Harold C. Riethman, Ph.D.

Associate Professor
Molecular and Cellular Oncogenesis Program
215-898-3872, Office
riethman@wistar.org
www.wistar.org/riethman

Introduction

The tips of human chromosomes, called telomeres, contain important genetic information and help control when cells divide. Harold C. Riethman, Ph.D., and colleagues are studying the DNA of telomeres to better understand diseases which result from damaged or rearranged telomeres. Deletion or rearrangement of small chromosome regions adjacent to telomeres (subtelomeric DNA) causes a range of disorders including mental retardation, muscular dystrophy, and heart defects. Dysfunctional telomeres are also associated with both the natural aging process and with cancer development. The Riethman lab has isolated and dissected DNA from human telomeres as part of the Human Genome Project. Molecular tools already developed as part of this effort are being used widely for detecting and characterizing subtelomeric DNA rearrangements that lead to human disease, and new tools and methods are being developed and applied to the analysis of telomere dysfunction and understanding the role of telomeres in aging and cancer.

Research Summary

Research in the Riethman laboratory focuses on analyzing the structure, function, and evolution of mammalian telomere regions.

Telomeres are dynamic and complex chromosomal structures. They are essential for genome stability and faithful chromosome replication, and mediate key biological activities including cell cycle regulation, cellular aging and immortalization, movements and localization of chromosomes within the nucleus, and transcriptional regulation of subtelomeric genes. The DNA at each human chromosome terminus is a simple repeat sequence tract (TTAGGG)n, typically 5 kb to 15 kb in length in somatic cells, that ends with a single-stranded extension of the G-strand of DNA  The lengths of the terminal repeat tracts are dynamically modulated in a tissue-specific and individual-specific manner; loss of this sequence tract is correlated with cellular and organismal aging, and continuous maintenance of this tract is essential for cellular immortalization and cancer progression. Adjacent to this “terminal repeat” is a subtelomeric repeat region comprised of a mosaic patchwork of segmentally duplicated DNA. This class of low-copy repeat DNA is characterized by very high sequence similarity (90 % to >99.5 %) between duplicated tracts, and variably-sized but often very large duplicated segment lengths (1 kb  to > 200 kb). The aggregate size of a subtelomeric repeat region varies according to the specific telomere; the shortest subtelomeric repeat region is 2 kb in length and the longest is greater than 500 kb.  At many individual telomeres, allelic differences in the sizes of subtelomeric repeat regions can be large, sometimes on the order of hundreds of kilobases in length.

The large copy number polymorphisms (CNPs) and unusual sequence organization has complicated the cloning, mapping, and sequencing of human subtelomeres. These same properties make subtelomere-associated regions especially prone to deletions, translocations, and other DNA rearrangements, and has led to their rapid evolution. For example, evolutionarily recent duplicative translocations of large subtelomeric DNA tracts has led to the generation of new gene families in primates, and to the formation of novel fusion transcripts with potentially new functions. In addition, germline instability of subtelomeric DNA leads to human diseases;  the deletion of a segment of subtelomeric repeat DNA near the 4q telomere is closely linked with the genetic disease FSHD, terminal deletions of 16p result in alpha-thalassemia, and an estimated 5-10% of all cases of idiopathic mental retardation are associated with cryptic rearrangements of subtelomeric DNA.  An important unanswered question is whether/how the large subtelomeric structural variations contribute to genome instability and to both normal and disease phenotypes.

Proper maintenance of the terminal (TTAGGG)n DNA tract is critical for cell division and genome stability. Telomere shortening and/or dysfuntion triggers cellular senescence or apoptosis in cells with intact checkpoint pathways. However, if these checkpoints are bypassed, continued telomere shortening leads to genome instability, crisis, and reactivation of telomere maintenance pathways which permit cellular immortalization and cancer.  Human aging correlates both with shortened telomeres and with a dramatic rise in cancer. More generally, individuals with telomeres shorter than age-matched controls are susceptible to a range of diseases and have a higher mortality. (TTAGGG)n tract lengths are telomere-specific and regulated in part by cis-acting subtelomeric DNA. Our lab is developing and refining methods for accurately tracking individual subtelomeric alleles in human populations, and for measuring (TTAGGG)n tract lengths at individual human telomeres.  This capability may reveal inborn individual-specific differences in telomere lengths that could affect susceptibility to aging and cancer, and would facilitate investigation of human telomere function and dysfunction in molecular detail. A key scientific question for us is whether/how cellular senescence relates to organismal aging and age-related pathologies including cancer and heart disease.

