc) and deletion mutants corresponding to Site 1 (ΔIk#1), Site 2 (ΔIk#2) or double mutants (ΔIK#1/2). The values correspond to the mean ± SE of triplicates and represent at least 3 independent experiments. *; Student´s T test, p < 0.05; NS; not significant. (C) Western blot showing silencing of Ikaros in RAW cells stably transduced with lentiviral particles containing a short hairpin (sh) specific for the Ikaros gene (shIkzf1). GAPDH levels were determined to ensure equal loads. (D) shIkaros cells and pLK0-transduced control cells were stimulated with 100 ng/mL of IFNγ or left stimulated for 24 h. The levels of MCJ were then determined by immunoblotting. (E) Levels of Ikaros in BMMs unstimulated or stimulated with 100 ng/mL of IFNγ for 24 h. The cells were l tested by immunoblotting using specific Ikaros antibodies. GAPDH levels were determined to ensure equal loads. (F) BMMs were stimulated with IFNγ in the presence or absence of TBB as before, and Ikaros binding was determined by chromatin immunoprecipitation. The values correspond to 1 of 2 experiments performed with similar results. (G) RAW cells and BMMs were stimulated with 100 ng/mL of IFNγ in the presence or absence of 50 μM of the CK2 inhibitor, 4,5,6,7-Tetrabromobenzotriazole (TBB), for 24 h, followed by MCJ protein level determination by immunoblotting. (G) RAW cells were co-transfected with the plasmids pGL3-MCJ-Luc plus pSV40-RenillaLuc. Four h later, the cells were stimulated with 100 ng/mL of IFNγ in the presence or absence of 50 μM TBB and 16 h later, assessed for luciferase levels. The values correspond to luciferase activity relative to Renilla luciferase in triplicate (mean ± SE) and represent one of at least 4 experiments performed. *; Student´s T test, p < 0.05; NS; not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4588509&req=5

f4: IFNγ induces the binding of Ikaros to the MCJ promoter through the activation of CK2.(A) Chromatin immunoprecipitation using an antibody specific for Ikaros. BMMs were stimulated with 100 ng/mL of IFNγ for 16 h or left unstimulated. Chromatin was processed and immunoprecipitated as described in Experimental Procedures. The values presented correspond to one experiment of 2 with similar results. (B) Luciferase activity driven by the 1 kb MCJ proximal promoter (MCJ-luc) and deletion mutants corresponding to Site 1 (ΔIk#1), Site 2 (ΔIk#2) or double mutants (ΔIK#1/2). The values correspond to the mean ± SE of triplicates and represent at least 3 independent experiments. *; Student´s T test, p < 0.05; NS; not significant. (C) Western blot showing silencing of Ikaros in RAW cells stably transduced with lentiviral particles containing a short hairpin (sh) specific for the Ikaros gene (shIkzf1). GAPDH levels were determined to ensure equal loads. (D) shIkaros cells and pLK0-transduced control cells were stimulated with 100 ng/mL of IFNγ or left stimulated for 24 h. The levels of MCJ were then determined by immunoblotting. (E) Levels of Ikaros in BMMs unstimulated or stimulated with 100 ng/mL of IFNγ for 24 h. The cells were l tested by immunoblotting using specific Ikaros antibodies. GAPDH levels were determined to ensure equal loads. (F) BMMs were stimulated with IFNγ in the presence or absence of TBB as before, and Ikaros binding was determined by chromatin immunoprecipitation. The values correspond to 1 of 2 experiments performed with similar results. (G) RAW cells and BMMs were stimulated with 100 ng/mL of IFNγ in the presence or absence of 50 μM of the CK2 inhibitor, 4,5,6,7-Tetrabromobenzotriazole (TBB), for 24 h, followed by MCJ protein level determination by immunoblotting. (G) RAW cells were co-transfected with the plasmids pGL3-MCJ-Luc plus pSV40-RenillaLuc. Four h later, the cells were stimulated with 100 ng/mL of IFNγ in the presence or absence of 50 μM TBB and 16 h later, assessed for luciferase levels. The values correspond to luciferase activity relative to Renilla luciferase in triplicate (mean ± SE) and represent one of at least 4 experiments performed. *; Student´s T test, p < 0.05; NS; not significant.

