is funded by the Drexel University College of Medicine Ageing Initiative Graduate College student Fellowship. Data availability The raw data and unique reagents and strains generated with this study are available from your corresponding author upon request. Code availability Not applicable. Declarations Conflicts of interestThe authors declare no discord of interest. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional statements in published maps and institutional affiliations.. in mediating cellular senescence. We statement here that Sp1 manifestation is decreased having a concomitant increase in senescence markers in response to DNA damage. Mutation of Sp1 at serine 101 to produce an ATM phospho-null mutant, or mutation of lysine 16 to create a sumo-null mutant, helps prevent the sumoylation and subsequent proteasomal degradation of Sp1 and results in a decrease in senescence. Conversely, depletion of Sp1 or mutation of Sp1 to produce an ATM phosphomimetic results in premature degradation of Sp1 and an increase in senescence markers. These data link a loss of genomic stability with senescence through the action of a DNA damage restoration element. Supplementary Information The online version consists of supplementary material available at 10.1007/s11357-021-00456-5. test, as specified in the number legends. ideals are indicated by non-significant (value greater than 0.05. All experiments were performed in triplicate, as specified in the number legends. Results Damage-induced degradation of Sp1 promotes cellular senescence Sp1 phosphorylated on serine 101 (Sp1pS101) localizes to DSBs and is necessary to facilitate DSB Malic enzyme inhibitor ME1 restoration via NHEJ [34, 35]. Its part appears to be self-employed of transcription as manifestation of a truncated Sp1 mutant (aa 1C182) that lacks its DNA binding website (Sp1-30?N), can still localize to DSBs and promote NHEJ restoration [34, 35]. Additionally, levels of Sp1 protein, but not RNA, decrease with age [17, 26C28]. This decrease is attributed to the accumulated effect of oxidative stress, resulting in part from deterioration in the antioxidant response in ageing cells. Sp1 is known to become phosphorylated at serine 101 (Sp1pS101) from the apical kinase in the DSB response, ataxia telangiectasia mutated (ATM) [33]. Inhibition of ATM reverses the degradation of Sp1 in response to damage-induced senescence, suggesting that DNA damage signaling is involved in senescence-related degradation of Sp1 [29]. Additionally, we have previously shown that in response to DNA damage, Sp1pS101 is definitely degraded in an S phase cell [36]. Consequently, we wanted to determine if damage-induced degradation of Sp1 promotes cellular senescence. hTert-BJ1 cells depleted of Sp1 using CRISPR/Cas9 were transduced with lentivirus expressing an empty-vector (Sp1?/?), or lentivirus expressing Flag-tagged Sp1WT, or Flag-tagged Sp1-30NWT under the control of its endogenous promoter (Supplemental Fig. 1a). These cells were then subjected to treatment with hydrogen peroxide (H2O2) or Adriamycin for 2?h, Malic enzyme inhibitor ME1 and lysates were then collected over a period of 24?h to assess p21 manifestation, a marker of cell cycle arrest, and lamin B1. Under wild-type conditions (cells expressing Sp1WT), we observe decreased manifestation of Sp1, which coincides with an increase of p21 manifestation and an Malic enzyme inhibitor ME1 increase in lamin B1, in response to DNA damage (Fig.?1a and Supplemental Fig. 2a). Additionally, cells expressing Sp1-30NWT, which lacks Sp1s DNA binding website and therefore cannot bind to DNA sequence specifically, displayed a decrease in Sp1 manifestation that also coincides with an increase in p21 manifestation and a decrease in lamin B1 in response to damage (Fig.?1a and Supplemental Fig. 2a). Sp1 offers been shown to play a transcriptional part in cellular senescence [16, 17, 27], but since manifestation of Sp1-30NWT, which cant bind DNA sequence specifically for transcription, Malic enzyme inhibitor ME1 does not enhance p21 manifestation in the absence of a DNA damaging agent suggesting that p21 manifestation was caused by damage-induced SLC2A2 senescence, but self-employed of Sp1s part like a transcription element. On the other hand, in Sp1?/? cells, we observe premature manifestation of p21 in the absence of DNA damage (Fig.?1a and Supplemental Fig. 2a). The addition of a DNA damaging agent further raises p21 manifestation in Sp1?/? cells. These Malic enzyme inhibitor ME1 results were mimicked in U2OS cells.