XPF參與ALT端粒上telomeric R-loop誘發的DNA雙股斷裂

Abstract

真核細胞中染色體尾端會有端粒(telomere)存在，目的是保護以及維持染色體末端的完整性。隨著細胞的分裂會使端粒長度縮短，當端粒短到某種程度後就無法繼續保護染色體，此時細胞中會有端粒延長機制來確保其長度。大部分的癌細胞是以端粒酶(telomerase)來延長，但有10~15%的癌細胞則是使用另一種機制，稱為alternative lengthening of telomeres (ALT)，目前可知主要是透過同源染色重組 (homologous recombination, HR) 來延長端粒，然而對於此機制仍不清楚。此外，在ALT細胞中有一種高度表現的長鏈非編碼RNA (long non-coding RNA, lncRNA) - TERRA (telomeric repeat-containing RNA)，被認為可能會參與ALT機制。因此本篇目的是要以TERRA-interacting protein為出發點，探討其蛋白質如何參與ALT 機制。其中之一enriched-TERRA-interacting protein為XPF (xeroderma pigmentosum, complementation group F)，是一種內切酶，主要參與細胞中DNA修復。我發現在U2OS細胞中，XPF和telomeric repeat-binding factor 2 (TRF2) 有高度重疊；並且，在U2OS細胞中高度表現RNase H時則會減少XPF被聚集到TRF2上。此結果表示telomeric R-loop會影響細胞中XPF聚集至端粒。進一步研究XPF在ALT機制中的角色，我利用siRNA在U2OS細胞中降低XPF表現量。結果顯示，在XPF knockdown (KD) U2OS細胞中，端粒上的RPA (single strand binding protein) 和γH2AX都會下降，telomere clustering也會下降。有研究指出，XPF和另一種內切酶-XPG (xeroderma pigmentosum complementation group G)會一起在轉錄過程中形成的R-loop上形成雙股DNA斷裂(DNA double-strand break, DSB)，並促進其修復。在我的結果也顯示出在XPF以及XPG KD U2OS細胞中，端粒上的γH2AX都有顯著下降。而之前研究顯示，在U2OS細胞中降低Fanconi anemia group M protein (FANCM) 會導致telomeric R-loop累積、端粒上的DSB增加以及C-circle的高度表現。而我發現當在FANCM-deficiency細胞中降低XPF表現量時，會使其增加的telomere intensity、DSB和C-circle都顯著下降。綜合上述結果可知在ALT細胞中，XPF參與ALT端粒上telomeric R-loop誘發的DNA雙股斷裂，並且會進一步影響由DNA break-induced replication fork所產生的C-circle。

Telomeres are important for protecting the integrity of chromosomes that contain vital information of DNA. A subset of human cancer cells maintains telomeres through the alternative lengthening of telomeres (ALT) pathway. Telomeric repeat-containing RNA (TERRA), a long non-coding RNA, transcribed from subtelomeric regions, is highly expressed in ALT cancer cells. However, the mechanism of ALT remains unclear. To study the role of TERRA-interacting protein in ALT, we knocked down (KD) several TERRA-interacting proteins in human ALT cells, U2OS, derived from osteosarcoma. One of those proteins is xeroderma pigmentosum complementation group F (XPF), which is a DNA repair protein encoded by ERCC4 gene and is responsible for nucleotide excision repair. We found that XPF is highly co-localized with telomeric repeat-binding factor 2 (TRF2) in ALT cells, but not in non-ALT cells. Depletion R-loop (DNA-RNA hybrids) formation by overexpressing wild type RNase H reduces XPF recruitment to telomeres, suggesting that telomeric R-loops trigger XPF localization to telomeres. To test the role of XPF in the ALT pathway, we depleted XPF by siRNAs and found that single strand binding protein (RPA) and γH2AX at telomeres are decreased. Moreover, knockdown of XPF suppresses telomere clustering in ALT cells. A pervious study has shown that XPF and xeroderma pigmentosum complementation group G (XPG), which is also an endonuclease, are involved in the generation of DSB from transcription-coupled R-loop and promote DNA repair. When we double KD XPF and XPG in U2OS cells, both KD cells were showed the decreased γH2AX at telomeres. Our recent studies have shown that knockdown of Fanconi anemia group M protein (FANCM) leads to accumulation of telomeric R-loops, DNA double-strand breaks at telomeres and c-circle formation in ALT cells. When XPF was depleted in FANCM-deficient cells, DNA double-strand breaks and telomere intensity were dramatically attenuated, and the c-circle formation was also reduced. These results imply that XPF mediates DNA double-strand breaks induced by telomeric R-loops in human ALT cancer cells.