Hyeung Kim

TPP1 is a homologue of ciliate TEBP-β and interacts with POT1 to recruit telomerase

Telomere dysfunction may result in chromosomal abnormalities, DNA damage responses, and even cancer. Early studies in lower organisms have helped to establish the crucial role of telomerase and telomeric proteins in maintaining telomere length and protecting telomere ends. In Oxytricha nova, telomere G-overhangs are protected by the TEBP-α/β heterodimer. Human telomeres contain duplex telomeric repeats with 3′ single-stranded G-overhangs, and may fold into a t-loop structure that helps to shield them from being recognized as DNA breaks. Additionally, the TEBP-α homologue, POT1, which binds telomeric single-stranded DNA (ssDNA), associates with multiple telomeric proteins (for example, TPP1, TIN2, TRF1, TRF2 and RAP1) to form the six-protein telosome/shelterin and other subcomplexes. These telomeric protein complexes in turn interact with diverse pathways to form the telomere interactome for telomere maintenance. However, the mechanisms by which the POT1-containing telosome communicates with telomerase to regulate telomeres remain to be elucidated. Here we demonstrate that TPP1 is a putative mammalian homologue of TEBP-β and contains a predicted amino-terminal oligonucleotide/oligosaccharide binding (OB) fold. TPP1–POT1 association enhanced POT1 affinity for telomeric ssDNA. In addition, the TPP1 OB fold, as well as POT1–TPP1 binding, seemed critical for POT1-mediated telomere-length control and telomere-end protection in human cells. Disruption of POT1–TPP1 interaction by dominant negative TPP1 expression or RNA interference (RNAi) resulted in telomere-length alteration and DNA damage responses. Furthermore, we offer evidence that TPP1 associates with the telomerase in a TPP1-OB-fold-dependent manner, providing a physical link between telomerase and the telosome/shelterin complex. Our findings highlight the critical role of TPP1 in telomere maintenance, and support a yin–yang model in which TPP1 and POT1 function as a unit to protect human telomeres, by both positively and negatively regulating telomerase access to telomere DNA.

TRF2 functions as a protein hub and regulates telomere maintenance by recognizing specific peptide motifs

In mammalian cells, the telomeric repeat binding factor (TRF) homology (TRFH) domain–containing telomeric proteins TRF1 and TRF2 associate with a collection of molecules necessary for telomere maintenance and cell-cycle progression. However, the specificity and the mechanisms by which TRF2 communicates with different signaling pathways remain largely unknown. Using oriented peptide libraries, we demonstrate that the TRFH domain of human TRF2 recognizes [Y/F]XL peptides with the consensus motif YYHKYRLSPL. Disrupting the interactions between the TRF2 TRFH domain and its targets resulted in telomeric DNA-damage responses. Furthermore, our genome-wide target analysis revealed phosphatase nuclear targeting subunit (PNUTS) and microcephalin 1 (MCPH1) as previously unreported telomere-associated proteins that directly interact with TRF2 via the [Y/F]XL motif. PNUTS and MCPH1 can regulate telomere length and the telomeric DNA-damage response, respectively. Our findings indicate that an array of TRF2 molecules functions as a protein hub and regulates telomeres by recruiting different signaling molecules via a linear sequence code.

Ten-Eleven Translocation 1 (Tet1) Is Regulated by O-Linked N-Acetylglucosamine Transferase (Ogt) for Target Gene Repression in Mouse Embryonic Stem Cells

Background: Ogt N-acetylglucosylates proteins and plays an important role in mouse ES cells. Results: The DNA demethylation enzyme Tet1 interacts with Ogt and is O-GlcNAcylated. Conclusion: Tet1 protein stability is positively regulated by O-GlcNAcylation, and its repression function on targeting genes is dependent on Ogt. Significance: Ogt-Tet1 interaction should further our understanding of how O-GlcNAcylation is integrated into ES cell regulatory networks. As a member of the Tet (Ten-eleven translocation) family proteins that can convert 5-methylcytosine (5mC) to 5-hydroxylmethylcytosine (5hmC), Tet1 has been implicated in regulating global DNA demethylation and gene expression. Tet1 is highly expressed in embryonic stem (ES) cells and appears primarily to repress developmental genes for maintaining pluripotency. To understand how Tet1 may regulate gene expression, we conducted large scale immunoprecipitation followed by mass spectrometry of endogenous Tet1 in mouse ES cells. We found that Tet1 could interact with multiple chromatin regulators, including Sin3A and NuRD complexes. In addition, we showed that Tet1 could also interact with the O-GlcNAc transferase (Ogt) and be O-GlcNAcylated. Depletion of Ogt led to reduced Tet1 and 5hmC levels on Tet1-target genes, whereas ectopic expression of wild-type but not enzymatically inactive Ogt increased Tet1 levels. Mutation of the putative O-GlcNAcylation site on Tet1 led to decreased O-GlcNAcylation and level of the Tet1 protein. Our results suggest that O-GlcNAcylation can positively regulate Tet1 protein concentration and indicate that Tet1-mediated 5hmC modification and target repression is controlled by Ogt.

