Dr David Clynes

My group is interested in exploring how ATRX prevents normal cells from elongating their telomeres via the ALT pathway, leading to tumorigenesis and trying to understand the role of ATRX in DNA replication and repair.

In addition we are interested in the identification of new drugs that could potentially be used to target ALT cancer cells.  The challenge of identifying new cancer drugs can be approached in different ways.  One way is understanding which gene and proteins underpin an abnormal characteristic of a cancer cell (in this case aberrant telomere lengthening or the lack of ATRX) to identify new targets for the rational design of drugs.  A second ways is screening pre-existing drug libraries and testing them for their ability to limit the growth of, or preferentially kill, ALT cancer cells that lack ATRX.

Name Department Institution Country
Prof Richard Gibbons FMedSci FRCP Nuffield Division of Clinical Laboratory Sciences Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Nguyen DT, Voon HPJ, Xella B, Scott C, Clynes D, Babbs C, Ayyub H, Kerry J, Sharpe JA, Sloane-Stanley JA et al. 2017. The chromatin remodelling factor ATRX suppresses R-loops in transcribed telomeric repeats. EMBO Rep, 18 (6), pp. 914-928. | Show Abstract | Read more

ATRX is a chromatin remodelling factor found at a wide range of tandemly repeated sequences including telomeres (TTAGGG)n ATRX mutations are found in nearly all tumours that maintain their telomeres via the alternative lengthening of telomere (ALT) pathway, and ATRX is known to suppress this pathway. Here, we show that recruitment of ATRX to telomeric repeats depends on repeat number, orientation and, critically, on repeat transcription. Importantly, the transcribed telomeric repeats form RNA-DNA hybrids (R-loops) whose abundance correlates with the recruitment of ATRX Here, we show loss of ATRX is also associated with increased R-loop formation. Our data suggest that the presence of ATRX at telomeres may have a central role in suppressing deleterious DNA secondary structures that form at transcribed telomeric repeats, and this may account for the increased DNA damage, stalling of replication and homology-directed repair previously observed upon loss of ATRX function.

Clynes D, Jelinska C, Xella B, Ayyub H, Scott C, Mitson M, Taylor S, Higgs DR, Gibbons RJ. 2015. Suppression of the alternative lengthening of telomere pathway by the chromatin remodelling factor ATRX. Nat Commun, 6 pp. 7538. | Show Abstract | Read more

Fifteen per cent of cancers maintain telomere length independently of telomerase by the homologous recombination (HR)-associated alternative lengthening of telomeres (ALT) pathway. A unifying feature of these tumours are mutations in ATRX. Here we show that expression of ectopic ATRX triggers a suppression of the pathway and telomere shortening. Importantly ATRX-mediated ALT suppression is dependent on the histone chaperone DAXX. Re-expression of ATRX is associated with a reduction in replication fork stalling, a known trigger for HR and loss of MRN from telomeres. A G-quadruplex stabilizer partially reverses the effect of ATRX, inferring ATRX may normally facilitate replication through these sequences that, if they persist, promote ALT. We propose that defective telomere chromatinization through loss of ATRX promotes the persistence of aberrant DNA secondary structures, which in turn present a barrier to DNA replication, leading to replication fork stalling, collapse, HR and subsequent recombination-mediated telomere synthesis in ALT cancers.

Chen E, Ahn JS, Massie CE, Clynes D, Godfrey AL, Li J, Park HJ, Nangalia J, Silber Y, Mullally A et al. 2014. JAK2V617F promotes replication fork stalling with disease-restricted impairment of the intra-S checkpoint response. Proc Natl Acad Sci U S A, 111 (42), pp. 15190-15195. | Show Abstract | Read more