Mentions: To identify the specific mechanism by which IFNγ represses MCJ gene transcription, we performed a search for potential transcription factor binding sites within the 1kb region of the mouse MCJ gene promoter using the tool TFSearch19. Two consensus binding sites for Ikaros (−350 to −361 and −706 to −717) were identified (Fig. S2A). Ikaros is known to act primarily as a repressor of gene expression20. To demonstrate whether Ikaros binds to these putative binding sites in the MCJ promoter and address whether binding was regulated by IFNγ, we performed chromatin immunoprecipitation (ChIP) assays in BMMs. Binding of Ikaros to both sites was almost undetectable in untreated BMMs (Fig. 4A). However, Ikaros binding to Site 1 (the most proximal to the transcription start site; Fig. S2A) was highly induced in cells treated with IFNγ (Fig. 4A). Ikaros binding to Site 2 (Fig. S2A), however, was not induced by IFNγ (Fig. 4A).


Ikaros mediates the DNA methylation-independent silencing of MCJ/DNAJC15 gene expression in macrophages.

Navasa N, Martin-Ruiz I, Atondo E, Sutherland JD, Angel Pascual-Itoiz M, Carreras-González A, Izadi H, Tomás-Cortázar J, Ayaz F, Martin-Martin N, Torres IM, Barrio R, Carracedo A, Olivera ER, Rincón M, Anguita J - Sci Rep (2015)

IFNγ induces the binding of Ikaros to the MCJ promoter through the activation of CK2.(A) Chromatin immunoprecipitation using an antibody specific for Ikaros. BMMs were stimulated with 100 ng/mL of IFNγ for 16 h or left unstimulated. Chromatin was processed and immunoprecipitated as described in Experimental Procedures. The values presented correspond to one experiment of 2 with similar results. (B) Luciferase activity driven by the 1 kb MCJ proximal promoter (MCJ-luc) and deletion mutants corresponding to Site 1 (ΔIk#1), Site 2 (ΔIk#2) or double mutants (ΔIK#1/2). The values correspond to the mean ± SE of triplicates and represent at least 3 independent experiments. *; Student´s T test, p < 0.05; NS; not significant. (C) Western blot showing silencing of Ikaros in RAW cells stably transduced with lentiviral particles containing a short hairpin (sh) specific for the Ikaros gene (shIkzf1). GAPDH levels were determined to ensure equal loads. (D) shIkaros cells and pLK0-transduced control cells were stimulated with 100 ng/mL of IFNγ or left stimulated for 24 h. The levels of MCJ were then determined by immunoblotting. (E) Levels of Ikaros in BMMs unstimulated or stimulated with 100 ng/mL of IFNγ for 24 h. The cells were l tested by immunoblotting using specific Ikaros antibodies. GAPDH levels were determined to ensure equal loads. (F) BMMs were stimulated with IFNγ in the presence or absence of TBB as before, and Ikaros binding was determined by chromatin immunoprecipitation. The values correspond to 1 of 2 experiments performed with similar results. (G) RAW cells and BMMs were stimulated with 100 ng/mL of IFNγ in the presence or absence of 50 μM of the CK2 inhibitor, 4,5,6,7-Tetrabromobenzotriazole (TBB), for 24 h, followed by MCJ protein level determination by immunoblotting. (G) RAW cells were co-transfected with the plasmids pGL3-MCJ-Luc plus pSV40-RenillaLuc. Four h later, the cells were stimulated with 100 ng/mL of IFNγ in the presence or absence of 50 μM TBB and 16 h later, assessed for luciferase levels. The values correspond to luciferase activity relative to Renilla luciferase in triplicate (mean ± SE) and represent one of at least 4 experiments performed. *; Student´s T test, p < 0.05; NS; not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588509&req=5

