LAP2α Represses Alternative Lengthening Of Telomeres(ALT) By Maintaining Telomere Binding Protein RAP1 And Telomeric Heterochromatin

Objective:To escape the senescence and death caused by continuous replication,cancer cells have to overcome the problem of telomere shortening.Cancer cells resist telomere shortening by activating telomere maintenance mechanism.Most of cancer cells rely on telomerase to elongate telomere.However,about 10%～15% of cancer cells elongate telomere in a telomerase-independent pathway,which is called alternative lengthening of telomeres pathway(ALT).ALT is a homologous mediated recombination dependent repair pathway,it uses telomere repeat DNA as template to synthesize new telomere DNA by homologous recombination.However,the precise molecular mechanism of ALT is still unclear.Previous studies have shown that lamin associated protein2 α(LAP2α)colocalized with telomere,and LAP2α also interacts with a variety of proteins involved in DNA damage repair.Our study aims to explore the effect of LAP2α on ALT pathway and elucidate the molecular mechanism of LAP2α on ALT,so as to provide a new direction and theoretical basis for further revealing the precise molecular mechanism of ALT.Methods:(1)Proximity ligation assay(PLA)was used to analyze the interaction between LAP2α and telomere binding protein.(2)Small RNA interference(si RNA)technology was used to construct cell lines with transient knockdown of LAP2α,and used this cell line as a tool to study the effect of LAP2α on telomere function.(3)Fluorescence in situ hybridization(FISH)was used to explore the effect of LAP2α knockdown on telomere function.(4)Interphase telomere fluorescence in situ hybridization(FISH)was used to define the effect of LAP2α on telomere clustering.(5)Chromosome orientation-fluorescence in situ hybridization(CO-FISH)was used to clarify the effect of LAP2α knockdown on telomere sister chromatid exchange(T-SCE).(6)Immunofluorescence-Fluorescence in situ hybridization(IF-FISH)and C-circle assay was used to detect the formation of APBs and C-circle after depletion of LAP2α.(7)Using the CRISPR/Cas9 system to induce sustained double-strand breaks of telomeric DNA in Hela cells,making the telomerase-positive Hela cells produce the hallmarks of ALT,and using this cell line as a tool to further verify the role of LAP2α in the occurrence of ALT.(8)Western blotting and RT-PCR were used to detect the change of telomere binding protein in LAP2α knockdown cells.(9)Chromatin immunoprecipitation assay(Ch IP)was used to analyze the effect of LAP2α knockdown on telomere heterochromatin level.(10)The expression of Terra was detected by RT-PCR in LAP2α knockdown cells.Results:(1)The results of PLA showed that LAP2α interacted with TRF1 and TRF2 in both telomerase positive and ALT positive cells.(2)FISH data showed that knockdown of LAP2α increased telomere signal free end(SFE)and fragile telomere in ALT positive cells.(3)Interphase telomere FISH showed that knockdown of LAP2α increased telomere clustering in ALT positive cells.(4)CO-FISH results showed that knockdown of LAP2α improved the level of sister chromatid exchange in the telomere region of U2 OS cells.(5)The results of IF-FISH and C-circle showed that the levels of APBs and C-circle was elevated after knockdown of LAP2α in ALT cells.(6)The CRISPR/Cas9 system successfully induced the production of C-circle and APBs in Hela cells,and depletion of LAP2α exacerbated the accumulation of C-circle in these cells.(7)Western blotting and RT-PCR results showed that knockdown of LAP2αdecreased the m RNA and protein levels of RAP1.(8)Ch IP assay showed that knockdown of LAP2α decreased the level of H3K9me3.(9)RT-PCR results showed that the expression of TERRA was increased in LAP2αknockdown ALT cells.Conclusion:LAP2α interacts with telomere binding proteins TRF1 and TRF2,and knockdown of LAP2α can cause telomere loss and increase the level of fragile telomere in ALT cells,which indicates that LAP2α is involved in regulating telomere function of ALT cells.Knockdown of LAP2α can promote the hallmarks of ALT,which suggests that LAP2α suppresses the occurrence of ALT.We also elucidate the molecular mechanism of LAP2α inhibiting the occurrence of ALT.Depletion of LAP2α will reduce the level of telomere binding protein RAP1,and the decrease of RAP1 will result in the decrease of heterochromatin level in telomere region,the increase of recruitment of homologous recombination repair related proteins to telomere and the increase of expression of TERRA,thus promoting the activity of ALT.