Human telomeric proteins occupy selective interstitial sites

Human telomeres are bound and protected by protein complexes assembled around the six core telomeric proteins RAP1, TRF1, TRF2, TIN2, TPP1, and POT1. The function of these proteins on telomeres has been studied extensively. Recently, increasing evidence has suggested possible roles for these proteins outside of telomeres. However, the non-canonical (extra-telomeric) function of human telomeric proteins remains poorly understood. To this end, we systematically investigated the binding sites of telomeric proteins along human chromosomes, by performing whole-genome chromatin immunoprecipitation (ChIP) for RAP1 and TRF2. ChIP sequencing (ChIP-seq) revealed that RAP1 and TRF2 could be found on a small number of interstitial sites, including regions that are proximal to genes. Some of these binding sites contain short telomere repeats, suggesting that telomeric proteins could directly bind to interstitial sites. Interestingly, only a small fraction of the available interstitial telomere repeat-containing regions were occupied by RAP1 and TRF2. Ectopically expressed TRF2 was able to occupy additional interstitial telomere repeat sites, suggesting that protein concentration may dictate the selective targeting of telomeric proteins to interstitial sites. Reducing RAP1 and TRF2 expression by RNA interference led to altered transcription of RAP1- and TRF2-targeted genes. Our results indicate that human telomeric proteins could occupy a limited number of interstitial sites and regulate gene transcription.

Genome-wide YFP Fluorescence Complementation Screen Identifies New Regulators for Telomere Signaling in Human Cells

Detection of low-affinity or transient interactions can be a bottleneck in our understanding of signaling networks. To address this problem, we developed an arrayed screening strategy based on protein complementation to systematically investigate protein-protein interactions in live human cells, and performed a large-scale screen for regulators of telomeres. Maintenance of vertebrate telomeres requires the concerted action of members of the Telomere Interactome, built upon the six core telomeric proteins TRF1, TRF2, RAP1, TIN2, TPP1, and POT1. Of the ∼12,000 human proteins examined, we identified over 300 proteins that associated with the six core telomeric proteins. The majority of the identified proteins have not been previously linked to telomere biology, including regulators of post-translational modifications such as protein kinases and ubiquitin E3 ligases. Results from this study shed light on the molecular niche that is fundamental to telomere regulation in humans, and provide a valuable tool to investigate signaling pathways in mammalian cells.

TIN2 Protein Dyskeratosis Congenita Missense Mutants Are Defective in Association with Telomerase

Dyskeratosis congenita (DC) is a progressive and heterogeneous congenital disorder that affects multiple systems and is characterized by bone marrow failure and a triad of abnormal skin pigmentation, nail dystrophy, and oral leukoplakia. One common feature for all DC patients is abnormally short telomeres and defects in telomere biology. Most of the known DC mutations have been found to affect core components of the telomerase holoenzyme. Recently, multiple mutations in the gene encoding the telomeric protein TIN2 have been identified in DC patients with intact telomerase genes, but the molecular mechanisms underlying TIN2 mutation-mediated DC remain unknown. Here, we demonstrate that ectopic expression of TIN2 with DC missense mutations in human cells led to accelerated telomere shortening, similar to the telomere phenotypes found in DC patients. However, this telomere shortening was not accompanied by changes in total telomerase activity, localization of TIN2, or telomere end protection status. Interestingly, we found TIN2 to participate in the TPP1-dependent recruitment of telomerase activity. Furthermore, DC mutations in TIN2 led to its decreased ability to associate with TERC and telomerase activity. Taken together, our data suggest that TIN2 mutations in DC may compromise the telomere recruitment of telomerase, leading to telomere shortening and the associated pathogenesis.

