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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 represses MCJ gene expression independently of DNA methylation.(A) RAW cells and BMMs were stimulated for 20 h with IFNγ and analyzed by qRT-PCR for MCJ mRNA levels. The results correspond to the average of 3 independent experiments. *; Student´s t test, p < 0.05. (B) RAW cells were co-transfected with plasmids containing the luciferase gene under the influence of the 1 kb proximal promoter region of the MCJ gene or the Renilla luciferase gene under the influence of the SV40 promoter. After 4 h, the cells were stimulated with 100 ng/mL of IFNγ or left unstimulated. Dual luciferase activity was assessed after 16 h of incubation. The promoterless vector, pGL3 was used as a control. *; Student´s t test, p < 0.05. (C) BMMs were left unstimulated or stimulated with 100 ng/mL of IFNγ in the absence or presence of 1 μM of decitabine (DEC) or Azacitidine (Aza). After 48 h, the cells were tested by Western blotting for the presence of MCJ. GAPDH levels were determined to ensure equal loading. (D) CpG-rich region in the MCJ gene analyzed by bisulfite sequencing. The primers used for amplification are noted in lower case. The percentage of methylated CpG residues in BMMs stimulated with 100 ng/mL of IFNγ or left untreated is marked in each of the 6 CpG residues. Black circles indicate 100% of the samples contained these residues methylated, while white circles represent 0%. The analysis corresponds to BMMs isolated from 6 mice. (E) CHIP analysis of BMM DNA immunoprecipitated with antibodies against the H3 marks corresponding to trimethylation of Lys 4 (H3K4m3) and 27 (H3K27m3) or H3 pan-acetylation (Pan Ac-H3). The binding leves are relative to total H3. The results correspond to the average ± SE of 3 independent experiments.
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f3: IFNγ represses MCJ gene expression independently of DNA methylation.(A) RAW cells and BMMs were stimulated for 20 h with IFNγ and analyzed by qRT-PCR for MCJ mRNA levels. The results correspond to the average of 3 independent experiments. *; Student´s t test, p < 0.05. (B) RAW cells were co-transfected with plasmids containing the luciferase gene under the influence of the 1 kb proximal promoter region of the MCJ gene or the Renilla luciferase gene under the influence of the SV40 promoter. After 4 h, the cells were stimulated with 100 ng/mL of IFNγ or left unstimulated. Dual luciferase activity was assessed after 16 h of incubation. The promoterless vector, pGL3 was used as a control. *; Student´s t test, p < 0.05. (C) BMMs were left unstimulated or stimulated with 100 ng/mL of IFNγ in the absence or presence of 1 μM of decitabine (DEC) or Azacitidine (Aza). After 48 h, the cells were tested by Western blotting for the presence of MCJ. GAPDH levels were determined to ensure equal loading. (D) CpG-rich region in the MCJ gene analyzed by bisulfite sequencing. The primers used for amplification are noted in lower case. The percentage of methylated CpG residues in BMMs stimulated with 100 ng/mL of IFNγ or left untreated is marked in each of the 6 CpG residues. Black circles indicate 100% of the samples contained these residues methylated, while white circles represent 0%. The analysis corresponds to BMMs isolated from 6 mice. (E) CHIP analysis of BMM DNA immunoprecipitated with antibodies against the H3 marks corresponding to trimethylation of Lys 4 (H3K4m3) and 27 (H3K27m3) or H3 pan-acetylation (Pan Ac-H3). The binding leves are relative to total H3. The results correspond to the average ± SE of 3 independent experiments.

Mentions: To determine if the downregulation of MCJ protein levels by IFNγ in macrophages was due to an effect on MCJ gene expression, we assessed MCJ mRNA levels in macrophages stimulated with IFNγ. The treatment with IFNγ resulted in a significant decrease in MCJ mRNA levels in RAW cells and BMMs (Fig. 3A). No previous studies have characterized the human or mouse MCJ gene promoter region and addressed transcriptional regulation. We identified a 1 kb region upstream of the start initiation site of the murine MCJ gene (Fig. S2A), that was capable to mediate high levels of transcription in RAW cells in luciferase reporter assays (Fig. 3B). Treatment with IFNγ caused a pronounced decrease in the transcriptional activity of this region of the MCJ promoter (Fig. 3B).


