Sir2 and Fun30 regulate ribosomal DNA replication timing via Mcm helicase positioning and nucleosome occupancy.

The association between late replication timing and low transcription rates in eukaryotic heterochromatin is well-known, yet the specific mechanisms underlying this link remain uncertain. In Saccharomyces cerevisiae, the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA arrays (rDNA). We have previously reported that in the absence of SIR2, a derepressed RNA PolII repositions MCM replicative helicases from their loading site at the ribosomal origin, where they abut well-positioned, high-occupancy nucleosomes, to an adjacent region with lower nucleosome occupancy. By developing a method that can distinguish activation of closely spaced MCM complexes, here we show that the displaced MCMs at rDNA origins have increased firing propensity compared to the non-displaced MCMs. Furthermore, we found that both, activation of the repositioned MCMs and low occupancy of the adjacent nucleosomes critically depend on the chromatin remodeling activity of FUN30. Our study elucidates the mechanism by which Sir2 delays replication timing, and it demonstrates, for the first time, that activation of a specific replication origin in vivo relies on the nucleosome context shaped by a single chromatin remodeler.

Identification of 1600 replication origins in S. cerevisiae.

There are approximately 500 known origins of replication in the yeast genome, and the process by which DNA replication initiates at these locations is well understood. In particular, these sites are made competent to initiate replication by loading of the Mcm replicative helicase prior to the start of S phase; thus, 'a site that binds Mcm in G1' might be considered to provide an operational definition of a replication origin. By fusing a subunit of Mcm to micrococcal nuclease, we previously showed that known origins are typically bound by a single Mcm double hexamer, loaded adjacent to the ARS consensus sequence (ACS). Here, we extend this analysis from known origins to the entire genome, identifying candidate Mcm binding sites whose signal intensity varies over at least three orders of magnitude. Published data quantifying single-stranded DNA (ssDNA) during S phase revealed replication initiation among the most abundant 1600 of these sites, with replication activity decreasing with Mcm abundance and disappearing at the limit of detection of ssDNA. Three other hallmarks of replication origins were apparent among the most abundant 5500 sites. Specifically, these sites: (1) appeared in intergenic nucleosome-free regions flanked on one or both sides by well-positioned nucleosomes; (2) were flanked by ACSs; and (3) exhibited a pattern of GC skew characteristic of replication initiation. We conclude that, if sites at which Mcm double hexamers are loaded can function as replication origins, then DNA replication origins are at least threefold more abundant than previously assumed, and we suggest that replication may occasionally initiate in essentially every intergenic region. These results shed light on recent reports that as many as 15% of replication events initiate outside of known origins, and this broader distribution of replication origins suggest that S phase in yeast may be less distinct from that in humans than widely assumed.

Identification of 1600 replication origins in S. cerevisiae.

There are approximately 500 known origins of replication in the yeast genome, and the process by which DNA replication initiates at these locations is well understood. In particular, these sites are made competent to initiate replication by loading of the Mcm replicative helicase prior to the start of S phase; thus, "a site to which MCM is bound in G1" might be considered to provide an operational definition of a replication origin. By fusing a subunit of Mcm to micrococcal nuclease, a technique referred to as "Chromatin Endogenous Cleavage", we previously showed that known origins are typically bound by a single Mcm double hexamer, loaded adjacent to the ARS consensus sequence (ACS). Here we extend this analysis from known origins to the entire genome, identifying candidate Mcm binding sites whose signal intensity varies over at least 3 orders of magnitude. Published data quantifying the production of ssDNA during S phase showed clear evidence of replication initiation among the most abundant 1600 of these sites, with replication activity decreasing in concert with Mcm abundance and disappearing at the limit of detection of ssDNA. Three other hallmarks of replication origins were apparent among the most abundant 5,500 sites. Specifically, these sites (1) appeared in intergenic nucleosome-free regions that were flanked on one or both sides by well-positioned nucleosomes; (2) were flanked by ACSs; and (3) exhibited a pattern of GC skew characteristic of replication initiation. Furthermore, the high resolution of this technique allowed us to demonstrate a strong bias for detecting Mcm double-hexamers downstream rather than upstream of the ACS, which is consistent with the directionality of Mcm loading by Orc that has been observed in vitro. We conclude that, if sites at which Mcm double-hexamers are loaded can function as replication origins, then DNA replication origins are at least 3-fold more abundant than previously assumed, and we suggest that replication may occasionally initiate in essentially every intergenic region. These results shed light on recent reports that as many as 15% of replication events initiate outside of known origins, and this broader distribution of replication origins suggest that S phase in yeast may be less distinct from that in humans than is widely assumed.

