Development and characterization of polyclonal antibodies against the linker region of the telomere-binding protein TRF2

Background: TRF2 (telomeric repeat binding factor 2) is an essential component of the telomere-binding protein complex shelterin. TRF2 induces the formation of a special structure of telomeric DNA and counteracts activation of DNA damage-response pathways telomeres. TRF2 has a poorly characterized linker region (udTRF2) between its homodimerization and DNA-binding domains. Some lines of evidence have shown that this region could be involved in TRF2 interaction with nuclear lamina. Results: In this study, the fragment of the TERF2 gene encoding udTRF2 domain of telomere-binding protein TRF2 was produced by PCR and cloned into the pET32a vector. The resulting plasmid pET32a-udTRF2 was used for the expression of the recombinant udTRF2 in E. coli RosettaBlue (DE3). The protein was isolated and purified using ammonium sulfate precipitation followed by ion-exchange chromatography. The purified recombinant protein udTRF2 was injected into guinea pigs to generate polyclonal antibodies. The ability of anti-udTRF2 antibodies to bind endogenous TRF2 in human skin fibroblasts was tested by western blotting and immunofluorescent staining. Conclusions: In this study, the recombinant protein udTRF2 and antibodies to it were generated. Both protein and antibodies will provide a useful tool for investigation of the functions of the udTRF2 domain and its role in the interaction between TRF2 and nuclear lamina.

Nucleolus-like body of mouse oocytes contains lamin A and B and TRF2 but not actin and topo II.

BACKGROUND: During the final stages of oocyte development, all chromosomes join in a limited nuclear volume for the final formation of a single complex chromatin structure - the karyosphere. In the majority of mammalian species, the chromosomes surround a round protein/fibrillar body known as the central body, or nucleolus-like body (NLB). Nothing seems to unite the inner portion of the karyosphere with the nucleolus except position at its remnants. Nevertheless, in this study we will use term NLB as the conventional one for karyosphere with the central body. At the morphological level, NLBs consist of tightly-packed fibres of 6-10 nm. The biochemical structure of this dense, compact NLB fibre centre remains uncertain. RESULTS: The aim of this study was to determine which proteins represent the NLB components at final stages of karyosphere formation in mouse oogenesis. To determine this, three antibodies (ABs) have been examined against different actin epitopes. Examination of both ABs against the actin N-end provided similar results: spots inside the nucleus. Double staining with AB against SC35 and actin revealed the colocalization of these proteins in IGCs (interchromatin granule clusters/nuclear speckles/SC35 domains). In contrast, examination of polyclonal AB against peptide at the C-end reveals a different result: actin is localized exclusively in connection with the chromatin. Surprisingly, no forms of actin or topoisomerase II are present as components of the NLB. It was discovered that: (1) lamin B is an NLB component from the beginning of NLB formation, and a major portion of it resides in the NLB at the end of oocyte development; (2) lamin A undergoes rapid movement into the NLB, and a majority of it remains in the NLB; (3) the telomere-binding protein TRF2 resides in the IGCs/nuclear speckles until the end of oocyte development, when significant part of it transfers to the NLB. CONCLUSIONS: NLBs do not contain actin or topo II. Lamin B is involved from the beginning of NLB formation. Both Lamin A and TRF2 exhibit rapid movement to the NLB at the end of oogenesis. This dynamic distribution of proteins may reflect the NLB's role in future chromatin organization post-fertilisation.

A telomere-binding protein (TRF2/MTBP) from mouse nuclear matrix with motives of an intermediate filament-type rod domain.

In previous work, we identified a telomeric DNA-binding protein (termed telomere-membrane binding protein, MTBP) in the envelope of the frog oocyte nucleus and raised antibodies against it. Here we present immunological evidence which suggests strongly that MTBP is identical with the vertebrate telomeric DNA-binding protein TRF2 (telomere-repeat factor 2). MTBP/TRF2 possesses motif which resembles rod domain characteristic of intermediate filament (IF) proteins as shown by immunological cross-reactivity with characteristic antibodies, as well as amino acid sequence homology. Anti-MTBP antibodies recognised a protein of the same M, as TRF2 in extracts of mouse nuclei and nuclear matrix as shown by ion-exchange chromatography, gel shift assays, and Western blots. This mouse MTBP analogue forms more stable complexes with the vertebrate telomeric DNA fragment (T(2)AG(3))(135) than with the corresponding fragment from Tetrahymena (T(2)G(4))(130). Proteins in each of these complexes are recognised by anti-MTBP antibody. In situ hybridization with the vertebrate telomeric DNA sequence (T(2)AG(3))(135) and immunofluorescence with anti-MTBP antibody had shown earlier that these are co-localised in the nucleus of mouse cells, and here MTBP is shown to be associated with the residual membrane of hepatocyte nuclei using Western blotting and immunofluorescence. Some immunofluorescence signal from MTBP is localized at chromosome extremities on metaphase plates from mouse cell culture, but the main signal is seen in patches scattered around the chromosomes which were identified as remnants of the nuclear envelope by double labelling with antibodies against lamin B. These observations suggest that MTBP/TRF2 is a good candidate for the attachment of telomeres to the nuclear envelope in somatic cells.