Inhibition of activated pericentromeric SINE/Alu repeat transcription in senescent human adult stem cells reinstates self-renewal

Abstract

Cellular aging is linked to deficiencies in efficient repair of DNA double strand breaks and authentic genome maintenance at the chromatin level. Aging poses a significant threat to adult stem cell function by triggering persistent DNA damage and ultimately cellular senescence. Senescence is often considered to be an irreversible process. Moreover, critical genomic regions engaged in persistent DNA damage accumulation are unknown. Here we report that 65% of naturally occurring repairable DNA damage in self-renewing adult stem cells occurs within transposable elements. Upregulation of Alu retrotransposon transcription upon ex vivo aging causes nuclear cytotoxicity associated with the formation of persistent DNA damage foci and loss of efficient DNA repair in pericentric chromatin. This occurs due to a failure to recruit of condensin I and cohesin complexes. Our results demonstrate that the cytotoxicity of induced Alu repeats is functionally relevant for the human adult stem cell aging. Stable suppression of Alu transcription can reverse the senescent phenotype, reinstating the cells' self-renewing properties and increasing their plasticity by altering so-called "master" pluripotency regulators.

Figure 1

Ex vivo aging of hADSCs is associated with formation of transcriptionally active persistent DNA damage foci and upregulation of transcriptional activity from Alu retrotransposons. (A) Immunohistochemical detection of senescence-associated β-galactosidase (SA-β-Gal) activity. Examples of hADSCs' morphological changes (10x magnification) shown in inserts. Bar graphs correspond to percentage of SA-β-Gal positive cells with progressive ex-vivo hADSC expansion, based on three independent experiments. Error bars are standard deviations from the mean. (B) DNA damage response (DDR) in senescent hADSCs. Representative immunostaining for the persistent γH2AX (green)/53BP1 (red) foci formation upon senescence of hADSCs. (C) Quantification of accumulation of persistent DNA damage foci with ex-vivo passaging of hADSCs. γH2AX was stained with affinity-purified rabbit polyclonal antibody. Histogram indicates the percentage of the cells with 1, 2, 3 or more than 3 foci. Representative examples are shown below. Foci formation was scored in self-renewing, SR (population doubling less than 17), pre-senescent, preSEN (population doubling more than 29, but less than 38) and senescent, SEN (population doubling greater than 39) hADSCs cultures. The growth curve of ADSC in shown in the Supplemental 1B. n = total number of nuclei counted in all 3 independent experiments. (D) Alu expression in SR and SEN hADSCs. Northern hybridization of self-renewing (SR) and senescent (SEN) hADSCs with Alu oligonucleotide probe. Alu and 7SL are indicated. Total RNA of 2 µg per lane was loaded as described in Materials and Methods. (E) Ribosomal small RNAs can be seen in the ethidium bromide stained gel for loading comparison. The ssRNA ladder sizes are indicated on the right. (F) Persistent γH2AX/53BP1 foci in senescent hADSC are associated with active transcription. Senescent hADSCs were incubated with hallogenated precursor FUr for 10 min in vivo to label nuclear RNA. After fixation, cells were immunolabelled with anti-BrdU antibody (red) to detect FUr incorporation sites in combination with anti-γH2AX (green). Arrows point to co-localization of the persistent DNA damage sites upon senescence with regions of high transcriptional activity. (G) Association of DNA damage foci with transription was further verified by confocal microscopy in co-immunostaing of DNA damage foci depicted by 53BP1 antibodies (green) with PML bodies (blue) and nascent RNA (red). Representative image of a single nucleus is shown. Spatial relationship between FUr incorporation sites, 53BP1 and PML bodies in a single 5 µm confocal section is shown. Image was analyzed by Imaris software and z₁, z₂ and z₃ planes are shown. Cartoon demonstrates the orientations of z₁, z₂ and z₃ planes within single z-section. Confocal sectioning confirms the tight association of nascent transcripts with persistent DNA damage sites in senescent hADSCs.