Recent Scientific Advances

Cloning, mapping, and collaborative DNA sequencing efforts culminated in reference sequences for each of the 41 genetically distinct human subtelomeric regions (Riethman et al. 2001, 2004). Sequence gaps that remain on the reference telomeres are generally small, well-defined, and for the most part restricted to regions directly adjacent to the terminal (TTAGGG)n tract. Distal subtelomere regions are highly enriched in recently-duplicated chromosome segments relative to the rest of the human genome. Our recent analysis of the substructure of this duplicated DNA has shown that (1) some duplicon blocks comprising the mosaic patchwork are very highly similar (98 to 99.5% identical), whereas others are less so; (2) Internal islands of (TTAGGG)n-like sequences, which are involved in controlling DNA replication and in enhanced recombination in model organisms,  are enriched > 25-fold in subtelomeric DNA relative to the rest of the genome and are almost always located at duplicon boundaries; (3) While most duplicon blocks have copies in both internal and in subtelomeric regions, a subset localizes exclusively to subtelomeres – these subtelomere-specific blocks may provide an opportunity to develop probes for tracking particular subtelomeric CNPs; (4) subterminal duplicon blocks, which are immediately adjacent to terminal (TTAGGG)n tracts and are potentially involved in cis-regulation of allele-specific (TTAGGG)n tract length, group into just 6 families of sequences, the detailed characterization of which may permit development of subterminal genotyping assays.

Transcripts annotated in our subtelomere assemblies frequently span 1-copy/duplicon and duplicon/duplicon boundaries, indicating that the ongoing evolutionary shuffling of human subtelomeric genomic DNA segments is providing opportunities for the duplication and generation of new genes. The overall transcript density in subtelomere regions is similar (within about 10%) to that which is found genome-wide, but there is wide variability in gene density among individual telomeres. Zinc finger-containing genes, olfactory receptor genes, and many additional transcript families of unknown function were found to be duplicated within and between multiple human telomere regions. Comparative genomic approaches with non-human primate subtelomeres could reveal how human subtelomeres evolved and point towards functionally important subtelomeric sequence elements.

Telomeric and subtelomeric DNA regions exhibit a very high level of structural variation that is likely to impact telomere function but has yet to be characterized in detail. A new resource of clone libraries and associated paired-end reads constructed as part of the Human Structural Variation initiative is being used along with a combination of computational and wet-lab  mapping methods to identify and characterize structural variants in distal human subtelomere regions. Terminal fosmids from these libraries are being identified, mapped and used to characterize and sequence (TTAGGG)n-adjacent subterminal DNA. Identification and characterization of telomeric structural variants will close many of the remaining gaps in the human genome sequence, and will in the long term reveal the universe of common germline subterminal allele structures that exist.  The global complement of variant subtelomeric alleles in a given individual may have important consequences for expression in gene-rich subtelomeric regions, and could contribute substantially to both natural human phenotypic variation and to disease phenotypes. Creating a database of allele-specific subterminal sequences will help fill a critical gap in our knowledge of telomere structure and its potential impact on length regulation and stability.  Appropriately regulated (TTAGGG)n tracts are critical for normal cell function;  individual (TTAGGG)n tract lengths in humans are allele-specific and regulated in part by cis-acting subtelomeric elements. The sequence information on subterminal alleles and allelic variants will open new avenues of telomere research and provide novel opportunities for developing PCR-based methods to track subterminal genotypes in populations and measure allele-specific telomere lengths.

The human subtelomeric reference sequence has been used to help develop diagnostic assays to detect subtelomeric DNA rearrangements. In collaboration with a group at Childrens Hospital of Philadelphia, we extended this work to fine-map deletions and duplications in subtelomeric regions of DNA from children with mental retardation and heart defects. Candidate genes for several of these developmental phenotypes have been identified by our analysis, and several recurrent subtelomeric breakpoint regions have been localized. Interestingly, several of these correspond to copy number variant sites and gaps in the reference sequence.

Pilot studies are being carried out to investigate telomere maintenance, subtelomeric epigenetic changes, cellular senescence and cellular immortalization in several contexts. Microarray analysis of gene expression is being carried out to investigate pathways involved in cellular senescence in human diploid fibroblasts and in human endothelial cells in the context of different oxidative stress levels. One goal of these studies is the identification of biomarkers of senescent cells that can be applied to characterization of in vivo senescence and oxidative stress in the contexts of cancer, atherosclerosis, and infertility. A second goal is to develop a more comprehensive and mechanistic understanding of cellular senescence. A collaboration with Kurt Barnhart’s group (Penn Ob/Gyn) is focused on identifying senescent and oxidative stress –associated markers useful for characterizing premature aging and infertility. We are analyzing a set of fibroblast cell line samples from Woody Wright (Univ of Texas-Southwestern) which are identical except for controlled telomere length variations induced by telomerase activation in several contexts, to test for gene expression specifically associated with very long or very short telomeres.

