Van Steensel Research Articles

Telomere identity crisis

Cells are designed to be intolerant of breaks in DNA, yet it is critical that cells do not identify the ends of linear chromosomes, called telomeres, as damaged DNA ends. Telomeres therefore must somehow prevent the recognition and subsequent repair of chromosome ends as double-strand breaks (DSBs), although how this is achieved is poorly understood. Chromosomal breaks can occur as a result of ionizing radiation, DNA replication across nicked DNA, or as intermediates of recombination. Whether these breaks are induced by the cell—for example, due to V(D)J recombination during lymphocyte development—or arise as a consequence of spontaneous damage, it is imperative that such lesions be repaired in order to prevent genomic rearrangements or even outright chromosome loss (Bassing and Alt 2004). The cell avoids such deleterious consequences by mounting a response, called the DNA damage checkpoint, which pauses the cell cycle, thereby permitting the efficient recruitment of a highly conserved set of proteins to the break (Zhou and Elledge 2000; Nyberg et al. 2002). In the budding yeast Saccharomyces cerevisiae, this process has been studied extensively using an experimental system that allows the creation of a single DSB at high frequency at a defined site, through the action of a sequence-specific endonuclease, HO (Rudin and Haber 1988). The DNA damage checkpoint is alerted to the presence of the HO-generated break by recruitment of sensor proteins, such as Tel1/ATM and Mec1/ATR, to the site of damage (Lisby et al. 2004; Garber et al. 2005). These kinases activate downstream effector proteins, Rad53/CHK2, Rad9, and Dun1, which in turn facilitate the activation of repair proteins. Repair of DSBs requires the recognition of these broken ends by the Ku70/80 heterodimer and the Mre11–Rad50–Xrs2 complex (MRX, or MRN in humans). The cell then has a choice between two pathways for DNA repair. If DSBs are repaired through the nonhomologous end-joining pathway (NHEJ), DNA ligase 4 is brought in to seal the two ends together. If the repair is accomplished by homologous recombination, the DSB is first processed by an exonuclease to reveal a single-stranded 3 overhang, which subsequently initiates recombination with homologous sequences present in the genome. Functional DNA damage checkpoints also act to inhibit cell cycle progression in the presence of damaged DNA. Even a single DSB is sufficient to cause the cell cycle to arrest until repair is completed (Sandell and Zakian 1993), thereby ensuring that cells do not progress through mitosis until the integrity of the genome has been restored. Despite the presence of a highly efficient machine for the recognition of DSBs, the ends of linear chromosomes are natural DNA termini that must somehow be masked from triggering the DNA damage checkpoint and subsequent repair events. This unique property of telomeres is owed to the sequence and structure of the telomere DNA itself, as well as to the proteins that localize to chromosome ends (Blackburn 2001; Smogorzewska and de Lange 2004). In most eukaryotes, telomeres are composed of tandem G-rich repeats that terminate with a 3 singlestranded overhang of the G-rich strand, often referred to as a G-tail. Disruption of this G-tail structure, due to either loss of the G-tail itself or exposure of the C-strand to nucleolytic attack, is a lethal event for the cell. Thus, cells have developed a dynamic protein assembly that maintains a telomere “cap.” In budding yeast, this cap depends in part on the essential single-strand telomerebinding protein Cdc13, along with several Cdc13-interacting factors. Loss of the Cdc13 complex exposes yeast telomeres to massive resection of the C-strand, with the resulting 20–30-kb region of exposed single-stranded DNA provoking arrest of the cell cycle (Weinert et al. 1994; Garvik et al. 1995; Booth et al. 2001). A second protein, Rap1, which is bound to duplex telomeric repeats, may aid in protection of the other strand of the telomere, the G-strand overhang: Depleting cells of Rap1 causes telomere fusions that are mediated through the NHEJ pathway (Pardo and Marcand 2005). Mammalian cells also possess a mechanism to protect the vulnerable G-tail, which similarly relies on a duplex telomere DNA-binding factor called TRF2. In TRF2-deficient cells, the integrity of the terminal G-strand overhang is disrupted, resulting in high frequencies of end-to-end fusions (van Steensel et al. 1998; Celli and de Lange 2005). This TRF2-mediated telomere capping activity may be aided by the ability of telomeres to fold into a looped structure, called the t-loop, wherein the 3 overhang invades the duplex region of telomere repeats (Griffith et al. 1999). Not unexpectedly, normal telomeres rarely interact Corresponding author. EMAIL lundblad@salk.edu; FAX (858) 457-4765. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ gad.1373205.

