Abstract Our long-term goal is to decipher the molecular mechanisms of telomerase regulation and telomere homeostasis during development. Telomerase elongates telomeres to compensate for their loss during cell proliferation. Its regulation is critical for human aging and susceptibilities to cancer and many age-related degenerative diseases. The TERT gene, encoding the human telomerase reverse transcriptase, is regulated primarily at the level of transcription. It is highly expressed in pluripotent stem cells, but stringently repressed in most somatic cells. Recent progresses on telomerase regulation in cancer cells have greatly improved our understanding of TERT gene activation during cancer development. However, the mechanisms of its repression in most differentiated cells and expression in certain somatic cells remain to be elucidated. Regulation of transcription during development and differentiation often involves distal elements and chromatin reorganization. We previously reported that the endogenous TERT gene was embedded in a condensed chromatin domain and stringently repressed in a histone deacetylase-dependent manner in somatic cells. To identify distal regulatory sequences required for establishing the repressive chromatin of the TERT locus and to understand its regulation in vivo, our laboratory has developed two innovative technical platforms in the past decade. The first is recombinase-mediated BAC targeting or RMBT method, for targeted integration of single-copy BAC reporters into specified chromosomal sites. This technique, together with the new CRISPR-mediated gene editing, enables us to study distal regulatory elements of the TERT gene in their genomic contexts. Consequently, we have discovered that a polymorphic tandem DNA repeat in intron 2 (VNTR2-1) functions as an enhancer for TERT transcription. In addition, we have engineered a humanized mTert allele (hmTert) for studying human-specific telomerase regulation in mice. In the next funding period, we plan to use these tools and focus on the following three directions: (1) Identify transcription factors that regulate TERT gene via VNTR2-1; (2) Identify key distal regulatory elements responsible for TERT repression; and (3) Study the roles of these distal elements in regulating telomere homeostasis in vivo using our mouse model with humanized telomeres. In short, using our unique tools, we will address some of the fundamental mechanisms critical to telomerase regulation and telomere homeostasis in humans, and ultimately telomere-associated human diseases.