Future Plans

A principal focus of the lab will remain analysis of human subtelomeric DNA structure, variation, and evolution. Cloning and collaborative sequencing of DNA segments responsible for subtelomeric CNPs is of fundamental importance for completing the human genome sequence and for understanding telomere biology. Comparative genomic analysis with fully sequenced non-human primate subtelomeres is critical for understanding the evolution of human subtelomeres and will shed light on functionally important subtelomere sequences, including new genes created by subtelomeric DNA shuffling.

Subterminal DNA genotypes associated with single-telomere (TTAGGG)n tracts lengths, along with subtelomeric CNP data and subtelomeric duplicon copy number information,  comprise aspects of human variation that may be directly relevant to aging, cancer, and other telomere-associated phenotypes. This variation cannot be ascertained by HapMappable SNPs.  In the next 3-5 years we plan to continue development of reagents to permit tracking of these sources of subtelomeric variation and to begin assessing correlations with phenotype in relevant human populations.

A new collaborative initiative (with the Lieberman, Skordalakes, Rauscher and M. Herlyn labs) is investigating telomere function in human stem cells. Our lab is focusing on the role of telomeric and subtelomeric epigenetic DNA changes in (TTAGGG)n tract length regulation and stability in human stem cells. Human telomere (TTAGGG)n tract length is known to be regulated at individual telomeres by cis-acting DNA elements; however, little is known about the epigenetic status of this DNA other than it is hypomethylated in the germ line, methylated de novo in somatic tissues, and may contain non-canonical DNA modifications.   Hypomethylation of subterminal DNA in the human germline (and suppression of meiotic recombination in the most distal 2-4 kb of telomere-adjacent DNA)  are characteristics of satellite heterochromatin, but the active transcription of genes within a few kb of some human (TTAGGG)n tracts suggest a very small telomeric heterochromatic region and a unique and precisely regulated heterochromatic-euchromatic boundary region in human subterminal DNA.  We have found from our sequence analyses of human telomeres that human subterminal DNA sequences typically contain clusters of very CpG-rich sequences which are arranged in variably sized and organized stretches among separate telomeres. We hypothesize that these sequences are among those differentially methylated in germline vs differentiated cells, that the methylation patterns will reflect the telomeric heterochromatin-euchromatin transition, and that the position of the heterochromatic-euchromatin boundary will in turn help to determine (TTAGGG)n length for individual telomeres. In stem cells, the subterminal chromatin structure reflected and/or determined by the methylation patterns may be required for the precise maintenance of (TTAGGG)n tract length by telomerase; the dynamic regulation of the chromatin may be critical for helping to determine pluripotency vs differentiation.  We are currently using bisulfite sequencing of human subterminal DNA to investigate subterminal methylation patterns in hES cells as they are induced to differentiate into melanocytes.

Selected Publications

Riethman, H., Xiang, Z., Paul, S., Morse, E., Hu, X.-L., Flint., J., Grady, D., Chi, H., and Moyzis, R.K. 2001. Integration of telomere sequences with the draft human genome sequence. Nature 409: 948-951.

Riethman, H.,  Ambrosini, A.,  Castaneda, C.,  Finklestein, J.,  Hu, X-L.,  Mudunuri, U., Paul, S., and Wei, J. 2004. Mapping and initial analysis of human subtelomeric sequence assemblies. Genome Research 14: 18-28.

Gibbs RA, Weinstock GM, …. Riethman H, … Collins F.  Rat Genome Sequencing Project Consortium. 2004. Genome sequence of the Brown Norway rat yields insights into mammalian evolution. Nature 428: 493-521.

International Human Genome Sequencing Consortium. 2004. Finishing the euchromatic sequence of the human genome. Nature 431: 931-945.

Riethman, H.,  Ambrosini, A.  and Paul, S. 2005.  Human subtelomere structure and variation.  Chromosome Research 13: 505-515.

Ambrosini A,  Paul S,  Hu S,  and Riethman H.  2007. Human Subtelomeric Duplicon Structure and Organization.  Genome Biology 2007, 8:R151.

DeScipio, C., Nancy B. Spinner, Maninder Kaur, Dinah Yaeger, Anthony Ambrosini, Sufen Hu, Simei Shan, Ian D. Krantz, and Harold Riethman. 2008. Fine-Mapping Subtelomeric Deletions and Duplications by Comparative Genomic Hybridization in 42 Individuals. Am J. Med Genet., In press