On the Interaction Between SMARCAL1 and BRG1.

SMARCAL1 and BRG1, both classified as ATP-dependent chromatin remodeling proteins, play a role in double-strand break DNA damage response pathways. Mutations in SMARCAL1 cause Schimke Immuno-osseous Dysplasia (SIOD) while mutations in BRG1 are associated with Coffin-Siris Syndrome (CSS4). In HeLa cells, SMARCAL1 and BRG1 co-regulate the expression of ATM, ATR, and RNAi genes on doxorubicin-induced DNA damage. Both the proteins are found to be simultaneously present on the promoter of these genes. Based on these results we hypothesized that SMARCAL1 and BRG1 interact with each other forming a complex. In this paper, we validate our hypothesis and show that SMARCAL1 and BRG1 do indeed interact with each other both in the absence and presence of doxorubicin. The formation of these complexes is dependent on the ATPase activity of both SMARCAL1 and BRG1. Using deletion constructs, we show that the HARP domains of SMARCAL1 mediate interaction with BRG1 while multiple domains of BRG1 are probably important for binding to SMARCAL1. We also show that SIOD-associated mutants fail to form a complex with BRG1. Similarly, CSS4-associated mutants of BRG1 fail to interact with SMARCAL1, thus, possibly contributing to the failure of the DNA damage response pathway and pathophysiology associated with SIOD and CSS4.

Altering mammalian transcription networking with ADAADi: An inhibitor of ATP-dependent chromatin remodeling.

Active DNA-dependent ATPase A Domain inhibitor (ADAADi) is the only known inhibitor of ATP-dependent chromatin remodeling proteins that targets the ATPase domain of these proteins. The molecule is synthesized by aminoglycoside phosphotransferase enzyme in the presence of aminoglycosides. ADAADi interacts with ATP-dependent chromatin remodeling proteins through motif Ia present in the conserved helicase domain, and thus, can potentially inhibit all members of this family of proteins. We show that mammalian cells are sensitive to ADAADi but with variable responses in different cell lines. ADAADi can be generated from a wide variety of aminoglycosides; however, cells showed differential response to ADAADi generated from various aminoglycosides. Using HeLa and DU145 cells as model system we have explored the effect of ADAADi on cellular functions. We show that the transcriptional network of a cell type is altered when treated with sub-lethal concentration of ADAADi. Although ADAADi has no known effects on DNA chemical and structural integrity, expression of DNA-damage response genes was altered. The transcripts encoding for the pro-apoptotic proteins were found to be upregulated while the anti-apoptotic genes were found to be downregulated. This was accompanied by increased apoptosis leading us to hypothesize that the ADAADi treatment promotes apoptotic-type of cell death by upregulating the transcription of pro-apoptotic genes. ADAADi also inhibited migration of cells as well as their colony forming ability leading us to conclude that the compound has effective anti-tumor properties.

BRG1 is a prognostic indicator and a potential therapeutic target for prostate cancer.

Brahma-related gene 1 (BRG1) is one of two mutually exclusive ATPases that function as the catalytic subunit of human SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling enzymes. BRG1 has been identified as a tumor suppressor in some cancer types but has been shown to be expressed at elevated levels, relative to normal tissue, in other cancers. Using TCGA (The Cancer Genome Atlas) prostate cancer database, we determined that BRG1 mRNA and protein expression is elevated in prostate tumors relative to normal prostate tissue. Only 3 of 491 (0.6%) sequenced tumors showed amplification of the locus or mutation in the protein coding sequence, arguing against the idea that elevated expression due to amplification or expression of a mutant BRG1 protein is associated with prostate cancer. Kaplan-Meier survival curves showed that BRG1 expression in prostate tumors inversely correlated with survival. However, BRG1 expression did not correlate with Gleason score/International Society of Urological Pathology (ISUP) Grade Group, indicating it is an independent predictor of tumor progression/patient outcome. To experimentally assess BRG1 as a possible therapeutic target, we treated prostate cancer cells with a biologic inhibitor called ADAADi (active DNA-dependent ATPase A Domain inhibitor) that targets the activity of the SNF2 family of ATPases in biochemical assays but showed specificity for BRG1 in prior tissue culture experiments. The inhibitor decreased prostate cancer cell proliferation and induced apoptosis. When directly injected into xenografts established by injection of prostate cancer cells in mouse flanks, the inhibitor decreased tumor growth and increased survival. These results indicate the efficacy of pursuing BRG1 as both an indicator of patient outcome and as a therapeutic target.

