Telomeres allow cells to distinguish natural chromosome ends from damaged DNA and protect the ends from degradation and fusion. In human cells, telomere protection depends on the TTAGGG repeat binding factor, TRF2 (refs 1-4), which has been proposed to remodel telomeres into large duplex loops (t-loops). Here we show by nanoelectrospray tandem mass spectrometry that RAD50 protein is present in TRF2 immunocomplexes. Protein blotting showed that a small fraction of RAD50, MRE11 and the third component of the MRE11 double-strand break (DSB) repair complex, the Nijmegen breakage syndrome protein (NBS1), is associated with TRF2. Indirect immunofluorescence demonstrated the presence of RAD50 and MRE11 at interphase telomeres. NBS1 was associated with TRF2 and telomeres in S phase, but not in G1 or G2. Although the MRE11 complex accumulated in irradiation-induced foci (IRIFs) in response to gamma-irradiation, TRF2 did not relocate to IRIFs and irradiation did not affect the association of TRF2 with the MRE11 complex, arguing against a role for TRF2 in DSB repair. Instead, we propose that the MRE11 complex functions at telomeres, possibly by modulating t-loop formation.

Human telomeres are protected by TRF2. Inhibition of this telomeric protein results in partial loss of the telomeric 3' overhang and chromosome end fusions formed through nonhomologous end-joining (NHEJ). Here we report that ERCC1/XPF-deficient cells retained the telomeric overhang after TRF2 inhibition, identifying this nucleotide excision repair endonuclease as the culprit in overhang removal. Furthermore, these cells did not accumulate telomere fusions, suggesting that overhang processing is a prerequisite for NHEJ of telomeres. ERCC1/XPF was also identified as a component of the telomeric TRF2 complex. ERCC1/XPF-deficient mouse cells had a novel telomere phenotype, characterized by Telomeric DNA-containing Double Minute chromosomes (TDMs). We speculate that TDMs are formed through the recombination of telomeres with interstitial telomere-related sequences and that ERCC1/XPF functions to repress this process. Collectively, these data reveal an unanticipated involvement of the ERCC1/XPF NER endonuclease in the regulation of telomere integrity and establish that TRF2 prevents NHEJ at telomeres through protection of the telomeric overhang from ERCC1/XPF.

An analytical approach is reported for the characterization of the specific glycans found on highly glycosylated proteins based on a combination of specific proteolysis and deglycosylation combined with two different mass spectrometric approaches, matrix-assisted laser desorption/ionization mass spectrometry, and nanoelectrospray mass spectrometry/tandem mass spectrometry using a hybrid quadrupole-time-of-flight tandem mass spectrometer. The high resolution and mass accuracy of the mass spectrometric data obtained on the hybrid instrument combined with the high parent mass capabilities are shown to be extremely useful in the site-specific assignment of heterogeneous glycans. Using this methodology, 25 of 26 consensus glycosylation sites on HIV-1(SF2) gp120, expressed in Chinese hamster ovary cells, could be assigned. Good correlations between the relative abundances of members of heterogeneous series in the matrix-assisted laser desorption/ionization mass spectra and the nanoelectrospray mass spectra were observed, indicating that the mass spectrometric data reflected the actual abundances of the members of the series. These data were incorporated with molecular modeling based on the solved structure of a mutant truncated, highly deglycosylated gp120 to propose a structural model for the completely glycosylated form.

Estrogen receptor (ER)-beta is thought to exert anti-proliferative effects in the normal prostate but supports prostate cancer (PCa) cell survival. We previously reported that the receptor's expression declined as PCa developed in the gland but reappeared in lymph node and bone metastases. To investigate whether hypermethylation was the underlying mechanism for these phenomena, we first identified two CpG islands (CGIs) encompassing 41 CpG dinucleotides, located separately in the untranslated exon 0N and the promoter region of ER-beta. Using immunostained, laser capture-microdissected samples from 56 clinical specimens, we demonstrated an inverse relationship exists between the extent of ER-beta CGI methylation and receptor expression in normal, hyperplastic, premalignant, and malignant foci of the prostate and in lymph node and bone metastases. Treatment of PCa cell lines (LNCaP and DU145), that express little ER-beta mRNA, with a demethylating agent increased levels of receptor expression thus corroborating our in vivo findings that methylation is involved in ER-beta silencing. Methylation centers in the promoter region and exon 0N were identified by hierarchical cluster analysis of bisulfite sequencing data obtained from 710 alleles. Methylation at these centers was insignificant in normal epithelium, reached 80 to 90% in grade 4/5 PCa, but declined to less than 20% in bone metastases. In addition, progressive methylation spreading from the exonic CGI to the promoter CGI, which correlated with loss of ER-beta expression, was detected in microdissected samples and in cell cultures. Using a new class of methylated oligonucleotides that mediate sequence-specific methylation in cellulo, we demonstrated that methylation of the promoter CGI, but not the exonic CGIs, led to transcriptional inactivation of ER-beta. Our results present the first evidence that epigenetic regulation of ER-beta is a reversible and tumor stage-specific process and that gene silencing via methylated oligonucleotides may have therapeutic potential in the treatment of advanced PCa.