Figure 2

Persistent γH2AX/53BP1 foci in senescent hADSCs are associated with Pol III transcription. Senescent hADSCs were either cultured in the presence of 10 µM inhibitor of Pol III transcriptional activity, tagetin, for 2 h at 37°C (+tagetin) or in the absence of the inhibitor treatment (-tagetin). Nuclear RNA was labeled by addition of 2 mM FUr to the culture for 10 min at 37°C. After fixation, cells were immunolabelled with anti-BrdU antibody (red) to detect FUr incorporation sites in combination with anti-53BP1 (green). Double labeling experiment revealed FUr incorporation sites exclusively localized with persistent DNA damage sites throughout entire depth of z-stack images. Tagetin inhibition of Pol III dependent transcription results in complete disappearance of FUr incorporation, and loss of compaction of the DNA damage sites as detected by more defuse 53BP1 staining. Fifteen optical sections are shown for confocal analysis of immunostained samples for each of the experimental conditions. Bottom images show the merged signals with DAPI staining.

Figure 3

Genome-wide location analysis of γH2AX. (A) Relative chromosomal distributions of γH2AX tags in self-renewing and senescent cells illustrated for chromosomes 10 and 21. γH2AX tag enrichment levels, calculated as the log₂ ratio of position-specific tag counts normalized by the genomic background, are shown for self-renewing (blue) and senescent (red) cells. Relative differences in γH2AX tag enrichment levels between cells, calculated as the absolute values of the differences in cell stage specific enrichment levels, are shown below the individual cell tracks. Below the difference tracks, the chromosomal locations of large clusters of γH2AX modified sites are shown for the self-renewing (blue) and senescent (red) cell types. (B) Differences in the relative γH2AX enrichment levels for self-renewing (SR) vs. senescent (SEN) cells across various genomic features. The absolute values of the normalized differences in γH2AX tag counts between cell types are shown on the y-axis. Blue bars show genomic features that have higher fractions in SR cells, and red bars show genomic features that have higher fractions in SEN cells. Error bars show the standard errors for the 4 replicates based on binomial distributions. (C) γH2AX enrichment levels in peritelomeric regions for self-renewing (SR-blue) and senescent (SEN-red) cell lines. Chromosome ends (telomeres) are shown at the origin of the x-axis, which then extends into the chromosome arms. Average γH2AX enrichment levels are calculated as the log₂ ratio of the position-specific tag counts normalized to the genomic background averaged over all chromosome arms. (D) γH2AX enrichment levels in pericentric regions for self-renewing (SR-blue) and senescent (SEN-red) cell lines. Centromeres are shown as a gap centered on the x-axis, which extends into the chromosome arms in either direction. Average γH2AX enrichment levels are calculated as the log₂ ratio of the position-specific tag counts normalized to the genomic background averaged over all chromosomes. (E) Fractional differences in the numbers of γH2AX sites within large clusters in pericentric regions between senescent vs. self-renewing cells. The fractional differences for γH2AX sites within large clusters between cell phenotypes are shown on the y-axis. Blue bars show chromosomes that have higher fractions of γH2AX sites within large clusters in SR cells, and red bars show chromosomes that have higher fractions of γH2AX sites within large clusters in SEN cells. Error bars show the standard errors for the four replicates based on binomial distributions.

Figure 4

Centromeric regions are associated with persistent DNA damage foci in senescent hADSCs. (A) Immunofluorescent labeling of self-renewing hADSCs. Cells were seeded on coverslips and co-stained with anti-CENP-A (green) and anti-53BP1 (red) antibodies. DAPI staining is shown in blue. Confocal image of representative interphase nucleus is shown as separate channels and as a merged image. Four µm z-slice was analyzed by Imaris software and z₁, z₂ and z₃ projections are shown. Self-renewing hADSCs show no focal damage associated sites. Centromeric areas are clearly visible. (B) Persistent DNA damage is associated with centromeres. Senescent hADSCs were seeded on coverslips and as in (A) and immunostaining was performed. An arrow depicts the co-localization of a centromeric region with persistent, senescence-associated γH2AX/53BP1 damage foci. Scale bar, 4 µm. (C) Quantification of CENP-A and 53BP1 co-localization in senescence. Senescent hADSCs were stained with antibodies against CENP-A (green) and 53BP1 (red) and DAPI (blue). Total of 200 cells were scored from three independent experiments. Error bars represent ± SAM. Example of higher magnification of the image is shown. Scale bar 1 µm. Images were analyzed by IMARIS software with optical sections representation as depicted on the left. Single 5 mm confocal section is shown. Image was analyzed by Imaris software and z₁, z₂ and z₃ planes are shown. Cartoon demonstrates the orientations of z₁, z₂ and z₃ planes within single z-section.

