Jiyue Zhu - US grants

DESCRIPTION (provided by applicant): Our long-term goal is to determine the mechanisms of telomerase regulation during development. The hTERT gene, which encodes the limiting subunit of human telomerase, is primarily regulated at the level of transcription. It is highly expressed in pluripotent stem cells, but stringently repressed in most adult somatic cells. Despite intensive investigation in the past decade, mechanisms of its repression, including cis-regulatory elements and trans-acting factors remain to be elucidated. We previously reported that the endogenous hTERT locus was embedded in a condensed chromatin domain in many somatic cells, while such a domain did not exist in the less repressed mouse TERT gene. Consistent with the vital role of chromatin in its tight regulation, we also found that an episomal hTERT locus in human fibroblasts was not subjected to repression, whereas a chromosomally integrated hTERT locus recapitulated its native regulation. Thus, we hypothesize that 1) the interplay between distal elements and core promoter in their native chromatin context is important for hTERT repression;and 2) partial loss of this repression leads to hTERT transcription during cellular immortalization. To study the mechanisms of hTERT repression, we developed a novel technical platform, the recombinase-mediated BAC targeting or RMBT method, for targeted integration of single-copy BAC reporters into specified chromosomal sites. Using this technique, we demonstrated that chromosomal integration of a BAC construct containing the hTERT locus resulted in the establishment of a surrogate chromatin setting in which the hTERT promoter was tightly repressed and recapitulated its endogenous gene in human fibroblasts. In this application, we plan to pursue the following specific aims: 1) Delineate cis elements involved in hTERT repression in human fibroblasts. 2) Identify and characterize protein factors involved in hTERT repression in human fibroblasts. 3) Determine cis elements that confer humanized regulation of the mTERT gene in mESCs. PUBLIC HEALTH RELEVANCE: Telomeres are ends of linear chromosomes and essential for long-term cell proliferation and survival. Telomerase, the enzyme that elongates telomeres, plays an important role in cancers and aging-related diseases. This grant application is proposed to determine the mechanisms of telomerase regulation in human somatic cells.

DESCRIPTION (provided by applicant): Noninvasive imaging methods that allow the tracking of critical steps in tumorigenesis and cancer progression in mouse models can impact importantly on efforts to understand tumorigenesis and to monitor the efficacy of anticancer drug candidates. Telomerase activation occurs in over 90% of human cancers as well as in mouse cancer models, and has been proposed be a diagnostic marker for cancer. Here, we propose to generate transgenic luciferase reporter mice with mouse and human genomic DNA constructs for monitoring the in vivo TERT expression, the rate-limiting step of telomerase activation. Our primary objective is to test the feasibility of using such reporters as imaging markers to monitor tumor progression in live animals. These reporters can potentially be used to image tumors in a broad spectrum of mouse cancer models and provide a standard platform for pre-clinical anticancer drug evaluation. In addition, direct comparison of mouse and human TERT promoter activities in these transgenic reporter mice may also reveal the molecular mechanisms underlying the different regulation of telomerase expression in mice and humans. Such difference may account for the discrepancies in tumor spectra between some human cancers and their mouse models.

DESCRIPTION (provided by applicant): Tumors shed their cells into the peripheral blood, generating metastases that confer their lethality. Circulating tumor cells (CTCs) have been detected in patients of many types of cancers, even those with no signs of clinically overt metastases. Because CTCs are accessible using minimally invasive procedures, the detection and characterization of CTCs are pivotal for early diagnosis, prognosis, and treatment monitoring, as well as for understanding of the molecular and cellular events that lead to metastases. Our objective is to establish a sensitive and reliable method for detecting and capturing CTCs of all tumor types, regardless of their tissue origins, in research and clinical settings. We hypothesize that the telomerase-selective adenoviruses specifically label cancer cells and provide a simple method for efficient CTC detection and isolation. Our specific aims are (1) to generate replication-competent adenoviral vectors with optimized hTERT promoters for selective labeling of viable cancer cells; (2) to generate and characterize a tet-regulatable telomerase-selective adenovirus for the detection and isolation of viable CTCs.

Abstract Telomeres function as an aging clock because most human somatic cells lack telomerase expression and their telomeres progressively shorten upon successive cell divisions. Accordingly, telomere shortening is a critical factor of human aging and telomerase activation is essential for the development of many human cancers. On the other hand, cells from some other organisms, including mice, do not exhibit telomere-mediated replicative aging. Mice possess long heterogeneous telomeres and ubiquitous telomerase activities in somatic tissues. This inter-species difference has become a bottleneck for addressing many fundamental questions in human aging and cancer biology using mouse models. Thus, the long-term goal of this project is to genetically engineer mouse strains with human-like telomere homeostasis and to test the hypothesis that short telomeres and repressed telomerase confer replicative aging in mice. Our recent studies have identified several distal regulatory elements critical for the repression of hTERT gene. In this proposal, we will test the hypothesis that incorporation of these elements into their corresponding mouse genomic sites leads to human-like TERT gene regulation in mice. We will first engineer mouse embryonic stem cells (ESCs) with a humanized mTERT locus (hmTERT) and then use these ESCs to generate mouse strains with human-like telomerase regulation. The regulation of hmTERT locus will be determined both during in vitro ESC differentiation and during mouse development. We anticipate that these mice will serve as an improved model for studying human aging and cancer.

