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SMAR1 binds to T(C/G) repeat and inhibits tumor progression by regulating miR-371-373 cluster

View Article: PubMed Central - PubMed

ABSTRACT

Chromatin architecture and dynamics are regulated by various histone and non-histone proteins. The matrix attachment region binding proteins (MARBPs) play a central role in chromatin organization and function through numerous regulatory proteins. In the present study, we demonstrate that nuclear matrix protein SMAR1 orchestrates global gene regulation as determined by massively parallel ChIP-sequencing. The study revealed that SMAR1 binds to T(C/G) repeat and targets genes involved in diverse biological pathways. We observe that SMAR1 binds and targets distinctly different genes based on the availability of p53. Our data suggest that SMAR1 binds and regulates one of the imperative microRNA clusters in cancer and metastasis, miR-371-373. It negatively regulates miR-371-373 transcription as confirmed by SMAR1 overexpression and knockdown studies. Further, deletion studies indicate that a ~200 bp region in the miR-371-373 promoter is necessary for SMAR1 binding and transcriptional repression. Recruitment of HDAC1/mSin3A complex by SMAR1, concomitant with alteration of histone marks results in downregulation of the miRNA cluster. The regulation of miR-371-373 by SMAR1 inhibits breast cancer tumorigenesis and metastasis as determined by in vivo experiments. Overall, our study highlights the binding of SMAR1 to T(C/G) repeat and its role in cancer through miR-371-373.

No MeSH data available.


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SMAR1 binds and recruits repressor complex upstream of miR-371-373 cluster.(A) Peak visualization of ChIP-seq predicted binding of SMAR1 at miR-371-373 promoter in HCT116 p53+/+ and HCT116 p53−/− cells. (B) MAR-Wiz analysis for the miR-371-373 promoter showing presence of a potential MAR. X-axis denotes base pairs and Y-axis denotes MAR potential. (C) Chromatin immunoprecipitation (ChIP) was performed in MCF-7 cells by α-SMAR1 antibody using miR-371-373 promoter specific primers. Rabbit IgG was used as a control. Gel image shown here is the cropped version of whole gel image given in Supplementary Fig. S7. (D) ChIP followed by quantitative real time PCR (qRT PCR) was performed for detecting enrichment of SMAR1, mSin3A, HDAC1 and H3K9me3 at the miR-371-373 promoter in four cell lines, HEK293, MCF-7, MDA-MB-231 and T47D. (E) ChIP followed by quantitative real time PCR was performed for detecting enrichment of RNA polymerase II at the miR-371-373 promoter in all four cell lines. (F) The presence of histone active mark H3K9Ac was detected by ChIP-qRT PCR at the miR-371-373 promoter in all four cell lines. (G) Electrophoretic mobility shift assay (EMSA) performed for determining the presence of SMAR1 binding upstream of the miR-371-373 cluster. (H) Diagrammatic representation of pluc371 and pluc371ΔMAR constructs. (I) Relative luciferase activity of pluc371 and pluc371ΔMAR constructs upon ectopic expression and knockdown of SMAR1 as compared to control.
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f4: SMAR1 binds and recruits repressor complex upstream of miR-371-373 cluster.(A) Peak visualization of ChIP-seq predicted binding of SMAR1 at miR-371-373 promoter in HCT116 p53+/+ and HCT116 p53−/− cells. (B) MAR-Wiz analysis for the miR-371-373 promoter showing presence of a potential MAR. X-axis denotes base pairs and Y-axis denotes MAR potential. (C) Chromatin immunoprecipitation (ChIP) was performed in MCF-7 cells by α-SMAR1 antibody using miR-371-373 promoter specific primers. Rabbit IgG was used as a control. Gel image shown here is the cropped version of whole gel image given in Supplementary Fig. S7. (D) ChIP followed by quantitative real time PCR (qRT PCR) was performed for detecting enrichment of SMAR1, mSin3A, HDAC1 and H3K9me3 at the miR-371-373 promoter in four cell lines, HEK293, MCF-7, MDA-MB-231 and T47D. (E) ChIP followed by quantitative real time PCR was performed for detecting enrichment of RNA polymerase II at the miR-371-373 promoter in all four cell lines. (F) The presence of histone active mark H3K9Ac was detected by ChIP-qRT PCR at the miR-371-373 promoter in all four cell lines. (G) Electrophoretic mobility shift assay (EMSA) performed for determining the presence of SMAR1 binding upstream of the miR-371-373 cluster. (H) Diagrammatic representation of pluc371 and pluc371ΔMAR constructs. (I) Relative luciferase activity of pluc371 and pluc371ΔMAR constructs upon ectopic expression and knockdown of SMAR1 as compared to control.