Chromosomal Mcm2-7 distribution and the genome replication program in species from yeast to humans.

The spatio-temporal program of genome replication across eukaryotes is thought to be driven both by the uneven loading of pre-replication complexes (pre-RCs) across the genome at the onset of S-phase, and by differences in the timing of activation of these complexes during S phase. To determine the degree to which distribution of pre-RC loading alone could account for chromosomal replication patterns, we mapped the binding sites of the Mcm2-7 helicase complex (MCM) in budding yeast, fission yeast, mouse and humans. We observed similar individual MCM double-hexamer (DH) footprints across the species, but notable differences in their distribution: Footprints in budding yeast were more sharply focused compared to the other three organisms, consistent with the relative sequence specificity of replication origins in S. cerevisiae. Nonetheless, with some clear exceptions, most notably the inactive X-chromosome, much of the fluctuation in replication timing along the chromosomes in all four organisms reflected uneven chromosomal distribution of pre-replication complexes.

Discovery of Selective SIRT2 Inhibitors as Therapeutic Agents in B-Cell Lymphoma and Other Malignancies.

Genetic ablation as well as pharmacological inhibition of sirtuin 2 (SIRT2), an NAD+-dependent protein deacylase, have therapeutic effects in various cancers and neurodegenerative diseases. Previously, we described the discovery of a dual SIRT1/SIRT2 inhibitor called cambinol (IC50 56 and 59 µM, respectively), which showed cytotoxic activity against cancer cells in vitro and a marked anti-proliferative effect in a Burkitt lymphoma mouse xenograft model. A number of recent studies have shown a protective effect of SIRT1 and SIRT3 in neurodegenerative and metabolic diseases as well as in certain cancers prompting us to initiate a medicinal chemistry effort to develop cambinol-based SIRT2-specific inhibitors devoid of SIRT1 or SIRT3 modulating activity. Here we describe potent cambinol-based SIRT2 inhibitors, several of which show potency of ~600 nM with >300 to >800-fold selectivity over SIRT1 and 3, respectively. In vitro, these inhibitors are found to be toxic to lymphoma and epithelial cancer cell lines. In particular, compounds 55 (IC50 SIRT2 0.25 µM and <25% inhibition at 50 µM against SIRT1 and SIRT3) and 56 (IC50 SIRT2 0.78 µM and <25% inhibition at 50 µM against SIRT1 and SIRT3) showed apoptotic as well as strong anti-proliferative properties against B-cell lymphoma cells.

Sir2 suppresses transcription-mediated displacement of Mcm2-7 replicative helicases at the ribosomal DNA repeats.

Repetitive DNA sequences within eukaryotic heterochromatin are poorly transcribed and replicate late in S-phase. In Saccharomyces cerevisiae, the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA arrays (rDNA). Despite the widespread association between transcription and replication timing, it remains unclear how transcription might impinge on replication, or vice versa. Here we show that, when silencing of an RNA polymerase II (RNA Pol II)-transcribed non-coding RNA at the rDNA is disrupted by SIR2 deletion, RNA polymerase pushes and thereby relocalizes replicative Mcm2-7 helicases away from their loading sites to an adjacent region with low nucleosome occupancy, and this relocalization is associated with increased rDNA origin efficiency. Our results suggest a model in which two of the major defining features of heterochromatin, transcriptional silencing and late replication, are mechanistically linked through suppression of polymerase-mediated displacement of replication initiation complexes.

Perturbed maintenance of transcriptional repression on the inactive X-chromosome in the mouse brain after Xist deletion.

BACKGROUND: The long noncoding RNA Xist is critical for initiation and establishment of X-chromosome inactivation during embryogenesis in mammals, but it is unclear whether its continued expression is required for maintaining X-inactivation in vivo. RESULTS: By using an inactive X-chromosome-linked MeCP2-GFP reporter, which allowed us to enumerate reactivation events in the mouse brain even when they occur in very few cells, we found that deletion of Xist in the brain after establishment of X-chromosome inactivation leads to reactivation in 2-5% of neurons and in a smaller fraction of astrocytes. In contrast to global loss of both H3 lysine 27 trimethylation (H3K27m3) and histone H2A lysine 119 monoubiquitylation (H2AK119ub1) we observed upon Xist deletion, alterations in CpG methylation were subtle, and this was mirrored by only minor alterations in X-chromosome-wide gene expression levels, with highly expressed genes more prone to both derepression and demethylation compared to genes with low expression level. CONCLUSION: Our results demonstrate that Xist plays a role in the maintenance of histone repressive marks, DNA methylation and transcriptional repression on the inactive X-chromosome, but that partial loss of X-dosage compensation in the absence of Xist in the brain is well tolerated.

