Cis-regulation of microRNA expression by scaffold/matrix-attachment regions.

microRNAs (miRNAs) spatio-temporally modulate gene expression; however, very little is known about the regulation of their expression. Here, we hypothesized that the well-known cis-regulatory elements of gene expression, scaffold/matrix-attachment regions (MARs) could modulate miRNA expression. Accordingly, we found MARs to be enriched in the upstream regions of miRNA genes. To determine their role in cell type-specific expression of miRNAs, we examined four individual miRNAs (let-7b, miR-17, miR-93 and miR-221) and the miR-17-92 cluster, known to be overexpressed in neuroblastoma. Our results show that MARs indeed define the cell-specific expression of these miRNAs by tethering the chromatin to nuclear matrix. This is brought about by cell type-specific binding of HMG I/Y protein to MARs that then promotes the local acetylation of histones, serving as boundary elements for gene activation. The binding, chromatin tethering and gene activation by HMG I/Y was not observed in fibroblast control cells but were restricted to neuroblastoma cells. This study implies that the association of MAR binding proteins to MARs could dictate the tissue/context specific regulation of miRNA genes by serving as a boundary element signaling the transcriptional activation.

Nuclear orphan receptor TLX induces Oct-3/4 for the survival and maintenance of adult hippocampal progenitors upon hypoxia.

Hypoxia promotes neural stem cell proliferation, the mechanism of which is poorly understood. Here, we have identified the nuclear orphan receptor TLX as a mediator for proliferation and pluripotency of neural progenitors upon hypoxia. We found an enhanced early protein expression of TLX under hypoxia potentiating sustained proliferation of neural progenitors. Moreover, TLX induction upon hypoxia in differentiating conditions leads to proliferation and a stem cell-like phenotype, along with coexpression of neural stem cell markers. Following hypoxia, TLX is recruited to the Oct-3/4 proximal promoter, augmenting the gene transcription and promoting progenitor proliferation and pluripotency. Knockdown of Oct-3/4 significantly reduced TLX-mediated proliferation, highlighting their interdependence in regulating the progenitor pool. Additionally, TLX synergizes with basic FGF to sustain cell viability upon hypoxia, since the knockdown of TLX along with the withdrawal of growth factor results in cell death. This can be attributed to the activation of Akt signaling pathway by TLX, the depletion of which results in reduced proliferation of progenitor cells. Cumulatively, the data presented here demonstrate a new role for TLX in neural stem cell proliferation and pluripotency upon hypoxia.

Heat-shock protein 70 binds to a novel sequence in 5' UTR of tumor suppressor SMAR1 and regulates its mRNA stability upon Prostaglandin A2 treatment.

Here, we report Prostaglandin A2 (PGA2) induced binding of HSP70 to a novel site on phi1 SMAR1 5' UTR which stabilizes the wild type transcript and leads to subsequent increase in SMAR1 protein levels. SMAR1 mediated cell cycle arrest is perturbed in PGA2-treated cells when HSP70 is knocked-down. Contrarily HSP70, unlike SMAR1, is overexpressed in breast cancers. We demonstrate that this is because of the inability of HSP70 to bind to the phi17 SMAR1 UTR variant which is the predominant form in breast cancers.

Nuclear matrix protein SMAR1 represses HIV-1 LTR mediated transcription through chromatin remodeling.

Nuclear Matrix and MARs have been implicated in the transcriptional regulation of host as well as viral genes but their precise role in HIV-1 transcription remains unclear. Here, we show that >98% of HIV sequences contain consensus MAR element in their promoter. We show that SMAR1 binds to the LTR MAR and reinforces transcriptional silencing by tethering the LTR MAR to nuclear matrix. SMAR1 associated HDAC1-mSin3 corepressor complex is dislodged from the LTR upon cellular activation by PMA/TNFalpha leading to an increase in the acetylation and a reduction in the trimethylation of histones, associated with the recruitment of RNA Polymerase II on the LTR. Overexpression of SMAR1 lead to reduction in LTR mediated transcription, both in a Tat dependent and independent manner, resulting in a decreased virion production. These results demonstrate the role of SMAR1 in regulating viral transcription by alternative compartmentalization of LTR between the nuclear matrix and chromatin.