Mentions: In addition to the potential protein coding gene targets of SMAR1, a number of microRNAs were also predicted. As previously demonstrated, one of the microRNAs miR-373, showed p53-independent regulation by SMAR1. miR-371-373 has been shown to play a central role in tumor progression as well as maintenance of stem cell pluripotency1629. Hence, we sought to elucidate the role of SMAR1 in the regulation of miR-371-373 in breast cancer tumorigenesis and metastasis. The ChIP-seq analysis predicted the binding of SMAR1 to a region upstream of miR-371, which is the first miRNA of the miR-371-373 cluster and the peak was only observed in HCT116 p53−/− cells while it was absent in HCT116 p53+/+ cells (Fig. 4A). The MAR-Wiz (http://genomecluster.secs.oakland.edu/MarWiz/) analysis performed in this region showed a significant MAR potential, further projecting the probability of MAR-binding proteins like SMAR1 to bind to this sequence (Fig. 4B). Next, we performed chromatin immunoprecipitation using α-SMAR1 antibody and the eluted DNA was amplified using primers spanning this predicted SMAR1 binding region. The ChIP-PCR showed specific amplification in the immune pulled fraction, thus confirming the occupancy of SMAR1 at the miR-371-373 locus and validating the ChIP-seq data (Fig. 4C).


SMAR1 binds to T(C/G) repeat and inhibits tumor progression by regulating miR-371-373 cluster
SMAR1 binds and recruits repressor complex upstream of miR-371-373 cluster.(A) Peak visualization of ChIP-seq predicted binding of SMAR1 at miR-371-373 promoter in HCT116 p53+/+ and HCT116 p53−/− cells. (B) MAR-Wiz analysis for the miR-371-373 promoter showing presence of a potential MAR. X-axis denotes base pairs and Y-axis denotes MAR potential. (C) Chromatin immunoprecipitation (ChIP) was performed in MCF-7 cells by α-SMAR1 antibody using miR-371-373 promoter specific primers. Rabbit IgG was used as a control. Gel image shown here is the cropped version of whole gel image given in Supplementary Fig. S7. (D) ChIP followed by quantitative real time PCR (qRT PCR) was performed for detecting enrichment of SMAR1, mSin3A, HDAC1 and H3K9me3 at the miR-371-373 promoter in four cell lines, HEK293, MCF-7, MDA-MB-231 and T47D. (E) ChIP followed by quantitative real time PCR was performed for detecting enrichment of RNA polymerase II at the miR-371-373 promoter in all four cell lines. (F) The presence of histone active mark H3K9Ac was detected by ChIP-qRT PCR at the miR-371-373 promoter in all four cell lines. (G) Electrophoretic mobility shift assay (EMSA) performed for determining the presence of SMAR1 binding upstream of the miR-371-373 cluster. (H) Diagrammatic representation of pluc371 and pluc371ΔMAR constructs. (I) Relative luciferase activity of pluc371 and pluc371ΔMAR constructs upon ectopic expression and knockdown of SMAR1 as compared to control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037395&req=5

