DAB2IP maintains intestinal epithelial homeostasis by modulating the DNA replication stress response

doi:10.21203/rs.3.rs-3631552/v1

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DAB2IP maintains intestinal epithelial homeostasis by modulating the DNA replication stress response

https://doi.org/10.21203/rs.3.rs-3631552/v1

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The proliferative activity of undifferentiated cells in intestinal crypts is essential for maintaining intestinal homeostasis and facilitating regeneration in response to both pathophysiological conditions and stress. DAB2IP (Disabled homolog 2 interacting protein), a tumor suppressor, plays a crucial role in regulating various oncogenic pathways. Our previous study unveiled that DAB2IP serves as a cell cycle regulator by facilitating PLK1-mediated mitosis progression. In this study, we uncovered a novel DAB2IP function as it mediates the maintenance and regeneration of intestinal epithelial homeostasis in normal conditions and following irradiation (IR)-induced gastrointestinal (GI) injury. Mechanistically, we identified that DAB2IP interacts with the histone acetyltransferase HBO1 and promotes PLK1-mediated phosphorylation of HBO1. This phosphorylation event leads to HBO-directed lysine 14 acetylation on histone 3 (H3K14Ac), the loading of the minichromosome maintenance protein (MCM) complex onto chromatin, progression of DNA replication, and maintenance of genome integrity. Furthermore, we found that ATR regulates Cdk1-mediated phosphorylation of DAB2IP and that phosphorylation of DAB2IP is crucial for the formation and activation of the HBO1-PLK1 complex. Finally, ablating DAB2IP phosphorylation results in increased genomic instability due to incomplete replication of genomic DNA, as shown via the accumulation of anaphase ultrafine bridges and 53BP1 nuclear bodies in the G1 cell cycle phase. Collectively, our findings highlight the regulatory role of DAP2IP in DNA replication-an essential process for maintaining genomic stability and intestinal homeostasis.

Biological sciences/Genetics/Gene regulation

Health sciences/Diseases/Gastrointestinal diseases

The tumor suppressor DAB2IP, also named AIP1 (ASK1 interacting protein), was initially discovered as a member of the RAS-GTPase activating protein (Ras-GAP) family [1, 2]. The GAP domain of DAB2IP is responsible for activating GTPase and inactivating the RAS protein [2]. In addition, DAB2IP acts as a scaffold to coordinate different signal transduction pathways involved in cell growth, apoptosis, and the epithelial-mesenchymal transition [3, 4]. DAB2IP directly interacts with both the PI3K regulatory subunit (p85) and Akt, which reduces the phosphorylation and activation of Akt [5]. Meanwhile, DAB2IP interacts with apoptosis signal-regulating kinase 1 (ASK1) and confers ASK1 activation and ASK1-mediated cell apoptosis [5]. DAB2IP is also involved in the inflammatory response. DAB2IP interacts with TNF receptor associated factor 2 (TRAF2) and abolishes TNF-α-induced NF-κB activation [6]. Mutant p53 has been found to competitively bind with DAB2IP, leading to the reactivation of NF-κB and the rewiring of the inflammatory microenvironment within tumors [7, 8]. Our previous research uncovered an additional function of DAB2IP, which is as a cell cycle regulator. We reported that DAB2IP was phosphorylated by Cdk1, and this phosphorylated form of DAB2IP interacts with and activates the mitotic regulator, polo-like kinase 1 (PLK1) [9, 10]. The loss of DAB2IP expression or the inhibition of its Cdk1-mediated phosphorylation causes significant abnormalities during mitosis. These include compromised kinetochore-microtubule attachment, impaired spindle assembly checkpoint, and subsequent chromosomal instability [9, 10]. However, the physiological role of DAB2IP in normal tissue function has not been fully clarified.

