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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.

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SMAR1 binds to T(C/G) repeat DNA sequence.(A) The dinucleotide T(C/G) DNA sequence predicted by the MEME suite as SMAR1 binding motif. (B) Isothermal titration calorimetry (ITC) was performed with SMAR1 protein (the macromolecule) and increasing amounts of (TG)10 sequence oligo (the ligand). (C) Fluorescent electrophoretic mobility shift assay (EMSA) performed using Cy5 tagged motif oligos (TG)10 and (TC)10 in combination with other competitive non-fluorophore tagged oligos (AT)10, (GC)10, (TCTG)5, (TG)10 and (TC)10 along with purified recombinant SMAR1 protein and poly (dI-dC). All the gels are run under same experimental condition. (D) The densitometry analysis of the supershift bands of interest of Fig. 2C.
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f2: SMAR1 binds to T(C/G) repeat DNA sequence.(A) The dinucleotide T(C/G) DNA sequence predicted by the MEME suite as SMAR1 binding motif. (B) Isothermal titration calorimetry (ITC) was performed with SMAR1 protein (the macromolecule) and increasing amounts of (TG)10 sequence oligo (the ligand). (C) Fluorescent electrophoretic mobility shift assay (EMSA) performed using Cy5 tagged motif oligos (TG)10 and (TC)10 in combination with other competitive non-fluorophore tagged oligos (AT)10, (GC)10, (TCTG)5, (TG)10 and (TC)10 along with purified recombinant SMAR1 protein and poly (dI-dC). All the gels are run under same experimental condition. (D) The densitometry analysis of the supershift bands of interest of Fig. 2C.

Mentions: In order to determine whether SMAR1 recognizes a particular DNA sequence as its binding site, the sequences corresponding to all peak co-ordinates were retrieved from the UCSC genome browser. The sequences thus obtained were analyzed for the presence of a consensus sequence using MEME-ChIP v4.10.1. The motif search revealed that a dinucleotide T(C/G) repeat sequence was enriched in sequences corresponding to peaks (E-value 1.4e−2329) (Fig. 2A). The distribution of length and frequency of occurrence of repeats has been shown in Supplementary Fig. S4. In order to validate the affinity of SMAR1 to this DNA sequence, we carried out an isothermal titration calorimetry to confirm the specificity of interaction between SMAR1 and its T(C/G) DNA binding repeat. The data indicated a linear correlation between the two with increasing amounts of oligo and SMAR1 protein, accompanied with a significant ΔH value (Fig. 2B). We carried out competitive electrophoretic mobility shift assay to further validate this interaction. The results showed a significant increase in SMAR1 binding to both fluorescently tagged oligos in a dose-dependent manner. It was also observed that only (TC)10 and (TG)10 could compete for binding to SMAR1 protein. The presence of any other untagged, dinucleotide oligos in the reaction mixture did not inhibit the binding of SMAR1 to the fluorophore-tagged oligos (Fig. 2C,D). These results indicate that SMAR1 prefers a dinucleotide T(C/G) DNA repeat sequence for accessing chromatin to exert its regulatory effects.


SMAR1 binds to T(C/G) repeat and inhibits tumor progression by regulating miR-371-373 cluster
SMAR1 binds to T(C/G) repeat DNA sequence.(A) The dinucleotide T(C/G) DNA sequence predicted by the MEME suite as SMAR1 binding motif. (B) Isothermal titration calorimetry (ITC) was performed with SMAR1 protein (the macromolecule) and increasing amounts of (TG)10 sequence oligo (the ligand). (C) Fluorescent electrophoretic mobility shift assay (EMSA) performed using Cy5 tagged motif oligos (TG)10 and (TC)10 in combination with other competitive non-fluorophore tagged oligos (AT)10, (GC)10, (TCTG)5, (TG)10 and (TC)10 along with purified recombinant SMAR1 protein and poly (dI-dC). All the gels are run under same experimental condition. (D) The densitometry analysis of the supershift bands of interest of Fig. 2C.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5037395&req=5

f2: SMAR1 binds to T(C/G) repeat DNA sequence.(A) The dinucleotide T(C/G) DNA sequence predicted by the MEME suite as SMAR1 binding motif. (B) Isothermal titration calorimetry (ITC) was performed with SMAR1 protein (the macromolecule) and increasing amounts of (TG)10 sequence oligo (the ligand). (C) Fluorescent electrophoretic mobility shift assay (EMSA) performed using Cy5 tagged motif oligos (TG)10 and (TC)10 in combination with other competitive non-fluorophore tagged oligos (AT)10, (GC)10, (TCTG)5, (TG)10 and (TC)10 along with purified recombinant SMAR1 protein and poly (dI-dC). All the gels are run under same experimental condition. (D) The densitometry analysis of the supershift bands of interest of Fig. 2C.
Mentions: In order to determine whether SMAR1 recognizes a particular DNA sequence as its binding site, the sequences corresponding to all peak co-ordinates were retrieved from the UCSC genome browser. The sequences thus obtained were analyzed for the presence of a consensus sequence using MEME-ChIP v4.10.1. The motif search revealed that a dinucleotide T(C/G) repeat sequence was enriched in sequences corresponding to peaks (E-value 1.4e−2329) (Fig. 2A). The distribution of length and frequency of occurrence of repeats has been shown in Supplementary Fig. S4. In order to validate the affinity of SMAR1 to this DNA sequence, we carried out an isothermal titration calorimetry to confirm the specificity of interaction between SMAR1 and its T(C/G) DNA binding repeat. The data indicated a linear correlation between the two with increasing amounts of oligo and SMAR1 protein, accompanied with a significant ΔH value (Fig. 2B). We carried out competitive electrophoretic mobility shift assay to further validate this interaction. The results showed a significant increase in SMAR1 binding to both fluorescently tagged oligos in a dose-dependent manner. It was also observed that only (TC)10 and (TG)10 could compete for binding to SMAR1 protein. The presence of any other untagged, dinucleotide oligos in the reaction mixture did not inhibit the binding of SMAR1 to the fluorophore-tagged oligos (Fig. 2C,D). These results indicate that SMAR1 prefers a dinucleotide T(C/G) DNA repeat sequence for accessing chromatin to exert its regulatory effects.

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