f4: IFNγ induces the binding of Ikaros to the MCJ promoter through the activation of CK2.(A) Chromatin immunoprecipitation using an antibody specific for Ikaros. BMMs were stimulated with 100 ng/mL of IFNγ for 16 h or left unstimulated. Chromatin was processed and immunoprecipitated as described in Experimental Procedures. The values presented correspond to one experiment of 2 with similar results. (B) Luciferase activity driven by the 1 kb MCJ proximal promoter (MCJ-luc) and deletion mutants corresponding to Site 1 (ΔIk#1), Site 2 (ΔIk#2) or double mutants (ΔIK#1/2). The values correspond to the mean ± SE of triplicates and represent at least 3 independent experiments. *; Student´s T test, p < 0.05; NS; not significant. (C) Western blot showing silencing of Ikaros in RAW cells stably transduced with lentiviral particles containing a short hairpin (sh) specific for the Ikaros gene (shIkzf1). GAPDH levels were determined to ensure equal loads. (D) shIkaros cells and pLK0-transduced control cells were stimulated with 100 ng/mL of IFNγ or left stimulated for 24 h. The levels of MCJ were then determined by immunoblotting. (E) Levels of Ikaros in BMMs unstimulated or stimulated with 100 ng/mL of IFNγ for 24 h. The cells were l tested by immunoblotting using specific Ikaros antibodies. GAPDH levels were determined to ensure equal loads. (F) BMMs were stimulated with IFNγ in the presence or absence of TBB as before, and Ikaros binding was determined by chromatin immunoprecipitation. The values correspond to 1 of 2 experiments performed with similar results. (G) RAW cells and BMMs were stimulated with 100 ng/mL of IFNγ in the presence or absence of 50 μM of the CK2 inhibitor, 4,5,6,7-Tetrabromobenzotriazole (TBB), for 24 h, followed by MCJ protein level determination by immunoblotting. (G) RAW cells were co-transfected with the plasmids pGL3-MCJ-Luc plus pSV40-RenillaLuc. Four h later, the cells were stimulated with 100 ng/mL of IFNγ in the presence or absence of 50 μM TBB and 16 h later, assessed for luciferase levels. The values correspond to luciferase activity relative to Renilla luciferase in triplicate (mean ± SE) and represent one of at least 4 experiments performed. *; Student´s T test, p < 0.05; NS; not significant.
Mentions: To identify the specific mechanism by which IFNγ represses MCJ gene transcription, we performed a search for potential transcription factor binding sites within the 1kb region of the mouse MCJ gene promoter using the tool TFSearch19. Two consensus binding sites for Ikaros (−350 to −361 and −706 to −717) were identified (Fig. S2A). Ikaros is known to act primarily as a repressor of gene expression20. To demonstrate whether Ikaros binds to these putative binding sites in the MCJ promoter and address whether binding was regulated by IFNγ, we performed chromatin immunoprecipitation (ChIP) assays in BMMs. Binding of Ikaros to both sites was almost undetectable in untreated BMMs (Fig. 4A). However, Ikaros binding to Site 1 (the most proximal to the transcription start site; Fig. S2A) was highly induced in cells treated with IFNγ (Fig. 4A). Ikaros binding to Site 2 (Fig. S2A), however, was not induced by IFNγ (Fig. 4A).

Bottom Line: IFNγ, a protective cytokine against cardiac inflammation during Lyme borreliosis, represses MCJ transcription in macrophages.The transcriptional regulator, Ikaros, binds to the MCJ promoter in a Casein kinase II-dependent manner, and mediates the repression of MCJ expression.These results identify the MCJ gene as a transcriptional target of IFNγ and provide evidence of the dynamic adaptation of normal tissues to changes in the environment as a way to adapt metabolically to new conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary and Animal Sciences. University of Massachusetts Amherst. Amherst, MA 01003.

ABSTRACT
MCJ (DNAJC15) is a mitochondrial protein that regulates the mitochondrial metabolic status of macrophages and their response to inflammatory stimuli. CpG island methylation in cancer cells constitutes the only mechanism identified for the regulation of MCJ gene expression. However, whether DNA methylation or transcriptional regulation mechanisms are involved in the physiological control of this gene expression in non-tumor cells remains unknown. We now demonstrate a mechanism of regulation of MCJ expression that is independent of DNA methylation. IFNγ, a protective cytokine against cardiac inflammation during Lyme borreliosis, represses MCJ transcription in macrophages. The transcriptional regulator, Ikaros, binds to the MCJ promoter in a Casein kinase II-dependent manner, and mediates the repression of MCJ expression. These results identify the MCJ gene as a transcriptional target of IFNγ and provide evidence of the dynamic adaptation of normal tissues to changes in the environment as a way to adapt metabolically to new conditions.

No MeSH data available.


Related in: MedlinePlus