Figure 5

Pericentromere-associated persistent DNA damage in senescent hADSCs correlates with transcriptional upregulation of Alu retrotransposons and defects in recruitment of cohesin and condensin I complexes. (A) On the left: pericentric region of human chromosome 10. Red box on the chromosome ideogram is depicting the region (Chr10: 41,800,000–42,050,000) given in higher magnification. Annotation for the repeats in this location is given under the tracks according to the UCSC genome browser. Tracks for distribution of γH2AX nucleosomes are shown in blue for self-renewing, and in red for senescent hADSCs. Large γH2AX clusters are shown as solid boxes. On the right: schematic diagram showing the relative positions of Alu retrotransposons to the large senescence-associated, persistent γH2AX cluster from this genomic location. Grey and black boxes are indicative of the positions of MIR and Alu repeats. Representative example of transcriptional activity of these repeats as assessed by strand-specific RT-PCR is shown. Graph represents the quantitation of the RT-PCR results from seven independent experiments. Upregulation of transcriptional activity from Alu repeat in senescent cells correlates with formation of persistent DNA damage cluster upon senescence. Data shown are mean ± SEM **p = 0.00.014 (B) Cartoons are representing the composition of cohesin, condensin I and Eco1-dependent conversion of cohesin to cohesive state. (C) Loss of cohesin and condensin I in the pericentric location of persistent DNA damage in senescent hADSC. ChIP analysis of the pericentric repeats on chromosome 10 in self-renewing (blue bars) and senescent (red bars) hADSCs. Same repeats as in (A) were assessed as locations for recruitment of TFIIIC, Eco1 as well as components of cohesin (Rad21) and condensin I (CAP-H) complexes (n = 3, ±SEM). Schematic representation of the subunits of the cohesin and condensin I complexes, as well as a cartoon of previously reported function of Eco1, are shown. *p < 0.02, **p < 0.2.

Figure 6

Stable knockdown of generic Alu transcript in senescent human adult stem cells restores cell's proliferative properties and induces iPS-like phenotype. (A) Model of Alu retrotransposon. Secondary structure of generic Alu RNA. Regions for shRNA design are shown in blue. (B) Representative example of the efficiency of lentiviral transduction of hADSCs depicted by GFP. (C) Nothern blot hybridization of the RNA recovered from hADSCs cells stably expressing sh-RNA against Alu. Senescent hADSCs were infected with lentiGFP sh-193Alu, lentiGFP sh-132Alu or control no sh-RNA insert lentiGFP. RNA was isolated after 24 hrs post transduction and northern hybridization was performed with a Alu specific oligonucleotide. Senescent hADSCs stably expressing sh-132Alu show near complete knockdown of the Alu transcripts. (D) Proliferative properties of senescent hADSCs were reinstated in the cells upon stable knockdown of Alu transcripts. ³[H] thymidine uptake is shown. Senescent cells (wt) or senescent cells transduced with lentiGFP (control) or lentiGFP sh-132Alu were pulse-labeled with 1 µCi of ³[H] thymidine for 24 hrs either 24 or 96 h post infection. Data shown are mean ± SEM for triplicate measurements. (E) Immunostaining of prio senescent lentiGFP sh-132Alu ADSCs with proliferation marker Ki67. (F) Senescent ADSC cultures infected with lentiGFP sh-132Alu demonstrate a significant loss in senescence-associated SA-B-Gal activity and persistent DNA damage foci, indicated by gH2AX staining in GFP-expressing cells. (G) Expression of pluripotency markers Nanog and Oct4 was measured by qPCR analysis in senescent hADSCs (wt) and in hADSC with reversed-senescent phenotype upon stable knockdown of Alu transcription (lentiGFP sh-132Alu). RNA was isolated from the cells 96 h post infection. Results are expressed as relative quantity (DCt). Samples were normalized against β--actin. Data are shown as mean ± SEM (n = 3) ***p = 6.98e-05, *p = 0.03. (H) Model of Alu transcriptional toxicity upon ex-vivo human ADSCs aging.