Fun30 and Rtt109 Mediate Epigenetic Regulation of the DNA Damage Response Pathway in C. albicans.

Fun30, an ATP-dependent chromatin remodeler from S. cerevisiae, is known to mediate both regulation of gene expression as well as DNA damage response/repair. The Fun30 from C. albicans has not yet been elucidated. We show that C. albicans Fun30 is functionally homologous to both S. cerevisiae Fun30 and human SMARCAD1. Further, C. albicans Fun30 can mediate double-strand break end resection as well as regulate gene expression. This protein regulates transcription of RTT109, TEL1, MEC1, and SNF2-genes that encode for proteins involved in DNA damage response and repair pathways. The regulation mediated by C. albicans Fun30 is dependent on its ATPase activity. The expression of FUN30, in turn, is regulated by histone H3K56 acetylation catalyzed by Rtt109 and encoded by RTT109. The RTT109Hz/FUN30Hz mutant strain shows sensitivity to oxidative stress and resistance to MMS as compared to the wild-type strain. Quantitative PCR showed that the sensitivity to oxidative stress results from downregulation of MEC1, RAD9, MRC1, and RAD5 expression; ChIP experiments showed that Fun30 but not H3K56ac regulates the expression of these genes in response to oxidative stress. In contrast, upon treatment with MMS, the expression of RAD9 is upregulated, which is modulated by both Fun30 and H3K56 acetylation. Thus, Fun30 and H3K56 acetylation mediate the response to genotoxic agents in C. albicans by regulating the expression of DNA damage response and repair pathway genes.

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.

Transcriptional Regulation of Atp-Dependent Chromatin Remodeling Factors: Smarcal1 and Brg1 Mutually Co-Regulate Each Other.

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.

SMARCAL1 Negatively Regulates C-Myc Transcription By Altering The Conformation Of The Promoter Region.

SMARCAL1, a member of the SWI2/SNF2 protein family, stabilizes replication forks during DNA damage. In this manuscript, we provide the first evidence that SMARCAL1 is also a transcriptional co-regulator modulating the expression of c-Myc, a transcription factor that regulates 10-15% genes in the human genome. BRG1, SMARCAL1 and RNAPII were found localized onto the c-myc promoter. When HeLa cells were serum starved, the occupancy of SMARCAL1 on the c-myc promoter increased while that of BRG1 and RNAPII decreased correlating with repression of c-myc transcription. Using Active DNA-dependent ATPase A Domain (ADAAD), the bovine homolog of SMARCAL1, we show that the protein can hydrolyze ATP using a specific region upstream of the CT element of the c-myc promoter as a DNA effector. The energy, thereby, released is harnessed to alter the conformation of the promoter DNA. We propose that SMARCAL1 negatively regulates c-myc transcription by altering the conformation of its promoter region during differentiation.

Ligand-induced conformation changes drive ATP hydrolysis and function in SMARCAL1.

Mutations and deletions in SMARCAL1, an SWI2/SNF2 protein, cause Schimke immuno-osseous dysplasia (SIOD). SMARCAL1 preferentially binds to DNA molecules possessing double-stranded to single-stranded transition regions and mediates annealing helicase activity. The protein is critical for alleviating replication stress and maintaining genome integrity. In this study, we have analysed the ATPase activity of three mutations ­ A468P, I548N and S579L ­ present in SIOD patients. These mutations are present in RecA-like domain I of the protein. Analysis using active DNA-dependent ATPase A domain (ADAAD), an N-terminal deleted construct of bovine SMARCAL1, showed that all three mutants were unable to hydrolyse ATP. Conformational studies indicated that the α-helix and ß-sheet content of the mutant proteins was altered compared to the wild-type protein. Molecular simulation studies confirmed that major structural changes had occurred in the mutant proteins. These changes included alteration of a loop region connecting motif Ia and II. As motif Ia has been implicated in DNA binding, ligand binding studies were done using fluorescence spectroscopy. These studies revealed that the Kd for protein-DNA interaction in the presence of ATP was indeed altered in the case of mutant proteins compared to the wild-type. Finally, in vivo studies were done to complement the in vitro and in silico studies. The results from these experiments demonstrate that mutations in human SMARCAL1 that result in loss in ATPase activity lead to increased replication stress and therefore possibly manifestation of SIOD.