Limits...
AP-1 mediated transcriptional repression of matrix metalloproteinase-9 by recruitment of histone deacetylase 1 in response to interferon β.

Mittelstadt ML, Patel RC - PLoS ONE (2012)

Bottom Line: Using reporter assays with promoter deletion constructs we show that IFNβ's inhibitory effects require a region of the promoter between -154 and -72, which contains an AP-1 binding site.Chromatin immunoprecipitation (ChIP) studies indicate that IFNβ increases histone deacetylase (HDAC)-1 recruitment to the MMP-9 promoter and reduces histone H3 acetylation, in addition to reduced NF-κB recruitment.Altogether, our results establish that the repression of MMP-9 transcription in response to IFNβ occurs by the recruitment of HDAC1 via the proximal AP-1 binding site.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of South Carolina, Columbia, South Carolina, United States of America.

ABSTRACT
Matrix metalloproteinase-9 (MMP-9) is a 92 kDa zinc-dependant endopeptidase that degrades components of the extracellular matrix. Increased expression of MMP-9 is implicated in many pathological conditions including metastatic cancer, multiple sclerosis, and atherosclerosis. Although it has been widely noted that interferon-β (IFNβ) downregulates both the basal and phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 expression at the transcriptional level, the molecular mechanism of this repression is poorly understood. In the present study we identify a novel mechanism for repression of MMP-9 transcription by IFNβ in HT1080 fibrosarcoma cells. Using reporter assays with promoter deletion constructs we show that IFNβ's inhibitory effects require a region of the promoter between -154 and -72, which contains an AP-1 binding site. Chromatin immunoprecipitation (ChIP) studies indicate that IFNβ increases histone deacetylase (HDAC)-1 recruitment to the MMP-9 promoter and reduces histone H3 acetylation, in addition to reduced NF-κB recruitment. ChIP analysis shows that IFNβ induced HDAC1 recruitment to the MMP-9 promoter and IFNβ mediated transcriptional repression is lost when the AP-1 binding site is inactivated by a point mutation. Altogether, our results establish that the repression of MMP-9 transcription in response to IFNβ occurs by the recruitment of HDAC1 via the proximal AP-1 binding site.

Show MeSH

Related in: MedlinePlus

Chromatin Immunoprecipitation (ChIP) analysis of the MMP-9 promoter.(A) HDAC1 recruitment on MMP-9 promoter in response to IFNβ. HT1080 cells were treated with IFNβ for 12 hrs, PMA for 4 hrs, or both. ChIP assays, using antibodies specific to p65, histone deacetylase (HDAC)-1, or acetylated histone-3 (aH3) were performed, followed by PCR performed within the linear range of amplification (determined to be 38 cycles). Input chromatin (1%) was removed from samples prior to immunoprecipitation and subjected to PCR to control for any variation in starting material. Band intensities were normalized to input and the relative band intensities were calculated. The relative band intensities are shown under each band with respect to the strongest band being represented as 1. Data shown is representative of four independent experiments. (B) HDAC1 recruitment on MMP-9 promoter in response to IFNβ is prevented in vivo by rendering AP-1 site non-functional. HT1080 cells were stably transfected with −660 bp MMP-9 promoter luciferase reporter constructs, either with or without proximal AP-1 site mutated. Resulting stable lines were treated with IFNβ for 12 hrs, PMA for 4 hrs, or both. Chromatin immunoprecipitation (ChIP) assays, using antibodies specific to histone deacetylase (HDAC)-1, or acetylated histone-3 (aH3) were performed, followed by PCR. PCR primers were designed to include the region of interest of the MMP-9 promoter and a section of the luciferase reporter, to select against amplification of endogenous wild type MMP-9 promoter region. Band intensities were normalized to input and the relative band intensities were calculated. The relative band intensities are shown under each band with respect to the strongest band being represented as 1. Data shown is representative of three independent experiments. (C) AP-1 binding to the proximal site is required for HDAC1 induced repression of MMP-9 HT1080 cells were co-transfected in 6-well dishes in triplicates with either −154 MMP-9 promoter deletion construct containing a point mutation in the proximal AP-1 site, which renders the binding site non-functional, or with wild type −154 MMP-9 construct and varying amounts of a HDAC1 expression construct. Cell extracts were assayed for firefly luciferase activities, and normalized by protein concentration to account for any cell concentration differences between samples. P-values were calculated using statistical analysis software. As indicated above the bars, * indicates P-values of significance (50 ng  =  0.000698 and 100 ng  =  0.000885, significant difference) and # indicates P-values of no significance (50 ng  =  0.0763 and 100 ng  =  0.882, no significant difference) with significant values being <0.01.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3412826&req=5