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome.

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have been directed toward elucidation of the location and timing of initiation of genome duplication rather than its completion. However, it is critical that we understand regions of the genome that are last to complete replication, because these regions suffer elevated levels of chromosomal breakage and mutation, and they have been associated with both disease and aging. Here we describe how we have extended a technique that has been used to monitor replication initiation to instead identify those regions of the genome last to complete replication. This approach is based on a combination of flow cytometry and high throughput sequencing. Although this report focuses on the application of this technique to yeast, the approach can be used with any cells that can be sorted in a flow cytometer according to DNA content.

Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome.

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control in model organisms. Technologies including short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR) have enabled such screens in diploid mammalian cells. Here we describe a large-scale shRNA screen for regulators of X-chromosome inactivation (XCI), using a murine cell line with firefly luciferase and hygromycin resistance genes knocked in at the C-terminus of the methyl CpG binding protein 2 (MeCP2) gene on the inactive X-chromosome (Xi). Reactivation of the construct in the reporter cell line conferred survival advantage under hygromycin B selection, enabling us to screen a large shRNA library and identify hairpins that reactivated the reporter by measuring their post-selection enrichment using next-generation sequencing. The enriched hairpins were then individually validated by testing their ability to activate the luciferase reporter on Xi.

Screen for reactivation of MeCP2 on the inactive X chromosome identifies the BMP/TGF-ß superfamily as a regulator of XIST expression.

Rett syndrome (RS) is a debilitating neurological disorder affecting mostly girls with heterozygous mutations in the gene encoding the methyl-CpG-binding protein MeCP2 on the X chromosome. Because restoration of MeCP2 expression in a mouse model reverses neurologic deficits in adult animals, reactivation of the wild-type copy of MeCP2 on the inactive X chromosome (Xi) presents a therapeutic opportunity in RS. To identify genes involved in MeCP2 silencing, we screened a library of 60,000 shRNAs using a cell line with a MeCP2 reporter on the Xi and found 30 genes clustered in seven functional groups. More than half encoded proteins with known enzymatic activity, and six were members of the bone morphogenetic protein (BMP)/TGF-ß pathway. shRNAs directed against each of these six genes down-regulated X-inactive specific transcript (XIST), a key player in X-chromosome inactivation that encodes an RNA that coats the silent X chromosome, and modulation of regulators of this pathway both in cell culture and in mice demonstrated robust regulation of XIST. Moreover, we show that Rnf12, an X-encoded ubiquitin ligase important for initiation of X-chromosome inactivation and XIST transcription in ES cells, also plays a role in maintenance of the inactive state through regulation of BMP/TGF-ß signaling. Our results identify pharmacologically suitable targets for reactivation of MeCP2 on the Xi and a genetic circuitry that maintains XIST expression and X-chromosome inactivation in differentiated cells.

SIR2 suppresses replication gaps and genome instability by balancing replication between repetitive and unique sequences.

Replication gaps that persist into mitosis likely represent important threats to genome stability, but experimental identification of these gaps has proved challenging. We have developed a technique that allows us to explore the dynamics by which genome replication is completed before mitosis. Using this approach, we demonstrate that excessive allocation of replication resources to origins within repetitive regions, induced by SIR2 deletion, leads to persistent replication gaps and genome instability. Conversely, the weakening of replication origins in repetitive regions suppresses these gaps. Given known age- and cancer-associated changes in chromatin accessibility at repetitive sequences, we suggest that replication gaps resulting from misallocation of replication resources underlie age- and disease-associated genome instability.

Development of pyrazolone and isoxazol-5-one cambinol analogues as sirtuin inhibitors.

