The Long Linker Region of Telomere-Binding Protein TRF2 Is Responsible for Interactions with Lamins.

Telomere-binding factor 2 (TRF2) is part of the shelterin protein complex found at chromosome ends. Lamin A/C interacts with TRF2 and influences telomere position. TRF2 has an intrinsically disordered region between the ordered dimerization and DNA-binding domains. This domain is referred to as the long linker region of TRF2, or udTRF2. We suggest that udTRF2 might be involved in the interaction between TRF2 and lamins. The recombinant protein corresponding to the udTRF2 region along with polyclonal antibodies against this region were used in co-immunoprecipitation with purified lamina and nuclear extracts. Co-immunoprecipitation followed by Western blots and mass spectrometry indicated that udTRF2 interacts with lamins, preferably lamins A/C. The interaction did not involve any lamin-associated proteins, was not dependent on the post-translation modification of lamins, nor did it require their higher-order assembly. Besides lamins, a number of other udTRF2-interacting proteins were identified by mass spectrometry, including several heterogeneous nuclear ribonucleoproteins (hnRNP A2/B1, hnRNPA1, hnRNP A3, hnRNP K, hnRNP L, hnRNP M), splicing factors (SFPQ, NONO, SRSF1, and others), helicases (DDX5, DHX9, and Eif4a3l1), topoisomerase I, and heat shock protein 71, amongst others. Some of the identified interactors are known to be involved in telomere biology; the roles of the others remain to be investigated. Thus, the long linker region of TRF2 (udTRF2) is a regulatory domain responsible for the association between TRF2 and lamins and is involved in interactions with other proteins.

Actin depolymerization disrupts karyosphere capsule integrity but not residual transcription in late oocytes of the grass frog Rana temporaria.

Late diplotene oocytes are characterized by an essential decrease in transcriptional activity. At this time, chromosomes condense and form a compact structure named a karyosphere. The karyosphere of grass frogs Rana temporaria is surrounded by a fibrillar karyosphere capsule (KC). One of the main protein constituents of R. temporaria KC is actin. In this study, we used antibodies against different actin epitopes to trace different forms of actin in the KC. We also investigated the effect of F-actin depolymerization on the oocyte nuclear structures and transcription of chromatin DNA and rDNA in the amplified nucleoli. It was determined that disruption of actin filaments leads to chromosome shrinkage, nucleoli fusion, and distortion of the KC structure, but does not inhibit residual transcription in both the karyosphere and the nucleoli.

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.

Telomere Repeat-Binding Factor 2 Is Responsible for the Telomere Attachment to the Nuclear Membrane.

Telomeres are nucleoprotein structures that specify ends of eukaryotic chromosomes. They enable complete DNA replication, protect chromosomes from end-to-end fusions, and help organize chromatin structure. These functions are mediated by special telomeric proteins. TRF2 (telomeric repeat-binding factor 2) is an essential component of shelterin, a telomere-binding protein complex. TRF2 induces formation of a special structure of telomeric DNA, counteracts activation of double-strand break response pathway and ataxia telangiectasia mutated kinase pathway at telomeres. Some line of evidence implicates TRF2 in interactions with the nuclear envelope (NE). TRF2 is tightly bound to the nuclear membrane in frog oocytes nucleus, and it was found colocalized with NE or its remnants in mouse cells. Computer analysis of TRF2 amino acid sequence has shown that TRF2 possesses motifs, which resemble rod domain characteristic of intermediate filament proteins. These observations suggest that TRF2 is a good candidate for the attachment of telomeres to the NE in somatic cells.