Regulation of ATM and ATR by SMARCAL1 and BRG1.

The G2/M checkpoint is activated on DNA damage by the ATM and ATR kinases that are regulated by post-translational modifications. In this paper, the transcriptional co-regulation of ATM and ATR by SMARCAL1 and BRG1, both members of the ATP-dependent chromatin remodeling protein family, is described. SMARCAL1 and BRG1 co-localize on the promoters of ATM and ATR; downregulation of SMARCAL1 and BRG1 results in transcriptional repression of ATM/ATR and overriding of the G2/M checkpoint leading to mitotic abnormalities. On doxorubicin-induced DNA damage, SMARCAL1 and BRG1 are upregulated and these two proteins in turn, upregulate the expression of ATM/ATR. The transcriptional response to DNA damage is feedback regulated by phospho-ATM as it binds to the promoters of SMARCAL1, BRG1, ATM and ATR on DNA damage. The regulation of ATM/ATR is rendered non-functional in Schimke Immuno-Osseous Dysplasia where SMARCAL1 is mutated and in Coffin-Siris Syndrome where BRG1 is mutated. Thus, an intricate transcriptional regulation of DNA damage response genes mediated by SMARCAL1 and BRG1 is present in mammalian cells.

RecA-like domain 2 of DNA-dependent ATPase A domain, a SWI2/SNF2 protein, mediates conformational integrity and ATP hydrolysis.

ATP-dependent chromatin remodeling proteins use the energy released from ATP hydrolysis to reposition nucleosomes in DNA-dependent processes. These proteins are classified as SF2 helicases. SMARCAL1, a member of this protein family, is known to modulate both DNA repair and transcription by specifically recognizing DNA molecules possessing double-strand to single-strand transition regions. Mutations in this gene cause a rare autosomal recessive disorder known as Schimke Immuno-Osseous Dysplasia (SIOD).Structural studies have shown that the ATP-dependent chromatin remodeling proteins possess two RecA-like domains termed as RecA-like domain 1 and RecA-like domain 2. Using Active DNA-dependent ATPase A domain (ADAAD), the bovine homolog of SMARCAL1, as a model system we had previously shown that the RecA-like domain 1 containing helicase motifs Q, I, Ia, II, and III are sufficient for ligand binding; however, the Rec A-like domain 2 containing motifs IV, V, and VI are needed for ATP hydrolysis. In the present study, we have focused on the motifs present in the RecA-like domain 2. Our studies demonstrate that the presence of an aromatic residue in motif IV is needed for interaction with DNA in the presence of ATP. We also show that the motif V is required for the catalytic efficiency of the protein and motif VI is needed for interaction with DNA in the presence of ATP. Finally, we show that the SIOD-associated mutation, R820H, present in motif VI results in loss of ATPase activity, and therefore, reduced response to DNA damage.

BRG1 and SMARCAL1 transcriptionally co-regulate DROSHA, DGCR8 and DICER in response to doxorubicin-induced DNA damage.

Recent investigations have emphasized the role of miRNA biogenesis proteins in the synthesis of non-coding RNA when double-strand DNA breaks are induced by ionizing radiations. However, the role of these non-coding RNA and their regulation in response to doxorubicin-induced DNA damage is not known. In this paper, BRG1 and SMARCAL1, members of the ATP-dependent chromatin remodelling family, are shown to co-regulate the transcription of DROSHA, DGCR8, and DICER in response to double-strand DNA breaks induced by doxorubicin. Both BRG1 and SMARCAL1 are needed for the upregulation of the three miRNA biogenesis genes as absence of BRG1 results in downregulation of DGCR8 and DICER while absence of SMARCAL1 results in downregulation of DROSHA. These two proteins act in coordination to upregulate expression of DROSHA, DGCR8, and DICER when cells are treated with doxorubicin. This transcriptional regulation of the miRNA biogenesis proteins is needed for the formation of 53BP1 foci as downregulation of either BRG1 or SMARCAL1 reduced the number of 53BP1 foci in DNA damaged cells. The foci formation was restored when the downregulated cells were treated with ncRNA purified from doxorubicin treated HeLa cells. From the results obtained, we conclude that the regulation of miRNA biogenesis proteins by SMARCAL1 and BRG1 is needed for the formation of non-coding RNA and thus, 53BP1 foci in response to doxorubicin-induced DNA damage.