pone-0042152-g004: Chromatin Immunoprecipitation (ChIP) analysis of the MMP-9 promoter.(A) HDAC1 recruitment on MMP-9 promoter in response to IFNβ. HT1080 cells were treated with IFNβ for 12 hrs, PMA for 4 hrs, or both. ChIP assays, using antibodies specific to p65, histone deacetylase (HDAC)-1, or acetylated histone-3 (aH3) were performed, followed by PCR performed within the linear range of amplification (determined to be 38 cycles). Input chromatin (1%) was removed from samples prior to immunoprecipitation and subjected to PCR to control for any variation in starting material. Band intensities were normalized to input and the relative band intensities were calculated. The relative band intensities are shown under each band with respect to the strongest band being represented as 1. Data shown is representative of four independent experiments. (B) HDAC1 recruitment on MMP-9 promoter in response to IFNβ is prevented in vivo by rendering AP-1 site non-functional. HT1080 cells were stably transfected with −660 bp MMP-9 promoter luciferase reporter constructs, either with or without proximal AP-1 site mutated. Resulting stable lines were treated with IFNβ for 12 hrs, PMA for 4 hrs, or both. Chromatin immunoprecipitation (ChIP) assays, using antibodies specific to histone deacetylase (HDAC)-1, or acetylated histone-3 (aH3) were performed, followed by PCR. PCR primers were designed to include the region of interest of the MMP-9 promoter and a section of the luciferase reporter, to select against amplification of endogenous wild type MMP-9 promoter region. Band intensities were normalized to input and the relative band intensities were calculated. The relative band intensities are shown under each band with respect to the strongest band being represented as 1. Data shown is representative of three independent experiments. (C) AP-1 binding to the proximal site is required for HDAC1 induced repression of MMP-9 HT1080 cells were co-transfected in 6-well dishes in triplicates with either −154 MMP-9 promoter deletion construct containing a point mutation in the proximal AP-1 site, which renders the binding site non-functional, or with wild type −154 MMP-9 construct and varying amounts of a HDAC1 expression construct. Cell extracts were assayed for firefly luciferase activities, and normalized by protein concentration to account for any cell concentration differences between samples. P-values were calculated using statistical analysis software. As indicated above the bars, * indicates P-values of significance (50 ng  =  0.000698 and 100 ng  =  0.000885, significant difference) and # indicates P-values of no significance (50 ng  =  0.0763 and 100 ng  =  0.882, no significant difference) with significant values being <0.01.

Mentions: Although it is known that transiently transfected DNA acquires immediate nucleosomal assembly on the plasmid, it is likely that the placement of histones along the endogenous gene is structurally dissimilar to that of the transiently introduced gene [16]. To circumvent this, the endogenous protein occupation status of the MMP-9 promoter was studied using chromatin immunoprecipitation (ChIP) assay. ChIP assays were performed using antibodies against p65 subunit of NF-κB, HDAC1, and acetylated histone H3. PCR analysis was used to probe immunoprecipitated samples for the presence of the MMP-9 promoter region surrounding the proximal AP-1 binding site (primers were designed to amplify the region of interest between −154 bp and +20 bp). PCR analysis of the control (input) indicated that the soluble chromatin samples obtained from each treatment condition contained equal amounts of chromatin fragments of the MMP-9 promoter region of interest (Figure 4A, input bands). In untreated cells, p65 association with the MMP-9 promoter was not detectable. As expected, in PMA treated cells, p65 shows a strong association with the MMP-9 promoter, corresponding to the requirement of NF-κB activation and binding at the −600 region of the promoter for transcriptional induction of MMP-9 in response to PMA. However, in cells treated with both PMA and IFNβ, p65 association with the MMP-9 promoter is significantly decreased. Thus, similar to IFNγ, it seems likely that IFNβ also induces binding of IRF1 to a site which competes with NF-κB’s binding at the MMP-9 promoter [13]. HDAC1 contributes to general histone deacetylation and condensed chromatin, so it is not surprising that there is a basal level of association of HDAC1 with the MMP-9 promoter under uninduced conditions (Figure 4A). In PMA treated cells, HDAC1 association with the MMP-9 promoter is undetectable; but with the addition of IFNβ, HDAC1 is recruited back to the proximal region of the MMP-9 promoter. Concurrently, the acetylation status of the MMP-9 proximal promoter region is reflective of the amount of HDAC1 present: as HDAC1 association is diminished, acetylation of histone H3 is increased and when HDAC1 returns to the MMP-9 promoter under IFNβ treatment, acetylation of histone H3 is decreased. Thus, histone H3 acetylation increases with PMA treatment and then decreases with IFN treatment (Figure 4A). It is likely that in addition to HDAC1, other histone deacetylases are also recruited to the MMP-9 proximal promoter region in a very similar manner. At this point, we have not examined involvement of other HDAC family members in regulation of MMP-9 promoter in response to IFNβ.