Preface to Pachyonychia Congenita Symposium Proceedings

Preface to Pachyonychia Congenita Symposium Proceedings

A Ligand-mediated Hydrogen Bond Network Required for the Activation of the Mineralocorticoid Receptor

Ligand binding is the first step in hormone regulation of mineralocorticoid receptor (MR) activity. Here, we report multiple crystal structures of MR (NR3C2) bound to both agonist and antagonists. These structures combined with mutagenesis studies reveal that maximal receptor activation involves an intricate ligand-mediated hydrogen bond network with Asn770 which serves dual roles: stabilization of the loop preceding the C-terminal activation function-2 helix and direct contact with the hormone ligand. In addition, most activating ligands hydrogen bond to Thr945 on helix 10. Structural characterization of the naturally occurring S810L mutant explains how stabilization of a helix 3/helix 5 interaction can circumvent the requirement for this hydrogen bond network. Taken together, these results explain the potency of MR activation by aldosterone, the weak activation induced by progesterone and the antihypertensive agent spironolactone, and the binding selectivity of cortisol over cortisone.

Myc Antagonizes Ras-mediated Growth Arrest in Leukemia Cells through the Inhibition of the Ras-ERK-p21Cip1 Pathway

Even though RAS usually acts as a dominant transforming oncogene, in primary fibroblasts and some established cell lines Ras inhibits proliferation. This can explain the virtual absence of RAS mutations in some types of tumors, such as chronic myeloid leukemia (CML). We report that in the CML cell line K562 Ras induces p21Cip1 expression through the Raf-MEK-ERK pathway. Because K562 cells are deficient for p15INK4b, p16INK4a, p14ARF, and p53, this would be the main mechanism whereby Ras up-regulates p21 expression in these cells. Accordingly, we also found that Ras suppresses K562 growth by signaling through the Raf-ERK pathway. Because c-Myc and Ras cooperate in cell transformation and c-Myc is up-regulated in CML, we investigated the effect of c-Myc on Ras activity in K562 cells. c-Myc antagonized the induction of p21Cip1 mediated by oncogenic H-, K-, and N-Ras and by constitutively activated Raf and ERK2. Activation of the p21Cip1 promoter by Ras was dependent on Sp1/3 binding sites in K562. However, mutational analysis of the p21 promoter and the use of a Gal4-Sp1 chimeric protein strongly suggest that c-Myc affects Sp1 transcriptional activity but not the binding of Sp1 to the p21 promoter. c-Myc-mediated impairment of Ras activity on p21 expression required a transactivation domain, a DNA binding region, and a Max binding region. Moreover, the effect was independent of Miz1 binding to c-Myc. Consistent with its effect on p21Cip1 expression, c-Myc rescued cell growth inhibition induced by Ras. The data suggest that in particular tumor types, such as those associated with CML, c-Myc contributes to tumorigenesis by inhibiting Ras antiproliferative activity.

Importance of TRF1 for Functional Telomere Structure

Telomeres are comprised of telomeric DNA sequences and associated binding molecules. Their structure functions to protect the ends of linear chromosomes and ensure chromosomal stability. One of the mammalian telomere-binding factors, TRF1, localizes telomeres by binding to double-stranded telomeric DNA arrays. Because the overexpression of wild-type and dominant-negative TRF1 induces progressive telomere shortening and elongation in human cells, respectively, a proposed major role of TRF1 is that of a negative regulator of telomere length. Here we report another crucial function of TRF1 in telomeres. In conditional mouse TRF1 null mutant embryonic stem cells, TRF1 deletion induced growth defect and chromosomal instability. Although no clear telomere shortening or elongation was observed in short term cultured TRF1-deficient cells, abnormal telomere signals were observed, and TRF1-interacting telomere-binding factor, TIN2, lost telomeric association. Furthermore, another double-stranded telomeric DNA-binding factor, TRF2, also showed decreased telomeric association. Importantly, end-to-end fusions with detectable telomere signals at fusion points accumulated in TRF1-deficient cells. These results strongly suggest that TRF1 interacts with other telomere-binding molecules and integrates into the functional telomere structure.