SMAR1 regulates free radical stress through modulation of AKR1a4 enzyme activity.

Tumor suppressor SMAR1 is known to be involved in regulation of cell cycle and apoptotic genes transcription. It also directly interacts and stabilizes p53 through phosphorylation at serine-15 residue. Although the functions of SMAR1 are mainly restricted to the nucleus, we report its novel function with the cytoplasm. We show that SMAR1 directly interacts and inhibits AKR1a4 enzyme activity. Interestingly, AKR1a4 enzyme activity is elevated in higher grades of breast cancer where SMAR1 expression is drastically downregulated. Higher AKR1a4 activity protects the cancer cells from anticancer drugs and free radical stress. Through increased metabolism, ARK1a4 helps fulfilling higher energy needs required by cancer cell. The present study delineates yet another facet of tumor suppressor activity of SMAR1 in the cytoplasm. We also depict that upon stress, ATM kinase leads to dissociation of SMAR1-AKR1a4 complex through nuclear translocation of SMAR1 causing elevated AKR1a4 activity. Nuclear SMAR1 causes cell cycle arrest giving ample time for DNA damage repair, while AKR1a4 scavenges the excess free radicals which may further cause DNA damage. Thus, we propose a novel mechanism of regulation of oxidative stress by ATM through modulation of SMAR1-AKR1a4 complex. Further, we show that a small peptide derived from SMAR1 induces free radical stress by inhibiting AKR1a4 enzyme activity, which can be a potential anticancer therapeutic agent.

SMAR1 forms a ternary complex with p53-MDM2 and negatively regulates p53-mediated transcription.

The intra-cellular level of tumor suppressor protein p53 is tightly controlled by an autoregulatory feedback loop between the protein and its negative regulator MDM2. The role of MDM2 in down-regulating the p53 response in unstressed conditions and in the post-stress recovery phase is well documented. However, interplay between the N-terminal phosphorylations and C-terminal acetylations of p53 in this context remains unclear. Here, we show that an MAR binding protein SMAR1 interacts with MDM2 and the Ser15 phosphorylated form of p53, forming a ternary complex in the post stress-recovery phase. This triple complex formation between p53, MDM2 and SMAR1 results in recruitment of HDAC1 to deacetylate p53. The deacetylated p53 binds poorly to the target promoter (p21), which results in switching off the p53 response, essential for re-entry into the cell cycle. Interestingly, the knock-down of SMAR1 using siRNA leads to a prolonged cell-cycle arrest in the post stress recovery phase due to ablation of p53-MDM2-HDAC1 interaction. Thus, the results presented here for the first time highlight the role of SMAR1 in masking the active phosphorylation site of p53, enabling the deacetylation of p53 by HDAC1-MDM2 complex, thereby regulating the p53 transcriptional response during stress rescue.

Tumor suppressor SMAR1 downregulates Cytokeratin 8 expression by displacing p53 from its cognate site.

Intermediary filaments play a crucial role in transformation of cells to a malignant phenotype. Here, we report that tumor suppressor SMAR1 downregulates Cytokeratin 8 gene expression by modulating p53-mediated transactivation of this gene. Moreover, the cell surface cytokeratin expression was downregulated leading to a decreased migration and invasiveness of cells. We further validated these results using genotoxic stress agents that lead to an increase in the levels of SMAR1 protein. This subsequently represses the transcription of Cytokeratin 8 gene by local chromatin condensation mediated by histone methylation and deacetylation. Evaluation of SMAR1 and Cytokeratin 8 proteins in different grades of cancer using tissue microarray point out at the inverse expression profiles of these genes (i.e. low levels of SMAR1 correlating with high expression of Cytokeratin 8) in higher grades of breast cancer. Therefore, the results presented here highlight the mechanism of Cytokeratin 8 gene regulation by interplay of tumor suppressor proteins SMAR1 and p53.