AP-1 mediated transcriptional repression of matrix metalloproteinase-9 by recruitment of histone deacetylase 1 in response to interferon β.

Mittelstadt ML, Patel RC - PLoS ONE (2012)

Chromatin Immunoprecipitation (ChIP) analysis of the MMP-9 promoter.(A) HDAC1 recruitment on MMP-9 promoter in response to IFNβ. HT1080 cells were treated with IFNβ for 12 hrs, PMA for 4 hrs, or both. ChIP assays, using antibodies specific to p65, histone deacetylase (HDAC)-1, or acetylated histone-3 (aH3) were performed, followed by PCR performed within the linear range of amplification (determined to be 38 cycles). Input chromatin (1%) was removed from samples prior to immunoprecipitation and subjected to PCR to control for any variation in starting material. Band intensities were normalized to input and the relative band intensities were calculated. The relative band intensities are shown under each band with respect to the strongest band being represented as 1. Data shown is representative of four independent experiments. (B) HDAC1 recruitment on MMP-9 promoter in response to IFNβ is prevented in vivo by rendering AP-1 site non-functional. HT1080 cells were stably transfected with −660 bp MMP-9 promoter luciferase reporter constructs, either with or without proximal AP-1 site mutated. Resulting stable lines were treated with IFNβ for 12 hrs, PMA for 4 hrs, or both. Chromatin immunoprecipitation (ChIP) assays, using antibodies specific to histone deacetylase (HDAC)-1, or acetylated histone-3 (aH3) were performed, followed by PCR. PCR primers were designed to include the region of interest of the MMP-9 promoter and a section of the luciferase reporter, to select against amplification of endogenous wild type MMP-9 promoter region. Band intensities were normalized to input and the relative band intensities were calculated. The relative band intensities are shown under each band with respect to the strongest band being represented as 1. Data shown is representative of three independent experiments. (C) AP-1 binding to the proximal site is required for HDAC1 induced repression of MMP-9 HT1080 cells were co-transfected in 6-well dishes in triplicates with either −154 MMP-9 promoter deletion construct containing a point mutation in the proximal AP-1 site, which renders the binding site non-functional, or with wild type −154 MMP-9 construct and varying amounts of a HDAC1 expression construct. Cell extracts were assayed for firefly luciferase activities, and normalized by protein concentration to account for any cell concentration differences between samples. P-values were calculated using statistical analysis software. As indicated above the bars, * indicates P-values of significance (50 ng  =  0.000698 and 100 ng  =  0.000885, significant difference) and # indicates P-values of no significance (50 ng  =  0.0763 and 100 ng  =  0.882, no significant difference) with significant values being <0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412826&req=5