Which end: dissecting Ku's function at telomeres and double-strand breaks.

The ends of chromosomes are active places. These natural DNA termini must be protected from degradation, recombination, and end-to-end fusions—events that would be ultimately fatal for the genome. In addition, the enzyme telomerase ensures that telomeres in proliferating cells, such as single-celled organisms like budding yeast or the mammalian germ line, are completely replicated. Over the past decade, a plethora of telomereassociated proteins have been identified, based on their ability to regulate telomerase access, provide protection from nucleases and recombinases, and control telomere length (for review, see McEachern et al. 2000; de Lange 2002). In the budding yeast, at least a dozen proteins interact with just the terminal single-stranded overhang, with an additional dozen or more proteins that are associated with the duplex region of the telomere. At mammalian telomeres, which are bound by additional factors that are apparently not present in yeast, the situation is even more complex. To make the task of understanding events that take place at telomeres even more challenging, many telomere-associated proteins perform more than one biochemical activity at chromosome ends. For example, in human cells, the telomere repeat binding factor TRF2 protects chromosomes from end-to-end fusions, and also acts as a negative regulator of telomere length (van Steensel et al. 1998; Loayza and De Lange 2003). The yeast telomere end-binding factor Cdc13 similarly performs several discrete tasks: Cdc13 both positively and negatively regulates telomere elongation by telomerase, as well as shields chromosome termini from unregulated degradation by nucleases (Garvik et al. 1995; Nugent et al. 1996; Booth et al. 2001; Chandra et al. 2001). Analyses of these Cdc13-mediated processes have been greatly aided by separation-of-function mutations that appear to selectively impair each activity. In an analogous manner, recent genetic studies of the catalytic subunit of telomerase have helped to identify discrete regions that mediate enzyme processivity, interaction with other telomerase subunits, or recruitment of the telomerase complex to chromosome termini (Peng et al. 2001; Armbruster et al. 2003; Friedman et al. 2003; Kim et al. 2003). One factor that is critical to several aspects of telomere biology, yet has lagged behind in this genetic dissection, is the Ku heterodimer. Composed of 70and 80-kD subunits, Ku was first appreciated for its role in double-strand break (DSB) repair (Taccioli et al. 1994). In both mammalian and yeast cells, DSBs that arise via developmentally regulated site-specific rearrangements [e.g., V(D)J recombination in vertebrate cells], or are induced experimentally or as a result of environmental assaults such as irradiation, must be readily repaired to maintain genome stability. Ku plays a central role in this process by binding to DNA double-strand ends and recruiting additional factors, which are required to complete repair via nonhomologous end-joining (for review, see Jackson 2002). Somewhat paradoxically, Ku also functions at telomeres, even though these DNA termini are not normally substrates for end-joining reactions. In fact, telomeres must be specifically protected from end-to-end fusions to maintain genomic stability. Furthermore, the role that the Ku heterodimer plays at telomeres is complex, as evidenced by the fact yku70or yku80budding yeast strains, although still viable, exhibit an impressively large number of phenotypes, indicative of severe telomere dysfunction. In these Ku-defective strains, the structure of the extreme single-stranded termini is altered by increased exposure to nucleolytic activity (Gravel et al. 1998; Polotnianka et al. 1998; Maringele and Lydall 2002). Yeast strains that lack Ku also have substantially shortened telomeres (Boulton and Jackson 1996; Porter et al. 1996), in part because of this enhanced nuclease action (A.A. Bertuch and V. Lundblad, in prep.), but primarily because of impaired regulation of telomerase (Peterson et al. 2001; Stellwagen et al. 2003). Additionally, loss of Ku function in yeast disrupts the transcriptional silencing of telomere-proximal genes, by altering the composition of factors that comprise telomeric heterochromatin (Laroche et al. 1998; Nugent et al. 1998; Mishra and Shore 1999). Analysis of yku70or yku80yeast null strains, therefore, has firmly established that the Ku heterodimer is a multifunctional player that contributes to telomere Corresponding author. E-MAIL abertuch@bcm.tmc.edu; FAX (713) 798-3457. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ gad.1146603.