f4: SMAR1 binds and recruits repressor complex upstream of miR-371-373 cluster.(A) Peak visualization of ChIP-seq predicted binding of SMAR1 at miR-371-373 promoter in HCT116 p53+/+ and HCT116 p53−/− cells. (B) MAR-Wiz analysis for the miR-371-373 promoter showing presence of a potential MAR. X-axis denotes base pairs and Y-axis denotes MAR potential. (C) Chromatin immunoprecipitation (ChIP) was performed in MCF-7 cells by α-SMAR1 antibody using miR-371-373 promoter specific primers. Rabbit IgG was used as a control. Gel image shown here is the cropped version of whole gel image given in Supplementary Fig. S7. (D) ChIP followed by quantitative real time PCR (qRT PCR) was performed for detecting enrichment of SMAR1, mSin3A, HDAC1 and H3K9me3 at the miR-371-373 promoter in four cell lines, HEK293, MCF-7, MDA-MB-231 and T47D. (E) ChIP followed by quantitative real time PCR was performed for detecting enrichment of RNA polymerase II at the miR-371-373 promoter in all four cell lines. (F) The presence of histone active mark H3K9Ac was detected by ChIP-qRT PCR at the miR-371-373 promoter in all four cell lines. (G) Electrophoretic mobility shift assay (EMSA) performed for determining the presence of SMAR1 binding upstream of the miR-371-373 cluster. (H) Diagrammatic representation of pluc371 and pluc371ΔMAR constructs. (I) Relative luciferase activity of pluc371 and pluc371ΔMAR constructs upon ectopic expression and knockdown of SMAR1 as compared to control.
Mentions: In addition to the potential protein coding gene targets of SMAR1, a number of microRNAs were also predicted. As previously demonstrated, one of the microRNAs miR-373, showed p53-independent regulation by SMAR1. miR-371-373 has been shown to play a central role in tumor progression as well as maintenance of stem cell pluripotency1629. Hence, we sought to elucidate the role of SMAR1 in the regulation of miR-371-373 in breast cancer tumorigenesis and metastasis. The ChIP-seq analysis predicted the binding of SMAR1 to a region upstream of miR-371, which is the first miRNA of the miR-371-373 cluster and the peak was only observed in HCT116 p53−/− cells while it was absent in HCT116 p53+/+ cells (Fig. 4A). The MAR-Wiz (http://genomecluster.secs.oakland.edu/MarWiz/) analysis performed in this region showed a significant MAR potential, further projecting the probability of MAR-binding proteins like SMAR1 to bind to this sequence (Fig. 4B). Next, we performed chromatin immunoprecipitation using α-SMAR1 antibody and the eluted DNA was amplified using primers spanning this predicted SMAR1 binding region. The ChIP-PCR showed specific amplification in the immune pulled fraction, thus confirming the occupancy of SMAR1 at the miR-371-373 locus and validating the ChIP-seq data (Fig. 4C).

View Article: PubMed Central - PubMed

ABSTRACT

Chromatin architecture and dynamics are regulated by various histone and non-histone proteins. The matrix attachment region binding proteins (MARBPs) play a central role in chromatin organization and function through numerous regulatory proteins. In the present study, we demonstrate that nuclear matrix protein SMAR1 orchestrates global gene regulation as determined by massively parallel ChIP-sequencing. The study revealed that SMAR1 binds to T(C/G) repeat and targets genes involved in diverse biological pathways. We observe that SMAR1 binds and targets distinctly different genes based on the availability of p53. Our data suggest that SMAR1 binds and regulates one of the imperative microRNA clusters in cancer and metastasis, miR-371-373. It negatively regulates miR-371-373 transcription as confirmed by SMAR1 overexpression and knockdown studies. Further, deletion studies indicate that a ~200 bp region in the miR-371-373 promoter is necessary for SMAR1 binding and transcriptional repression. Recruitment of HDAC1/mSin3A complex by SMAR1, concomitant with alteration of histone marks results in downregulation of the miRNA cluster. The regulation of miR-371-373 by SMAR1 inhibits breast cancer tumorigenesis and metastasis as determined by in vivo experiments. Overall, our study highlights the binding of SMAR1 to T(C/G) repeat and its role in cancer through miR-371-373.

No MeSH data available.


Related in: MedlinePlus