Biomedical subjects

Diego Loayza

Publications and source records attributed to Diego Loayza.

5 recordsLinked to original sources

PubMed2004

TIN2 binds TRF1 and TRF2 simultaneously and stabilizes the TRF2 complex on telomeres.

Human telomeres contain two related telomeric DNA-binding proteins, TRF1 and TRF2. The TRF1 complex contains the TRF1 interacting partner, TIN2, as well as PIP1 and POT1 and regulates telomere-length homeostasis. The TRF2 complex is primarily involved in telomere protection and contains the TRF2 interacting partner human (h)Rap1 as well as several factors involved in the DNA damage response. A prior report showed that conditional deletion of murine TRF1 reduced the presence of TRF2 on telomeres. Here we showed that TRF2 is also lost from human telomeres upon TRF1 depletion with small interfering RNA prompting a search for the connection between the TRF1 and TRF2 complexes. Using mass spectrometry and co-immunoprecipitation, we found that TRF1, TIN2, PIP1, and POT1 are associated with the TRF2-hRap1 complex. Gel filtration identified a TRF2 complex containing TIN2 and POT1 but not TRF1 indicating that TRF1 is not required for this interaction. Co-immunoprecipitation, Far-Western assays, and two-hybrid assays showed that TIN2, but not POT1 or PIP1, interacts directly with TRF2. Furthermore, TIN2 was found to bind TRF1 and TRF2 simultaneously, showing that TIN2 can link these telomeric proteins. This connection appeared to stabilize TRF2 on the telomeres as the treatment of cells with TIN2 small interfering RNA resulted in a decreased presence of TRF2 and hRap1 at chromosome ends. The TIN2-mediated cooperative binding of TRF1 and TRF2 to telomeres has important implications for the mechanism of telomere length regulation and protection.

Antigens, Surface↗

PubMed2004

POT1-interacting protein PIP1: a telomere length regulator that recruits POT1 to the TIN2/TRF1 complex.

Human telomere length is controlled by a negative feedback loop based on the binding of TRF1 to double-stranded telomeric DNA. The TRF1 complex recruits POT1, a single-stranded telomeric DNA-binding protein necessary for cis-inhibition of telomerase. By mass spectrometry, we have identified a new telomeric protein, which we have named POT1-interacting protein 1 (PIP1). PIP1 bound both POT1 and the TRF1-interacting factor TIN2 and could tether POT1 to the TRF1 complex. Reduction of PIP1 or POT1 levels with shRNAs led to telomere elongation, indicating that PIP1 contributes to telomere length control through recruitment of POT1.

Carrier Proteins↗

PubMed2004

Telomerase regulation at the telomere: a binary switch.

Telomerase is known to preferentially elongate the shortest telomeres in a cell. Using an elegant yeast assay, Texeira et al. (2004 [this issue of Cell]) address what aspect of telomerase action is regulated by telomere length: the frequency or the extent of telomere elongation. They show that short telomeres are elongated more frequently than long telomeres, arguing that telomeres switch between two states, one that allows telomere extension and one that does not.

Crosses, Genetic↗

PubMed2004

DNA binding features of human POT1: a nonamer 5'-TAGGGTTAG-3' minimal binding site, sequence specificity, and internal binding to multimeric sites.

The human telomeric protein POT1 is known to bind single-stranded telomeric DNA in vitro and to participate in the regulation of telomere maintenance by telomerase in vivo. We examined the in vitro DNA binding features of POT1. We report that deleting the oligosaccharide/oligonucleotide-binding fold of POT1 abrogates its DNA binding activity. The minimal binding site (MBS) for POT1 was found to be the telomeric nonamer 5'-TAGGGTTAG-3', and the optimal substrate is [TTAGGG](n (n > or = 2)). POT1 displays exceptional sequence specificity when binding to MBS, tolerating changes only at position 7 (T7A). Whereas POT1 binding to MBS or [TTAGGG](2) was enhanced by the proximity of a 3' end, POT1 was able to bind to a [TTAGGG](5) array when positioned internally. These data indicate that POT1 has a strong sequence preference for the human telomeric repeat tract and predict that POT1 can bind both the 3' telomeric overhang and the displaced TTAGGG repeats at the base of the t-loop.

Animals↗

PubMed2003

POT1 as a terminal transducer of TRF1 telomere length control.

Human telomere maintenance is essential for the protection of chromosome ends, and changes in telomere length have been implicated in ageing and cancer. Human telomere length is regulated by the TTAGGG-repeat-binding protein TRF1 and its interacting partners tankyrase 1, TIN2 and PINX1 (refs 5-9). As the TRF1 complex binds to the duplex DNA of the telomere, it is unclear how it can affect telomerase, which acts on the single-stranded 3' telomeric overhang. Here we show that the TRF1 complex interacts with a single-stranded telomeric DNA-binding protein--protection of telomeres 1 (POT1)--and that human POT1 controls telomerase-mediated telomere elongation. The presence of POT1 on telomeres was diminished when the amount of single-stranded DNA was reduced. Furthermore, POT1 binding was regulated by the TRF1 complex in response to telomere length. A mutant form of POT1 lacking the DNA-binding domain abrogated TRF1-mediated control of telomere length, and induced rapid and extensive telomere elongation. We propose that the interaction between the TRF1 complex and POT1 affects the loading of POT1 on the single-stranded telomeric DNA, thus transmitting information about telomere length to the telomere terminus, where telomerase is regulated.

Amino Acid Sequence↗