pone-0042152-g004: Chromatin Immunoprecipitation (ChIP) analysis of the MMP-9 promoter.(A) HDAC1 recruitment on MMP-9 promoter in response to IFNβ. HT1080 cells were treated with IFNβ for 12 hrs, PMA for 4 hrs, or both. ChIP assays, using antibodies specific to p65, histone deacetylase (HDAC)-1, or acetylated histone-3 (aH3) were performed, followed by PCR performed within the linear range of amplification (determined to be 38 cycles). Input chromatin (1%) was removed from samples prior to immunoprecipitation and subjected to PCR to control for any variation in starting material. Band intensities were normalized to input and the relative band intensities were calculated. The relative band intensities are shown under each band with respect to the strongest band being represented as 1. Data shown is representative of four independent experiments. (B) HDAC1 recruitment on MMP-9 promoter in response to IFNβ is prevented in vivo by rendering AP-1 site non-functional. HT1080 cells were stably transfected with −660 bp MMP-9 promoter luciferase reporter constructs, either with or without proximal AP-1 site mutated. Resulting stable lines were treated with IFNβ for 12 hrs, PMA for 4 hrs, or both. Chromatin immunoprecipitation (ChIP) assays, using antibodies specific to histone deacetylase (HDAC)-1, or acetylated histone-3 (aH3) were performed, followed by PCR. PCR primers were designed to include the region of interest of the MMP-9 promoter and a section of the luciferase reporter, to select against amplification of endogenous wild type MMP-9 promoter region. Band intensities were normalized to input and the relative band intensities were calculated. The relative band intensities are shown under each band with respect to the strongest band being represented as 1. Data shown is representative of three independent experiments. (C) AP-1 binding to the proximal site is required for HDAC1 induced repression of MMP-9 HT1080 cells were co-transfected in 6-well dishes in triplicates with either −154 MMP-9 promoter deletion construct containing a point mutation in the proximal AP-1 site, which renders the binding site non-functional, or with wild type −154 MMP-9 construct and varying amounts of a HDAC1 expression construct. Cell extracts were assayed for firefly luciferase activities, and normalized by protein concentration to account for any cell concentration differences between samples. P-values were calculated using statistical analysis software. As indicated above the bars, * indicates P-values of significance (50 ng  =  0.000698 and 100 ng  =  0.000885, significant difference) and # indicates P-values of no significance (50 ng  =  0.0763 and 100 ng  =  0.882, no significant difference) with significant values being <0.01.
Mentions: Although it is known that transiently transfected DNA acquires immediate nucleosomal assembly on the plasmid, it is likely that the placement of histones along the endogenous gene is structurally dissimilar to that of the transiently introduced gene [16]. To circumvent this, the endogenous protein occupation status of the MMP-9 promoter was studied using chromatin immunoprecipitation (ChIP) assay. ChIP assays were performed using antibodies against p65 subunit of NF-κB, HDAC1, and acetylated histone H3. PCR analysis was used to probe immunoprecipitated samples for the presence of the MMP-9 promoter region surrounding the proximal AP-1 binding site (primers were designed to amplify the region of interest between −154 bp and +20 bp). PCR analysis of the control (input) indicated that the soluble chromatin samples obtained from each treatment condition contained equal amounts of chromatin fragments of the MMP-9 promoter region of interest (Figure 4A, input bands). In untreated cells, p65 association with the MMP-9 promoter was not detectable. As expected, in PMA treated cells, p65 shows a strong association with the MMP-9 promoter, corresponding to the requirement of NF-κB activation and binding at the −600 region of the promoter for transcriptional induction of MMP-9 in response to PMA. However, in cells treated with both PMA and IFNβ, p65 association with the MMP-9 promoter is significantly decreased. Thus, similar to IFNγ, it seems likely that IFNβ also induces binding of IRF1 to a site which competes with NF-κB’s binding at the MMP-9 promoter [13]. HDAC1 contributes to general histone deacetylation and condensed chromatin, so it is not surprising that there is a basal level of association of HDAC1 with the MMP-9 promoter under uninduced conditions (Figure 4A). In PMA treated cells, HDAC1 association with the MMP-9 promoter is undetectable; but with the addition of IFNβ, HDAC1 is recruited back to the proximal region of the MMP-9 promoter. Concurrently, the acetylation status of the MMP-9 proximal promoter region is reflective of the amount of HDAC1 present: as HDAC1 association is diminished, acetylation of histone H3 is increased and when HDAC1 returns to the MMP-9 promoter under IFNβ treatment, acetylation of histone H3 is decreased. Thus, histone H3 acetylation increases with PMA treatment and then decreases with IFN treatment (Figure 4A). It is likely that in addition to HDAC1, other histone deacetylases are also recruited to the MMP-9 proximal promoter region in a very similar manner. At this point, we have not examined involvement of other HDAC family members in regulation of MMP-9 promoter in response to IFNβ.

Bottom Line: Using reporter assays with promoter deletion constructs we show that IFNβ's inhibitory effects require a region of the promoter between -154 and -72, which contains an AP-1 binding site.Chromatin immunoprecipitation (ChIP) studies indicate that IFNβ increases histone deacetylase (HDAC)-1 recruitment to the MMP-9 promoter and reduces histone H3 acetylation, in addition to reduced NF-κB recruitment.Altogether, our results establish that the repression of MMP-9 transcription in response to IFNβ occurs by the recruitment of HDAC1 via the proximal AP-1 binding site.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of South Carolina, Columbia, South Carolina, United States of America.

ABSTRACT
Matrix metalloproteinase-9 (MMP-9) is a 92 kDa zinc-dependant endopeptidase that degrades components of the extracellular matrix. Increased expression of MMP-9 is implicated in many pathological conditions including metastatic cancer, multiple sclerosis, and atherosclerosis. Although it has been widely noted that interferon-β (IFNβ) downregulates both the basal and phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 expression at the transcriptional level, the molecular mechanism of this repression is poorly understood. In the present study we identify a novel mechanism for repression of MMP-9 transcription by IFNβ in HT1080 fibrosarcoma cells. Using reporter assays with promoter deletion constructs we show that IFNβ's inhibitory effects require a region of the promoter between -154 and -72, which contains an AP-1 binding site. Chromatin immunoprecipitation (ChIP) studies indicate that IFNβ increases histone deacetylase (HDAC)-1 recruitment to the MMP-9 promoter and reduces histone H3 acetylation, in addition to reduced NF-κB recruitment. ChIP analysis shows that IFNβ induced HDAC1 recruitment to the MMP-9 promoter and IFNβ mediated transcriptional repression is lost when the AP-1 binding site is inactivated by a point mutation. Altogether, our results establish that the repression of MMP-9 transcription in response to IFNβ occurs by the recruitment of HDAC1 via the proximal AP-1 binding site.

Show MeSH
Related in: MedlinePlus