Sirtuins are a family of NAD(+)-dependent protein deacetylases that play critical roles in epigenetic regulation, stress responses, and cellular aging in eukaryotic cells. In an effort to identify small molecule inhibitors of sirtuins for potential use as chemotherapeutics as well as tools to modulate sirtuin activity, we previously identified a nonselective sirtuin inhibitor called cambinol (IC50 ≈ 50 µM for SIRT1 and SIRT2) with in vitro and in vivo antilymphoma activity. In the current study, we used saturation transfer difference (STD) NMR experiments with recombinant SIRT1 and 20 to map parts of the inhibitor that interacted with the protein. Our ongoing efforts to optimize cambinol analogues for potency and selectivity have resulted in the identification of isoform selective analogues: 17 with >7.8-fold selectivity for SIRT1, 24 with >15.4-fold selectivity for SIRT2, and 8 with 6.8- and 5.3-fold selectivity for SIRT3 versus SIRT1 and SIRT2, respectively. In vitro cytotoxicity studies with these compounds as well as EX527, a potent and selective SIRT1 inhibitor, suggest that antilymphoma activity of this compound class may be predominantly due to SIRT2 inhibition.

Enterocyte-specific inactivation of SIRT1 reduces tumor load in the APC(+/min) mouse model.

SIRT1 is a mammalian NAD(+)-dependent histone deacetylase implicated in metabolism, development, aging and tumorigenesis. Prior studies that examined the effect of enterocyte-specific overexpression and global deletion of SIRT1 on polyp formation in the intestines of APC(+/min) mice, a commonly used model for intestinal tumorigenesis, yielded conflicting results, supporting either tumor-suppressive or tumor-promoting roles for SIRT1, respectively. In order to resolve the controversy emerging from these prior in vivo studies, in the present report we examined the effect of SIRT1 deficiency confined to the intestines, avoiding the systemic perturbations such as growth retardation seen with global SIRT1 deletion. We crossed APC(+/min) mice with mice bearing enterocyte-specific inactivation of SIRT1 and examined polyp development in the progeny. We found that SIRT1-inactivation reduced total polyp surface (9.3 mm(2) vs. 23.3 mm(2), p = 0.01), average polyp size (0.24 mm(2) vs. 0.51 mm(2), p = 0.005) and the number of polyps >0.5 mm in diameter (14 vs. 23, p = 0.04), indicating that SIRT1 affects both the number and size of tumors. Additionally, tumors in SIRT1-deficient mice exhibited markedly increased numbers of cells undergoing apoptosis, suggesting that SIRT1 contributes to tumor growth by enabling survival of tumor cells. Our results indicate that SIRT1 acts as a tumor promoter in the APC(+/min) mouse model of intestinal tumorigenesis.

LST8 regulates cell growth via target-of-rapamycin complex 2 (TORC2).

The evolutionarily conserved serine/threonine protein kinase target-of-rapamycin (TOR) controls cell growth as a core component of TOR complexes 1 (TORC1) and 2 (TORC2). Although TORC1 is the more central growth regulator, TORC2 has also been shown to affect cell growth. Here, we demonstrate that Drosophila LST8, the only conserved TOR-binding protein present in both TORC1 and TORC2, functions exclusively in TORC2 and is not required for TORC1 activity. In mutants lacking LST8, expression of TOR and RAPTOR, together with their upstream activator Rheb, was sufficient to provide TORC1 activity and stimulate cell and organ growth. Furthermore, using an lst8 knockout mutation, we show that TORC2 regulates cell growth cell autonomously. Surprisingly, however, TORC2 does not regulate cell growth via its best-characterized target, AKT. Our findings support the possible application of TORC2-specific drugs in cancer therapy.

TOR-mediated autophagy regulates cell death in Drosophila neurodegenerative disease.

Target of rapamycin (TOR) signaling is a regulator of cell growth. TOR activity can also enhance cell death, and the TOR inhibitor rapamycin protects cells against proapoptotic stimuli. Autophagy, which can protect against cell death, is negatively regulated by TOR, and disruption of autophagy by mutation of Atg5 or Atg7 can lead to neurodegeneration. However, the implied functional connection between TOR signaling, autophagy, and cell death or degeneration has not been rigorously tested. Using the Drosophila melanogaster visual system, we show in this study that hyperactivation of TOR leads to photoreceptor cell death in an age- and light-dependent manner and that this is because of TOR's ability to suppress autophagy. We also find that genetically inhibiting TOR or inducing autophagy suppresses cell death in Drosophila models of Huntington's disease and phospholipase C (norpA)-mediated retinal degeneration. Thus, our data indicate that TOR induces cell death by suppressing autophagy and provide direct genetic evidence that autophagy alleviates cell death in several common types of neurodegenerative disease.