Summary DNA double strand breaks (DSBs) initiate extensive local and global alterations in chromatin structure, many of which depend on the ATM kinase. Histone H2A ubiquitylation (uH2A) on chromatin surrounding DSBs is one example, thought to be important for recruitment of repair proteins. uH2A is also implicated in transcriptional repression; an intriguing yet untested hypothesis is that this function is conserved in the context of DSBs. Using a novel reporter that allows for visualization of repair protein recruitment and local transcription in single cells, we describe an ATM-dependent transcriptional silencing program in cis to DSBs. ATM prevents RNA polymerase II elongation dependent chromatin decondensation at regions distal to DSBs. Silencing is partially dependent on E3 ubiquitin ligases RNF8 and RNF168, while reversal of silencing relies on the uH2A deubiquitylating enzyme USP16. These findings give insight into the role of post-translational modifications in mediating cross talk between diverse processes occurring on chromatin.

Understanding gene expression requires the ability to follow the fate of individual molecules. Here we use a cellular system for monitoring messenger RNA (mRNA)expression to characterize the movement in real time of single mRNA-protein complexes (mRNPs) in the nucleus of living mammalian cells. This mobility was not directed but was governed by simple diffusion. Some mRNPs were partially corralled throughout the nonhomogenous nuclear environment, but no accumulation at subnuclear domains was observed. Following energy deprivation, energyindependent motion of mRNPs was observed in a highly ATP-dependent nuclear environment; movements were constrained to chromatin-poor domains and excluded by newly formed chromatin barriers. This observation resolves a controversy, showing that the energetic requirements of nuclear mRNP trafficking are consistent with a diffusional model. Recent technological developments have facilitated imaging of single RNA molecules in the cytoplasm of living cells (1). We have developed a cellular system in which the expression of a transgene array can be followed sequentially in single living cells, and we have previously analyzed the particular chromatin-related modifications occurring at this specific locus from a silenced state throughout its transcriptional activation (2). Here we use this system to address the mechanism by which individual mRNA transcripts move within the nucleoplasm after release from the transcription site.In this system, a genetic locus, its transcribed mRNAs, and the translated protein were rendered visible in cells after electroporation with the cyan fluorescent protein (CFP) or the red fluorescent protein (RFP)-lac repressor protein (marks the genomic locus), yellow fluorescent protein (YFP)-MS2 (labels the mRNA), and pTet-On (for transcriptional induction) (3). Transcriptional activation by doxycycline induced the unfolding of the integrated locus (4) and the recruitment of the YFP-MS2 protein to the locus as a result of the specific labeling of the nascent transcripts bearing the MS2 stem loops. Minutes after induction (15 to 30 min), the MS2 signal began accumulating in the nucleoplasm in a particulate pattern suggestive of mRNA-protein complexes (mRNPs). At later times (1 to 2 hours after induction), mRNPs were detected in the cytoplasm in conjunction with the Correspondence to: Robert H. Singer, rhsinger@aecom.yu.edu. The presence of the nascent RNAs at the site of transcription was verified using fluorescent in situ hybridization (FISH) on fixed cells with two different probes either to the MS2 repeats (located in the middle of the transcript) or to the β globin exon (3′ end). Both probes hybridized at the active locus, indicating that the complete pre-mRNA transcripts were retained at the transcription site before release ( fig. S2A to D). Colocalization of the mRNA signal (FISH) with the YFP signal demonstrated that the particles visualized in living cells were mRNPs (3). RNA quantification with single-molecule sensitivity was performed on deconvol...

We have developed an inducible system to visualize gene expression at the levels of DNA, RNA and protein in living cells. The system is composed of a 200 copy transgene array integrated into a euchromatic region of chromosome 1 in human U2OS cells. The condensed array is heterochromatic as it is associated with HP1, histone H3 methylated at lysine 9, and several histone methyltransferases. Upon transcriptional induction, HP1alpha is depleted from the locus and the histone variant H3.3 is deposited suggesting that histone exchange is a mechanism through which heterochromatin is transformed into a transcriptionally active state. RNA levels at the transcription site increase immediately after the induction of transcription and the rate of synthesis slows over time. Using this system, we are able to correlate changes in chromatin structure with the progression of transcriptional activation allowing us to obtain a real-time integrative view of gene expression.

DNA double-strand breaks (DSBs) are repaired via nonhomologous end-joining (NHEJ) or homologous recombination (HR), but cellular repair processes remain elusive. We show here that the ATP-dependent chromatin-remodeling factors, ACF1 and SNF2H, accumulate rapidly at DSBs and are required for DSB repair in human cells. If the expression of ACF1 or SNF2H is suppressed, cells become extremely sensitive to X-rays and chemical treatments producing DSBs, and DSBs remain unrepaired. ACF1 interacts directly with KU70 and is required for the accumulation of KU proteins at DSBs. The KU70/80 complex becomes physically more associated with the chromatin-remodeling factors of the CHRAC complex, which includes ACF1, SNF2H, CHRAC15, and CHRAC17, after treatments producing DSBs. Furthermore, the frequency of NHEJ as well as HR induced by DSBs in chromosomal DNA is significantly decreased in cells depleted of either of these factors. Thus, ACF1 and its complexes play important roles in DSBs repair.