Regulation of ATM and ATR by SMARCAL1 and BRG1 [preprint]

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/BRG1 results in transcriptional repression of ATM/ATR and therefore, overriding of the G2/M checkpoint leading to mitotic abnormalities. On doxorubicin-induced DNA damage, SMARCAL1 and BRG1 are upregulated and in turn, upregulate the expression of ATM/ATR. Phosphorylation of ATM/ATR is needed for the transcriptional upregulation of SMARCAL1 and BRG1, and therefore, of ATM and ATR on DNA damage. The regulation of ATM/ATR is rendered non-functional if SMARCAL1 and/or BRG1 are absent or if the two proteins are mutated such that they are unable to hydrolyze ATP, as in for example in Schimke Immuno-Osseous Dysplasia and Coffin-Siris Syndrome. Thus, an intricate transcriptional regulation of DNA damage response genes mediated by SMARCAL1 and BRG1 is present in mammalian cells

Ras hyperactivation versus overexpression : Lessons from Ras dynamics in Candida albicans

We thank Prof. Neta Dean for the CIp10ADH1-Cherry plasmid and Prof. Aaron Mitchell for the BWP17 strain. We gratefully acknowledge Prof. Sudipta Maiti, TIFR, Mumbai, India for providing the data acquisition software. We also appreciate the feedback and discussions with Dr. Rohini Muthuswami, SLS, JNU as well as from the Protein Society group, New Delhi while this study was taking shape. We thank Prof. Alok Bhattacharya for Cytochalasin D. The GC-MS and fluorescence lifetime measurements were carried out at the Advanced Instrumentation Research Facility (AIRF), JNU. Confocal images were recorded either at the central instrumentation facility (CIF), SLS, JNU or at AIRF, JNU. This work was supported by project grants from Department of Biotechnology (DBT, Project grant no. BT/PR20410/BRB/10/1542/2016) and Department of Science and Technology (DST, Project grant no. SB/SO/BB-011/2014), India to S.S.K; and project grants from Department of Information Technology, (DIT, Project grant no. 12(4)/2007-PDD), India to S.S. for FCS setup. In addition, both S.S. and S.S.K. thank DBT-BUILDER for funding support (Project grant no. BT/PR5006/INF/153/2012). S.S.K. also acknowledges funding support from UGC Resource Networking grant to the School of Life Sciences. We thank DST-PURSE and JNU for assistance with funding for publication. G.S.V. and S.C.S. received a fellowship from UGC; V.A.P., B.Y., P.J., N.P., M.F.K. acknowledge CSIR for fellowships. S.L.S. received a fellowship from ICMR. D.T.H. and M.F.K. thank DBT-BUILDER for funding.Peer reviewedPublisher PD

Casein kinase 2-mediated phosphorylation of Brahma-related gene 1 controls myoblast proliferation and contributes to SWI/SNF complex composition

Transcriptional regulation is modulated in part by chromatin-remodeling enzymes that control gene accessibility by altering chromatin compaction or nucleosome positioning. Brahma-related gene 1 (Brg1), a catalytic subunit of the mammalian SWI/SNF chromatin-remodeling enzymes, is required for both myoblast proliferation and differentiation, and the control of Brg1 phosphorylation by calcineurin, PKCbeta1, and p38 regulates the transition to differentiation. However, we hypothesized that Brg1 activity might be regulated by additional kinases. Here, we report that Brg1 is also a target of casein kinase 2 (CK2), a serine/threonine kinase, in proliferating myoblasts. We found that CK2 interacts with Brg1, and mutation of putative phosphorylation sites to non-phosphorylatable (Ser to Ala, SA) or phosphomimetic residues (Ser to Glu, SE) reduced Brg1 phosphorylation by CK2. Although BRG1-deleted myoblasts that ectopically express the SA-Brg1 mutant proliferated similarly to the parental cells or cells ectopically expressing wild-type (WT) Brg1, ectopic expression of the SE-Brg1 mutant reduced proliferation and increased cell death, similar to observations from cells lacking Brg1. Moreover, pharmacological inhibition of CK2 increased myoblast proliferation. Furthermore, the Pax7 promoter, which controls expression of a key transcription factor required for myoblast proliferation, was in an inaccessible chromatin state in the SE-Brg1 mutant, suggesting that hyperphosphorylated Brg1 cannot remodel chromatin. WT-, SA-, and SE-Brg1 exhibited distinct differences in interacting with and affecting expression of the SWI/SNF subunits Baf155 and Baf170 and displayed differential sub-nuclear localization. Our results indicate that CK2-mediated phosphorylation of Brg1 regulates myoblast proliferation and provides insight into one mechanism by which composition of the mammalian SWI/SNF enzyme complex is regulated

CK2-Dependent Phosphorylation of the Brg1 Chromatin Remodeling Enzyme Occurs during Mitosis

Brg1 (Brahma-related gene 1) is one of two mutually exclusive ATPases that can act as the catalytic subunit of mammalian SWI/SNF (mSWI/SfigureNF) chromatin remodeling enzymes that facilitate utilization of the DNA in eukaryotic cells. Brg1 is a phospho-protein, and its activity is regulated by specific kinases and phosphatases. Previously, we showed that Brg1 interacts with and is phosphorylated by casein kinase 2 (CK2) in a manner that regulates myoblast proliferation. Here, we use biochemical and cell and molecular biology approaches to demonstrate that the Brg1-CK2 interaction occurred during mitosis in embryonic mouse somites and in primary myoblasts derived from satellite cells isolated from mouse skeletal muscle tissue. The interaction of CK2 with Brg1 and the incorporation of a number of other subunits into the mSWI/SNF enzyme complex were independent of CK2 enzymatic activity. CK2-mediated hyperphosphorylation of Brg1 was observed in mitotic cells derived from multiple cell types and organisms, suggesting functional conservation across tissues and species. The mitotically hyperphosphorylated form of Brg1 was localized with soluble chromatin, demonstrating that CK2-mediated phosphorylation of Brg1 is associated with specific partitioning of Brg1 within subcellular compartments. Thus, CK2 acts as a mitotic kinase that regulates Brg1 phosphorylation and subcellular localization