Reply to correspondence from van Steensel—“Poland anomaly: Not unilateral or bilateral, but mosaic”

Reply to correspondence from van Steensel—“Poland anomaly: Not unilateral or bilateral, but mosaic”

TANK2, a New TRF1-associated Poly(ADP-ribose) Polymerase, Causes Rapid Induction of Cell Death upon Overexpression

Tankyrase (TANK1) is a human telomere-associated poly(ADP-ribose) polymerase (PARP) that binds the telomere-binding protein TRF1 and increases telomere length when overexpressed. Here we report characterization of a second human tankyrase, tankyrase 2 (TANK2), which can also interact with TRF1 but has properties distinct from those of TANK1. TANK2 is encoded by a 66-kilobase pair gene (TNKS2) containing 28 exons, which express a 6.7-kilobase pair mRNA and a 1166-amino acid protein. The protein shares 85% amino acid identity with TANK1 in the ankyrin repeat, sterile alpha-motif, and PARP catalytic domains but has a unique N-terminal domain, which is conserved in the murine TNKS2 gene. TANK2 interacted with TRF1 in yeast and in vitro and localized predominantly to a perinuclear region, similar to the properties of TANK1. In contrast to TANK1, however, TANK2 caused rapid cell death when highly overexpressed. TANK2-induced death featured loss of mitochondrial membrane potential, but not PARP1 cleavage, suggesting that TANK2 kills cells by necrosis. The cell death was prevented by the PARP inhibitor 3-aminobenzamide. In vivo, TANK2 may differ from TANK1 in its intrinsic or regulated PARP activity or its substrate specificity.

Nobles, Ministerials, and Knights in the Archdiocese of Salzburg

Previous articleNext article No AccessNobles, Ministerials, and Knights in the Archdiocese of SalzburgJohn B. FreedJohn B. Freed Search for more articles by this author PDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by Speculum Volume 62, Number 3Jul., 1987 The journal of the Medieval Academy of America Article DOIhttps://doi.org/10.2307/2846383 Views: 11Total views on this site Citations: 9Citations are reported from Crossref Copyright 1987 The Medieval Academy of AmericaPDF download Crossref reports the following articles citing this article:Cullen J. Chandler Carolingian Catalonia, 89 (Dec 2018).https://doi.org/10.1017/9781108565745 The education of the cleric, II, (Jan 2015): 208–235.https://doi.org/10.1017/CBO9781316091364.010 Clergy of cathedral and collegiate churches, (Jan 2015): 269–309.https://doi.org/10.1017/CBO9781316091364.012Arie van Steensel Origins and Transformations. Recent Historiography on the Nobility in the Medieval Low Countries I, History Compass 12, no.33 (Mar 2014): 263–272.https://doi.org/10.1111/hic3.12137John D. Cotts People, Economy, and Social Relations, (Jan 2013): 80–106.https://doi.org/10.1007/978-1-137-29608-5_3Robert Stacey Nobles and knights, (Oct 1999): 11–25.https://doi.org/10.1017/CHOL9780521362894.003 Primary sources and secondary works arranged by chapter, (Oct 1999): 835–982.https://doi.org/10.1017/CHOL9780521362894.042John B. Freed Medieval German Social History: Generalizations and Particularism, Central European History 25, no.11 (Dec 2008): 1–26.https://doi.org/10.1017/S0008938900019683Julia Barrow German cathedrals and the monetary economy in the twelfth century, Journal of Medieval History 16, no.11 (Jan 2012): 13–38.https://doi.org/10.1016/0304-4181(90)90012-P