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1.
Epigenomes ; 6(4)2022 Oct 18.
Article in English | MEDLINE | ID: mdl-36278682

ABSTRACT

Cells respond to oxidative stress by elevating the levels of antioxidants, signaling, and transcriptional regulation, often implemented by chromatin remodeling proteins. The study presented here shows that the expression of PICH, a Rad54-like helicase belonging to the ATP-dependent chromatin remodeling protein family, is upregulated during oxidative stress in HeLa cells. We also show that PICH regulates the expression of Nrf2, a transcription factor regulating antioxidant response in both the absence and presence of oxidative stress. The overexpression of PICH in PICH-depleted cells restored Nrf2 as well as antioxidant gene expression. In turn, Nrf2 regulated the expression of PICH in the presence of oxidative stress. ChIP experiments showed that PICH is present on the Nrf2 as well as antioxidant gene promoters, suggesting that the protein might be regulating the expression of these genes directly by binding to the DNA sequences. In addition, Nrf2 and histone acetylation (H3K27ac) also played a role in activating transcription in the presence of oxidative stress. Both Nrf2 and H3K27ac were found to be present on PICH and antioxidant promoters. Their occupancy was dependent on the PICH expression as fold enrichment was found to be decreased in PICH-depleted cells. PICH ablation led to the reduced expression of Nrf2 and impaired antioxidant response, leading to increased ROS content and thus showing PICH is essential for the cell to respond to oxidative stress.

2.
J Vis Exp ; (180)2022 02 10.
Article in English | MEDLINE | ID: mdl-35225275

ABSTRACT

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent advancements in CD spectroscopy have enabled the study of DNA-protein interactions and conformational dynamics of DNA in different microenvironments in detail for a better understanding of transcriptional regulation in vivo. The area around a potential transcription zone needs to be unwound for transcription to occur. This is a complex process requiring the coordination of histone modifications, binding of the transcription factor to DNA, and other chromatin remodeling activities. Using CD spectroscopy, it is possible to study conformational changes in the promoter region caused by regulatory proteins, such as ATP-dependent chromatin remodelers, to promote transcription. The conformational changes occurring in the protein can also be monitored. In addition, queries regarding the affinity of the protein towards its target DNA and sequence specificity can be addressed by incorporating mutations in the target DNA. In short, the unique understanding of this sensitive and inexpensive method can predict changes in chromatin dynamics, thereby improving the understanding of transcriptional regulation.


Subject(s)
Chromatin Assembly and Disassembly , Chromatin , Circular Dichroism , DNA/chemistry , Spectrum Analysis , Transcription Factors/genetics , Transcription Factors/metabolism
3.
Biochim Biophys Acta Gene Regul Mech ; 1861(12): 1076-1092, 2018 12.
Article in English | MEDLINE | ID: mdl-30317028

ABSTRACT

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.


Subject(s)
Ataxia Telangiectasia Mutated Proteins/physiology , DNA Helicases/physiology , Mitosis/physiology , Nuclear Proteins/physiology , Transcription Factors/physiology , Gene Expression Regulation , HeLa Cells , Humans , Phosphorylation
4.
Biosci Rep ; 38(3)2018 06 29.
Article in English | MEDLINE | ID: mdl-29748240

ABSTRACT

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.


Subject(s)
Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Chromatin/enzymology , DNA Helicases/metabolism , DNA/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Transcription Factors/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Amino Acid Motifs , Animals , Binding Sites , Cattle , Chromatin/ultrastructure , Cloning, Molecular , DNA/genetics , DNA Helicases/chemistry , DNA Helicases/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , HeLa Cells , Humans , Hydrolysis , Kinetics , Protein Binding , Protein Interaction Domains and Motifs , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Transcription Factors/chemistry , Transcription Factors/genetics
5.
Biochim Biophys Acta Gene Regul Mech ; 1860(9): 936-951, 2017 Sep.
Article in English | MEDLINE | ID: mdl-28716689

ABSTRACT

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.


Subject(s)
DEAD-box RNA Helicases/genetics , DNA Damage/genetics , DNA Helicases/genetics , Nuclear Proteins/genetics , RNA-Binding Proteins/genetics , Ribonuclease III/genetics , Transcription Factors/genetics , Transcription, Genetic/genetics , Cell Line, Tumor , DNA Breaks, Double-Stranded/drug effects , DNA Damage/drug effects , Down-Regulation/drug effects , Down-Regulation/genetics , Doxorubicin/pharmacology , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , HeLa Cells , Humans , MicroRNAs/genetics , RNA, Untranslated/genetics , Up-Regulation/drug effects , Up-Regulation/genetics
6.
Sci Rep ; 6: 20532, 2016 Feb 04.
Article in English | MEDLINE | ID: mdl-26843359

ABSTRACT

The ATP-dependent chromatin remodeling factors regulate gene expression. However, it is not known whether these factors regulate each other. Given the ability of these factors to regulate the accessibility of DNA to transcription factors, we postulate that one ATP-dependent chromatin remodeling factor should be able to regulate the transcription of another ATP-dependent chromatin remodeling factor. In this paper, we show that BRG1 and SMARCAL1, both members of the ATP-dependent chromatin remodeling protein family, regulate each other. BRG1 binds to the SMARCAL1 promoter, while SMARCAL1 binds to the brg1 promoter. During DNA damage, the occupancy of SMARCAL1 on the brg1 promoter increases coinciding with an increase in BRG1 occupancy on the SMARCAL1 promoter, leading to increased brg1 and SMARCAL1 transcripts respectively. This is the first report of two ATP-dependent chromatin remodeling factors regulating each other.


Subject(s)
DNA Helicases/genetics , DNA Helicases/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic , Cell Nucleus/metabolism , Cytoplasm/metabolism , DNA Damage , Gene Expression Regulation , HeLa Cells , Humans , Promoter Regions, Genetic , Regulatory Elements, Transcriptional
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