Cancers result from the accumulation of genetic lesions, but the cellular consequences of driver mutations remain unclear, especially during the earliest stages of malignancy. The V617F mutation in the JAK2 non-receptor tyrosine kinase (JAK2V617F) is present as an early somatic event in most patients with myeloproliferative neoplasms (MPNs), and the study of these chronic myeloid malignancies provides an experimentally tractable approach to understanding early tumorigenesis. Introduction of exogenous JAK2V617F impairs replication fork progression and is associated with activation of the intra-S checkpoint, with both effects mediated by phosphatidylinositide 3-kinase (PI3K) signaling. Analysis of clonally derived JAK2V617F-positive erythroblasts from MPN patients also demonstrated impaired replication fork progression accompanied by increased levels of replication protein A (RPA)-containing foci. However, the associated intra-S checkpoint response was impaired in erythroblasts from polycythemia vera (PV) patients, but not in those from essential thrombocythemia (ET) patients. Moreover, inhibition of p53 in PV erythroblasts resulted in more gamma-H2Ax (γ-H2Ax)-marked double-stranded breaks compared with in like-treated ET erythroblasts, suggesting the defective intra-S checkpoint function seen in PV increases DNA damage in the context of attenuated p53 signaling. These results demonstrate oncogene-induced impairment of replication fork progression in primary cells from MPN patients, reveal unexpected disease-restricted differences in activation of the intra-S checkpoint, and have potential implications for the clonal evolution of malignancies.

Howe FS, Boubriak I, Sale MJ, Nair A, Clynes D, Grijzenhout A, Murray SC, Woloszczuk R, Mellor J. 2014. Lysine Acetylation Controls Local Protein Conformation by Influencing Proline Isomerization Molecular Cell, 55 (5), pp. 733-744. | Show Abstract | Read more

© 2014 The Authors. Gene transcription responds to stress and metabolic signals to optimize growth and survival. Histone H3 (H3) lysine 4 trimethylation (K4me3) facilitates state changes, but how levels are coordinated with the environment is unclear. Here, we show that isomerization of H3 at the alanine 15-proline 16 (A15-P16) peptide bond is influenced by lysine 14 (K14) and controls gene-specific K4me3 by balancing the actions of Jhd2, the K4me3 demethylase, and Spp1, a subunit of the Set1 K4 methyltransferase complex. Acetylation at K14 favors the A15-P16. trans conformation and reduces K4me3. Environmental stress-induced genes are most sensitive to the changes atK14 influencing H3 tail conformation and K4me3. By contrast, ribosomal protein genes maintain K4me3, required for their repression during stress, independently of Spp1, K14, and P16. Thus, the plasticity in control of K4me3, via signaling to K14 and isomerization at P16, informs distinct gene regulatory mechanisms and processes involving K4me3.

Clynes D, Jelinska C, Xella B, Ayyub H, Taylor S, Mitson M, Bachrati CZ, Higgs DR, Gibbons RJ. 2014. ATRX dysfunction induces replication defects in primary mouse cells. PLoS One, 9 (3), pp. e92915. | Show Abstract | Read more

The chromatin remodeling protein ATRX, which targets tandem repetitive DNA, has been shown to be required for expression of the alpha globin genes, for proliferation of a variety of cellular progenitors, for chromosome congression and for the maintenance of telomeres. Mutations in ATRX have recently been identified in tumours which maintain their telomeres by a telomerase independent pathway involving homologous recombination thought to be triggered by DNA damage. It is as yet unknown whether there is a central underlying mechanism associated with ATRX dysfunction which can explain the numerous cellular phenomena observed. There is, however, growing evidence for its role in the replication of various repetitive DNA templates which are thought to have a propensity to form secondary structures. Using a mouse knockout model we demonstrate that ATRX plays a direct role in facilitating DNA replication. Ablation of ATRX alone, although leading to a DNA damage response at telomeres, is not sufficient to trigger the alternative lengthening of telomere pathway in mouse embryonic stem cells.