Abstract Our long-term goal is to determine the mechanisms of telomerase regulation during development. The hTERT gene, encoding the limiting subunit of human telomerase, is primarily regulated at the level of transcription. It is highly expressed in pluripotent stem cells, but stringently repressed in most somatic cells. Regulation of transcription during development and differentiation often involves distal elements and chromatin reorganization, in addition to proximal promoter elements. We previously showed that the endogenous hTERT locus was embedded in a condensed chromatin domain in many somatic cells. Consistent with the vital role of chromatin in its tight regulation, we found that an episomal hTERT locus in human fibroblasts was not subjected to repression, whereas a chromosomally integrated hTERT locus recapitulated its native regulation. To study the mechanisms of hTERT repression, we developed a novel technical platform, the recombinase-mediated BAC targeting or RMBT method, for targeted integration of single-copy BAC reporters into specified chromosomal sites. This technique enables us to study the regulation of hTERT gene in its native genomic context. Here, we hypothesize that 1) the interplay between distal elements and core promoter in their native chromatin contexts is important for hTERT repression in somatic cells and reactivation in cancer cells; and 2) polymorphic regulatory elements of the hTERT gene impact human aging and tumorigenesis. We propose three specific aims: 1) determine the recruitment of corepressor complexes and their roles in hTERT repression; 2) determine the regulation of hTERT promoter by Ets family proteins in normal and cancer cells; 3) determine the function and genetic variation of a polymorphic element in hTERT regulation and human longevity.

Abstract Our long-term goal is to determine the mechanisms of telomerase regulation during development. The hTERT gene, encoding the limiting subunit of human telomerase, is primarily regulated at the level of transcription. It is highly expressed in pluripotent stem cells, but stringently repressed in most somatic cells. Regulation of transcription during development and differentiation often involves distal elements and chromatin reorganization, in addition to proximal promoter elements. We previously showed that the endogenous hTERT locus was embedded in a condensed chromatin domain in many somatic cells. Consistent with the vital role of chromatin in its tight regulation, we found that an episomal hTERT locus in human fibroblasts was not subjected to repression, whereas a chromosomally integrated hTERT locus recapitulated its native regulation. To study the mechanisms of hTERT repression, we developed a novel technical platform, the recombinase-mediated BAC targeting or RMBT method, for targeted integration of single-copy BAC reporters into specified chromosomal sites. This technique enables us to study the regulation of hTERT gene in its native genomic context. Here, we hypothesize that 1) the interplay between distal elements and core promoter in their native chromatin contexts is important for hTERT repression in somatic cells and reactivation in cancer cells; and 2) polymorphic regulatory elements of the hTERT gene impact human aging and tumorigenesis. We propose three specific aims: 1) determine the recruitment of corepressor complexes and their roles in hTERT repression; 2) determine the regulation of hTERT promoter by Ets family proteins in normal and cancer cells; 3) determine the function and genetic variation of a polymorphic element in hTERT regulation and human longevity.

Abstract The goal of this project is to develop a mouse model that acquires human-like telomere homeostasis for the study of human aging, cancer, and other age-related diseases. In humans, most somatic cells lack telomerase expression and cannot replenish their telomeres. Consequently, telomeres progressively shortened upon successive cell divisions and function as an aging clock. Accordingly, telomere shortening is a critical factor of human aging and telomerase activation is essential for the development of most human cancers. On the other hand, some other organisms, including laboratory mice, do not exhibit telomere-mediated replicative aging. Mice possess long telomeres and ubiquitous telomerase activity in adult tissues. This interspecies difference has become a bottleneck for addressing many fundamental questions in human aging and cancer biology using mouse models. To tackle this challenge, we have started to create a mouse strain with humanized telomere homeostasis. Here, we have engineered a humanized mouse Tert allele (hmTert) by using regulatory sequences from the human TERT gene (hTERT) to replace their mouse counterparts. We found that the hmTert gene regulation recapitulated that of the hTERT gene during mouse development and in mouse adult tissues. In this application, we propose the following three specific aims: 1) Create a mouse strain with hmTert alleles and human-like short telomeres; 2) Study replicative aging in mice with humanized telomere homeostasis; and 3) Determine lifespan and health-span of mice with humanized telomere homeostasis.