Chromatin proteins play important roles in gene expression a

Chromatin proteins play important roles in gene expression and DNA repair. The importance of chromatin composition on gene expression is exemplified by X-inactivation, where one of the two X chromosomes in female mammalian DHAP is transcriptionally silenced by heterochromatin , , , . In the ATR pathway, a histone acetyltransferase-containing protein complex termed TFTC preferentially binds UV-damaged DNA and acetylates histone H3 in vitro . ATM interactions with the histone acetyltransferase hMOF , histone H2AX , and the histone acetyltransferase TIP60 are important for the repair of DSBs. ATM kinase was shown to be activated when cells are exposed to agents that alter chromatin structure but do not cause detectable DSBs; this led to the proposal that DSBs activate ATM via chromatin changes . In , loss of Atm reduces the levels of heterochromatin protein 1 (HP1) at telomeres and increases HP1 abundance at an internal euchromatic region . In mammalian cells, ATM and ATR associate with histone deacetylases even in the absence of DNA damaging agents , . Here we show that disruption of Atm and Atr function destabilizes the silencing of a reporter gene on the mouse inactive X chromosome (Xi). In contrast, the reporter gene was not reactivated when cells with functionally intact Atm and Atr genes were subjected to considerable DNA damage. Materials and methods Preparation of the reporter cell line with GFP on the Xi has been described [24], [25]. 2-Aminopurine (Sigma Chemical Co., Saint Louis) was applied at 5mM to cell culture for 1 day [for immunostaining or gene reactivation with 5-aza-cytidine (5-AZ), Sigma Chemical Co., Saint Louis] for 3 days (gene reactivation without 5-AZ). Caffeine (Sigma Chemical Co., Saint Louis) was applied at 5mM to cell culture for 3 days. RNA FISH was performed as described in [26] using a 50-mer DNA probe complementary to map position 3064–3113, which is in Repeat C of Xist. Immunostaining was performed as described previously [27], with modifications. Briefly, for macroH2A staining, murine embryonic fibroblasts were grown on coverslips, and then treated with methanol for fixation, followed by TBST (0.1M Tris 8.0, 0.15M NaCl, and 0.5% Triton X-100) washes. Cells were immunostained with anti-macroH2A (Upstate Inc., Lake Placid) or anti-acetyl H4 (Serotec, UK) in 3% TBST milk at 4°C overnight. After TBS wash, FITC-conjugated secondary antibody (Sigma, Saint Louis) was applied for 30min. DAPI (Vector Laboratories, Burlingame) was used for counterstaining. Metaphase spread chromosomes were prepared by harvesting colcemid-treated cells followed by centrifugation onto coverslips. NIH image 1.63 software (http://rsb.info.nih.gov/nih-image/Default.html) was used to quantify the FITC signal of immunostained mitotic chromosome spreads. For each individual chromosome in a spread, the number of pixels occupied by each chromosome (DAPI) and the number of pixels displaying FITC signal were determined. SiRNA was made with Silencer Construction (Ambion, Austin) and transfections were performed as previously described [25]. Human counterpart of SiRNA sequence for Atm was in [28] and for Atr in [29]. SiRNA sequences were as follows: Atr/musAntisense (AAA CCA AGA CAG ATT CTC TGC CCT GTC TC), Atr/musSense (aaG CAG AGA ATC TGT CTT GGT CCT GTC TC), Atm/musAntisense (AAC ATA CTA CTC AAA GAC ATT CCT GTC TC), Atm/musSense (aaA ATG TCT TTG AGT AGT ATG CCT GTC TC), and SMC1/Antisense (AAG ACT TGA AGG AGA AGA TGA CCT GTC TC), SMC1/Sense (aaT CAT CTT CTC CTT CAA GTC CCT GTC TC). The control siRNA sequence is AAG GGC CTT CAG TAT GTC CTT CCT GTC TC, which is against Brca1 with a mismatch that prevents RNA interference. Nine microliter of oligofectamine (Invitrogen, Carlsbad) and 0.12μM siRNA (final concentration) were transfected into 6-well plates at 2×105 cells/well and assayed using flow cytometry 3 days later. To quantitate the levels of Atm and Atr transcripts in siRNA-treated cells, reporter cells were, in parallel, subjected to siRNA against Smc1. GFP-expressing cells from each of the three siRNA reactions were flow sorted and subjected to real-time RT-PCR for either Atm and Gadph or Atr and Gadph. The PCR primers used for these real-time RT-PCRs were Atr-reverse (TGT TCA CCC ATT CAA TAA TCC CAC), Atr-forward (TAA AAG GCT TGT AGA AGA CCC GAC), Atm-reverse (GTG CGC AGA CAG CAG AGT TCT CCA CGA TTC), Atm-forward (GAA GGC CTG GAT GCT GTG AAT CTG TGG GTT), and Gapdh reverse (CAT ACC AGG AAA TGA GCT TG), and Gapdh forward (ATG ACA TCA AGA AGG TGG TG). The conditions for RT-PCR were 95°C for 2min, then 40 cycles of 95°C for 15s, 56°C for 15s, and 72°C for 30s. For the reverse transcription reaction we used the reverse primers and M-MuLV reverse transcriptase (New England BioLabs, Beverly, MA) following manufacturers instructions. Real-time PCR was carried out by using iQ Syber-Green reaction mix from Bio-Rad (Hercules, CA) following manufacturers instructions. Atm and Atr mRNA levels thus obtained from siRNA treated cells were normalized against the Gapdh mRNA levels in the same reactions and then expressed a percent of the transcript level for Atm or Atr in cells treated with siRNA directed against Smc1.