Clynes D, Higgs DR, Gibbons RJ. 2013. The chromatin remodeller ATRX: a repeat offender in human disease. Trends Biochem Sci, 38 (9), pp. 461-466. | Show Abstract | Read more

The regulation of chromatin structure is of paramount importance for a variety of fundamental nuclear processes, including gene expression, DNA repair, replication, and recombination. The ATP-dependent chromatin-remodelling factor ATRX (α thalassaemia/mental retardation X-linked) has emerged as a key player in each of these processes. Exciting recent developments suggest that ATRX plays a variety of key roles at tandem repeat sequences within the genome, including the deposition of a histone variant, prevention of replication fork stalling, and the suppression of a homologous recombination-based pathway of telomere maintenance. Here, we provide a mechanistic overview of the role of ATRX in each of these processes, and propose how they may be connected to give rise to seemingly disparate human diseases.

Cited:

20

Scopus

Clynes D, Gibbons RJ. 2013. ATRX and the replication of structured DNA Current Opinion in Genetics and Development, 23 (3), pp. 289-294. | Show Abstract | Read more

Understanding the underlying molecular basis for disease can often be a prolonged and tortuous process with many false leads and blind alleys. Relating the cause of ATR-X syndrome to the function of the protein ATRX is a case in point. In this review we attempt to bring together the diverse biological phenomena associated with ATRX dysfunction with what has recently been discovered concerning the chromatin remodelling activity of this protein. This potentially casts light on how defective DNA replication/histone replacement can impact on transcription, telomere maintenance and also possibly chromosome segregation. © 2013 Elsevier Ltd.

Cited:

103

Scopus

Eustermann S, Yang JC, Law MJ, Amos R, Chapman LM, Jelinska C, Garrick D, Clynes D, Gibbons RJ, Rhodes D et al. 2011. Combinatorial readout of histone H3 modifications specifies localization of ATRX to heterochromatin Nature Structural and Molecular Biology, 18 (7), pp. 777-782. | Show Abstract | Read more

Accurate read-out of chromatin modifications is essential for eukaryotic life. Mutations in the gene encoding X-linked ATRX protein cause a mental-retardation syndrome, whereas wild-type ATRX protein targets pericentric and telomeric heterochromatin for deposition of the histone variant H3.3 by means of a largely unknown mechanism. Here we show that the ADD domain of ATRX, in which most syndrome-causing mutations occur, engages the N-terminal tail of histone H3 through two rigidly oriented binding pockets, one for unmodified Lys4 and the other for di- or trimethylated Lys9. In vivo experiments show this combinatorial readout is required for ATRX localization, with recruitment enhanced by a third interaction through heterochromatin protein-1 (HP1) that also recogniz es trimethylated Lys9. The cooperation of ATRX ADD domain and HP1 in chromatin recruitment results in a tripartite interaction that may span neighboring nucleosomes and illustrates how the 'histone-code' is interpreted by a combination of multivalent effector-chromatin interactions. © 2011 Nature America, Inc. All rights reserved.

Pinskaya M, Nair A, Clynes D, Morillon A, Mellor J. 2009. Nucleosome remodeling and transcriptional repression are distinct functions of Isw1 in Saccharomyces cerevisiae. Mol Cell Biol, 29 (9), pp. 2419-2430. | Show Abstract | Read more

The SANT domain is a nucleosome recognition module found in transcriptional regulatory proteins, including chromatin-modifying enzymes. It shows high functional degeneracy between species, varying in sequence and copy number. Here, we investigate functions in vivo associated with two SANT motifs, SANT and SLIDE, in the Saccharomyces cerevisiae Isw1 chromatin-remodeling ATPase. We show that differences in the primary structures of the SANT and SLIDE domains in yeast and Drosophila melanogaster reflect their different functions. In yeast, the SLIDE domain is required for histone interactions, while this is a function of the SANT domain in flies. In yeast, both motifs are required for optimal association with chromatin and for formation of the Isw1b complex (Isw1, Ioc2, and Ioc4). Moreover, nucleosome remodeling at the MET16 locus is defective in strains lacking the SANT or SLIDE domain. In contrast, the SANT domain is dispensable for the interaction between Isw1 and Ioc3 in the Isw1a complex. We show that, although defective in nucleosome remodeling, Isw1 lacking the SANT domain is able to repress transcription initiation at the MET16 promoter. Thus, chromatin remodeling and transcriptional repression are distinct activities of Isw1.