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 represses MCJ gene expression independently of DNA methylation.(A) RAW cells and BMMs were stimulated for 20 h with IFNγ and analyzed by qRT-PCR for MCJ mRNA levels. The results correspond to the average of 3 independent experiments. *; Student´s t test, p < 0.05. (B) RAW cells were co-transfected with plasmids containing the luciferase gene under the influence of the 1 kb proximal promoter region of the MCJ gene or the Renilla luciferase gene under the influence of the SV40 promoter. After 4 h, the cells were stimulated with 100 ng/mL of IFNγ or left unstimulated. Dual luciferase activity was assessed after 16 h of incubation. The promoterless vector, pGL3 was used as a control. *; Student´s t test, p < 0.05. (C) BMMs were left unstimulated or stimulated with 100 ng/mL of IFNγ in the absence or presence of 1 μM of decitabine (DEC) or Azacitidine (Aza). After 48 h, the cells were tested by Western blotting for the presence of MCJ. GAPDH levels were determined to ensure equal loading. (D) CpG-rich region in the MCJ gene analyzed by bisulfite sequencing. The primers used for amplification are noted in lower case. The percentage of methylated CpG residues in BMMs stimulated with 100 ng/mL of IFNγ or left untreated is marked in each of the 6 CpG residues. Black circles indicate 100% of the samples contained these residues methylated, while white circles represent 0%. The analysis corresponds to BMMs isolated from 6 mice. (E) CHIP analysis of BMM DNA immunoprecipitated with antibodies against the H3 marks corresponding to trimethylation of Lys 4 (H3K4m3) and 27 (H3K27m3) or H3 pan-acetylation (Pan Ac-H3). The binding leves are relative to total H3. The results correspond to the average ± SE of 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: IFNγ represses MCJ gene expression independently of DNA methylation.(A) RAW cells and BMMs were stimulated for 20 h with IFNγ and analyzed by qRT-PCR for MCJ mRNA levels. The results correspond to the average of 3 independent experiments. *; Student´s t test, p < 0.05. (B) RAW cells were co-transfected with plasmids containing the luciferase gene under the influence of the 1 kb proximal promoter region of the MCJ gene or the Renilla luciferase gene under the influence of the SV40 promoter. After 4 h, the cells were stimulated with 100 ng/mL of IFNγ or left unstimulated. Dual luciferase activity was assessed after 16 h of incubation. The promoterless vector, pGL3 was used as a control. *; Student´s t test, p < 0.05. (C) BMMs were left unstimulated or stimulated with 100 ng/mL of IFNγ in the absence or presence of 1 μM of decitabine (DEC) or Azacitidine (Aza). After 48 h, the cells were tested by Western blotting for the presence of MCJ. GAPDH levels were determined to ensure equal loading. (D) CpG-rich region in the MCJ gene analyzed by bisulfite sequencing. The primers used for amplification are noted in lower case. The percentage of methylated CpG residues in BMMs stimulated with 100 ng/mL of IFNγ or left untreated is marked in each of the 6 CpG residues. Black circles indicate 100% of the samples contained these residues methylated, while white circles represent 0%. The analysis corresponds to BMMs isolated from 6 mice. (E) CHIP analysis of BMM DNA immunoprecipitated with antibodies against the H3 marks corresponding to trimethylation of Lys 4 (H3K4m3) and 27 (H3K27m3) or H3 pan-acetylation (Pan Ac-H3). The binding leves are relative to total H3. The results correspond to the average ± SE of 3 independent experiments.
Mentions: To determine if the downregulation of MCJ protein levels by IFNγ in macrophages was due to an effect on MCJ gene expression, we assessed MCJ mRNA levels in macrophages stimulated with IFNγ. The treatment with IFNγ resulted in a significant decrease in MCJ mRNA levels in RAW cells and BMMs (Fig. 3A). No previous studies have characterized the human or mouse MCJ gene promoter region and addressed transcriptional regulation. We identified a 1 kb region upstream of the start initiation site of the murine MCJ gene (Fig. S2A), that was capable to mediate high levels of transcription in RAW cells in luciferase reporter assays (Fig. 3B). Treatment with IFNγ caused a pronounced decrease in the transcriptional activity of this region of the MCJ promoter (Fig. 3B).

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