Stabilization of SMAR1 mRNA by PGA2 involves a stem loop structure in the 5' UTR.

Prostaglandins are anticancer agents known to inhibit tumor cell proliferation both in vitro and in vivo by affecting the mRNA stability. Here we report that a MAR-binding protein SMAR1 is a target of Prostaglandin A2 (PGA2) induced growth arrest. We identify a regulatory mechanism leading to stabilization of SMAR1 transcript. Our results show that a minor stem and loop structure present in the 5' UTR of SMAR1 (1-UTR) is critical for nucleoprotein complex formation that leads to SMAR1 stabilization in response to PGA2. This results in an increased SMAR1 transcript and altered protein levels, that in turn causes downregulation of Cyclin D1 gene, essential for G1/S phase transition. We also provide evidence for the presence of a variant 5' UTR SMAR1 (17-UTR) in breast cancer-derived cell lines. This form lacks the minor stem and loop structure required for mRNA stabilization in response to PGA2. As a consequence of this, there is a low level of endogenous tumor suppressor protein SMAR1 in breast cancer-derived cell lines. Our studies provide a mechanistic insight into the regulation of tumor suppressor protein SMAR1 by a cancer therapeutic PGA2, that leads to repression of Cyclin D1 gene.

MARs and MARBPs: key modulators of gene regulation and disease manifestation.

The DNA in eukaryotic genome is compartmentalized into various domains by a series of loops tethered onto the base of nuclear matrix. Scaffold/Matrix attachment regions (S/MAR) punctuate these attachment sites and govern the nuclear architecture by establishing chromatin boundaries. In this context, specific proteins that interact with and bind to MAR sequences called MAR binding proteins (MARBPs), are of paramount importance, as these sequences spool the proteins that regulate transcription, replication, repair and recombination. Recent evidences also suggest a role for these cis-acting elements in viral integration, replication and transcription, thereby affecting host immune system. Owing to the complex nature of these nucleotide sequences, less is known about the MARBPs that bind to and bring about diverse effects on chromatin architecture and gene function. Several MARBPs have been identified and characterized so far and the list is growing. The fact that most the MARBPs exist in a co-repressor/co-activator complex and bring about gene regulation makes them quintessential for cellular processes. This participation in gene regulation means that any perturbation in the regulation and levels of MARBPs could lead to disease conditions, particularly those caused by abnormal cell proliferation, like cancer. In the present chapter, we discuss the role of MARs and MARBPs in eukaryotic gene regulation, recombination, transcription and viral integration by altering the local chromatin structure and their dysregulation in disease manifestation

Tumor suppressor SMAR1 activates and stabilizes p53 through its arginine-serine-rich motif.

Various stresses and DNA-damaging agents trigger transcriptional activity of p53 by post-translational modifications, making it a global regulatory switch that controls cell proliferation and apoptosis. Earlier we have shown that the novel MAR-associated protein SMAR1 interacts with p53. Here we delineate the minimal domain of SMAR1 (the arginine-serine-rich domain) that is phosphorylated by protein kinase C family proteins and is responsible for p53 interaction, activation, and stabilization within the nucleus. SMAR1-mediated stabilization of p53 is brought about by inhibiting Mdm2-mediated degradation of p53. We also demonstrate that this arginine-serine (RS)-rich domain triggers the various cell cycle modulating proteins that decide cell fate. Furthermore, phenotypic knock-down experiments using small interfering RNA showed that SMAR1 is required for activation and nuclear retention of p53. The level of phosphorylated p53 was significantly increased in the thymus of SMAR1 transgenic mice, showing in vivo significance of SMAR1 expression. This is the first report that demonstrates the mechanism of action of the MAR-binding protein SMAR1 in modulating the activity of p53, often referred to as the "guardian of the genome."