Walter W, Clynes D, Tang Y, Marmorstein R, Mellor J, Berger SL. 2008. 14-3-3 interaction with histone H3 involves a dual modification pattern of phosphoacetylation. Mol Cell Biol, 28 (8), pp. 2840-2849. | Show Abstract | Read more

Histone modifications occur in precise patterns and are proposed to signal the recruitment of effector molecules that profoundly impact chromatin structure, gene regulation, and cell cycle events. The linked modifications serine 10 phosphorylation and lysine 14 acetylation on histone H3 (H3S10phK14ac), modifications conserved from Saccharomyces cerevisiae to humans, are crucial for transcriptional activation of many genes. However, the mechanism of H3S10phK14ac involvement in these processes is unclear. To shed light on the role of this dual modification, we utilized H3 peptide affinity assays to identify H3S10phK14ac-interacting proteins. We found that the interaction of the known phospho-binding 14-3-3 proteins with H3 is dependent on the presence of both of these marks, not just phosphorylation alone. This is true of mammalian 14-3-3 proteins as well as the yeast homologues Bmh1 and Bmh2. The importance of acetylation in this interaction is also seen in vivo, where K14 acetylation is required for optimal Bmh1 recruitment to the GAL1 promoter during transcriptional activation.

Mellor J, Dudek P, Clynes D. 2008. A glimpse into the epigenetic landscape of gene regulation. Curr Opin Genet Dev, 18 (2), pp. 116-122. | Show Abstract | Read more

Post-translational modifications to histone proteins and methylation of DNA comprise the epigenome of a cell. The epigenome, which changes through development, controls access to our genes. Recent advances in DNA sequencing technology has led to genome-wide distribution data for a limited number of histone modifications in mammalian stem cells and some differentiated lineages. These studies reveal predictive correlations between histone modifications, different classes of gene and chromosomal features. Moreover, this glimpse into our epigenome challenges current ideas about regulation of gene expression. Many genes in stem cells are poised for expression with initiated RNA polymerase II at the promoter. This state is maintained by an epigenetic mark through multiple lineages until the gene is expressed.

Macdonald N, Welburn JPI, Noble MEM, Nguyen A, Yaffe MB, Clynes D, Moggs JG, Orphanides G, Thomson S, Edmunds JW et al. 2005. Molecular basis for the recognition of phosphorylated and phosphoacetylated histone h3 by 14-3-3. Mol Cell, 20 (2), pp. 199-211. | Show Abstract | Read more

Phosphorylation of histone H3 is implicated in transcriptional activation and chromosome condensation, but its immediate molecular function has remained obscure. By affinity chromatography of nuclear extracts against modified H3 tail peptides, we identified 14-3-3 isoforms as proteins that bind these tails in a strictly phosphorylation-dependent manner. Acetylation of lysines 9 and 14 does not impede 14-3-3 binding to serine 10-phosphorylated H3 tails. In vivo, 14-3-3 is inducibly recruited to c-fos and c-jun nucleosomes upon gene activation, concomitant with H3 phosphoacetylation. We have determined the structures of 14-3-3zeta complexed with serine 10-phosphorylated or phosphoacetylated H3 peptides. These reveal a distinct mode of 14-3-3/phosphopeptide binding and provide a structural understanding for the lack of effect of acetylation at lysines 9 and 14 on this interaction. 14-3-3 isoforms thus represent a class of proteins that mediate the effect of histone phosphorylation at inducible genes.

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