Phosphorylation of TPP1 regulates cell cycle-dependent telomerase recruitment

Telomere maintenance is essential for organisms with linear chromosomes and is carried out by telomerase during cell cycle. The precise mechanism by which cell cycle controls telomeric access of telomerase and telomere elongation in mammals remains largely unknown. Previous work has established oligonucleotide/oligosaccharide binding (OB) fold-containing telomeric protein TPP1, formerly known as TINT1, PTOP, and PIP1, as a key factor that regulates telomerase recruitment and activity. However, the role of TPP1 in cell cycle-dependent telomerase recruitment is unclear. Here, we report that human TPP1 is phosphorylated at multiple sites during cell cycle progression and associates with higher telomerase activity at late S/G2/M. Phosphorylation of Ser111 (S111) within the TPP1 OB fold appears important for cell cycle-dependent telomerase recruitment. Structural analysis indicates that phosphorylated S111 resides in the telomerase-interacting domain within the TPP1 OB fold. Mutations that disrupt S111 phosphorylation led to decreased telomerase activity in the TPP1 complex and telomere shortening. Our findings provide insight into the regulatory pathways and structural basis that control cell cycle-dependent telomerase recruitment and telomere elongation through phosphorylation of TPP1.

The Daxx/Atrx Complex Protects Tandem Repetitive Elements during DNA Hypomethylation by Promoting H3K9 Trimethylation.

In mammals, DNA methylation is essential for protecting repetitive sequences from aberrant transcription and recombination. In some developmental contexts (e.g., preimplantation embryos) DNA is hypomethylated but repetitive elements are not dysregulated, suggesting that alternative protection mechanisms exist. Here we explore the processes involved by investigating the role of the chromatin factors Daxx and Atrx. Using genome-wide binding and transcriptome analysis, we found that Daxx and Atrx have distinct chromatin-binding profiles and are co-enriched at tandem repetitive elements in wild-type mouse ESCs. Global DNA hypomethylation further promoted recruitment of the Daxx/Atrx complex to tandem repeat sequences, including retrotransposons and telomeres. Knockdown of Daxx/Atrx in cells with hypomethylated genomes exacerbated aberrant transcriptional de-repression of repeat elements and telomere dysfunction. Mechanistically, Daxx/Atrx-mediated repression seems to involve Suv39h recruitment and H3K9 trimethylation. Our data therefore suggest that Daxx and Atrx safeguard the genome by silencing repetitive elements when DNA methylation levels are low.

Actl6a Protects Embryonic Stem Cells From Differentiating Into Primitive Endoderm

Actl6a (actin‐like protein 6A, also known as Baf53a or Arp4) is a subunit shared by multiple complexes including esBAF, INO80, and Tip60‐p400, whose main components (Brg1, Ino80, and p400, respectively) are crucial for the maintenance of embryonic stem cells (ESCs). However, whether and how Actl6a functions in ESCs has not been investigated. ESCs originate from the epiblast (EPI) that is derived from the inner cell mass (ICM) in blastocysts, which also give rise to primitive endoderm (PrE). The molecular mechanisms for EPI/PrE specification remain unclear. In this study, we provide the first evidence that Actl6a can protect mouse ESCs (mESCs) from differentiating into PrE. While RNAi knockdown of Actl6a, which appeared highly expressed in mESCs and downregulated during differentiation, induced mESCs to differentiate towards the PrE lineage, ectopic expression of Actl6a was able to repress PrE differentiation. Our work also revealed that Actl6a could interact with Nanog and Sox2 and promote Nanog binding to pluripotency genes such as Oct4 and Sox2. Interestingly, cells depleted of p400, but not of Brg1 or Ino80, displayed similar PrE differentiation patterns. Mutant Actl6a with impaired ability to bind Tip60 and p400 failed to block PrE differentiation induced by Actl6a dysfunction. Finally, we showed that Actl6a could target to the promoters of key PrE regulators (e.g., Sall4 and Fgf4), repressing their expression and inhibiting PrE differentiation. Our findings uncover a novel function of Actl6a in mESCs, where it acts as a gatekeeper to prevent mESCs from entering into the PrE lineage through a Yin/Yang regulating pattern. Stem Cells 2015;33:1782–1793

Studying of Telomeric Protein–Protein Interactions by Bi-Molecular Fluorescence Complementation (BiFC) and Peptide Array-Based Assays

Studying protein-protein interactions is critical to our understanding of the signaling pathways. The Telomere Interactome is assembled around the telomeres and consists of proteins and factors from diverse pathways. Dissecting how this protein network contributes to telomere protection and length regulation requires the elucidation of the complex and dynamic interactions between the proteins within the interactome. Here, we focus on the Bi-molecular fluorescence complementation (BiFC) and peptide array methods that have proven vital in our studies of telomere protein interaction networks.