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 ...

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 ...

Fred Hutchinson Cancer Research Center

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.

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

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.

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

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.

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

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.

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

Wild-type microglia do not reverse pathology in mouse models of Rett syndrome.

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.

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

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-β 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-β 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.

Uyen Lao

Division of Clinical Research,
Clinical Research Division

Fred Hutchinson Cancer Research Center

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

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

Uyen Lao

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Division of Clinical Research,Clinical Research Division

Fred Hutchinson Cancer Research Center

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

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

Division of Clinical Research,
Clinical Research Division