The small intestinal epithelium, one of the most dynamic adult tissues, undergoes complete renewal approximately every 4–5 days in mice and 5–7 days in humans [11, 12]. This rapid turnover is driven by the presence of intestinal stem cells (ISCs) located at the base of intestinal crypts, interspersed between Paneth cells, and characterized by the expression of various Wnt target genes, including the G-protein coupled-receptor Lgr5 [13]. ISCs of the Lgr5⁺ subtype produce highly proliferative bipotent progenitor cells called transit amplifying (TA) cells, which undergo four to five rounds of cell division as they migrate along the crypt-villus axis [14]. Subsequently, TA cells differentiate into distinct functional cell types within the mature epithelium and are eventually eliminated through apoptosis at the uppermost portion of the villus [15, 16]. The proliferative activity of both ISCs and TA cells is vital for maintaining intestinal homeostasis and facilitating tissue renewal and regeneration under normal conditions as well as in response to stress [17]. Proper regulation of cell cycle progression critically controls the proliferative activity of ISCs and intestinal progenitor cells. A recent study indicates that DNA replication licensing in ISCs serves as a “checkpoint” mechanism that influences the decision-making process regarding their proliferative fate [18]. During the S phase of cell cycle, DNA replication origin licensing initiates, involving the loading of minichromosome maintenance (MCM) 2–7 complexes onto origins and subsequent origin firing. The origin firing includes the activation of DNA-bound MCM2-7 as the core of the DNA helicase complex, CMG (Cdc45, MCM2-7, GINS) [19]. Notably, it was reported that MCM2 binds to chromatin in cells located around the + 4/+5 cell position of the crypt, while Lgr5⁺ ISCs remain in an unlicensed state to allow for sufficient time to make decisions regarding their proliferative fate [18]. Finally, the highly proliferative nature of the intestinal epithelium makes it particularly vulnerable to DNA-damaging agents, such as ionizing radiation (IR). ISCs and intestinal progenitor cells undergo compensatory proliferation, a heightened proliferative capacity aimed at repairing the IR-induced damaged intestinal epithelium [20]. Although, there are parallels between intestinal homeostasis and intestinal regeneration following injury, much is to be deciphered in regard to the mechanistic regulation of these processes.

In this study, we created a mouse model with a knockout of the DAB2IP gene and reported that DAB2IP^−/− mice display noticeably shorter villi in the small intestine. Furthermore, we observed a significant impairment in the proliferative activity of intestinal crypts in DAB2IP^−/− mice under both normal conditions and after exposure to IR. Mechanically, DAB2IP confers PLK1-mediated activation of histone acetyltransferase HBO1. This activation, in turn, regulates DNA replication by targeting histone H3K14 in the vicinity of the replication origin. The loss of DAB2IP results in reduced chromatin loading and phosphorylation of MCM2, leading to decelerated DNA replication speed. Consequently, genomic instability is increased, and cells experience cell death due to incomplete replication of genomic DNA. These observations shed light on a previously unanticipated function of DAB2IP in the regulation of DNA replication. We postulate that replication stress and genomic instability caused by the loss of DAB2IP in undifferentiated intestinal cells poses a threat to the maintenance of intestinal epithelial homeostasis and hinder the process of regeneration following radiation-induced GI injury.

DAB2IP promotes intestinal homeostasis and regeneration after IR-induced intestinal injury

In a previous study, mice lacking DAB2IP display hyperplasia in colonic epithelium as indicated by increased dilated colonic crypts and disruption of the monolayer epithelial arrangement of the crypts [21]. To examine if DAB2IP maintains epithelial homeostasis in the small intestine, we generated DAB2IP knockout mice via CRISPR/Cas9 in the C57BL/6 background. Sequencing and immunoblotting validated the DAB2IP^+/+, DAB2IP^+/−, and DAB2IP^−/− genotypes and further studies were performed using this cohort of mice (Fig. S1A). DAB2IP^+/+, DAB2IP^+/−, and DAB2IP^−/− mice were tracked for growth and we found that all three genotypes (males and females) developed normally (data not shown), which is similar to previous observations [22]. Next, we examined the crypts of the small intestines of the DAB2IP^+/+, DAB2IP^+/−, and DAB2IP^−/− mice. First, we found that DAB2IP is expressed in small intestine crypts and that this is completely lost in the DAB2IP^−/− mice (Fig. S1B). DAB2IP^−/− mice exhibited shortened villi and reduced crypt depth when compared to WT mice under both normal conditions and after exposure to 9 Gy γ-ray total body irradiation (TBI) (Fig. 1A and B and Fig. S1C). Elevated apoptosis in undifferentiated cells within the intestinal crypts has been shown to impair epithelial cell turnover and shorten villi in normal conditions and in response cellular stress; therefore, we assessed via TUNEL staining if apoptosis was increased in the intestinal crypts in our cohort of mice with and without radiation [23, 24]. We observed a substantial increase in the number of TUNEL⁺ crypt cells in DAB2IP^−/− mice compared to the DAB2IP^+/+ and DAB2IP^+/− mice under normal conditions and in response to TBI (Fig. 1C and D). To expand on this, we examined if intestinal self-renewal was decreased in DAB2IP^−/− mice by staining the crypts for the proliferation marker Ki67 in normal cycling cells and in response to DNA damage. We found that the number of Ki67⁺ cells per crypt was significantly decreased in the DAB2IP^−/− mice compared to the DAB2IP^+/+ and DAB2IP^+/− mice under normal conditions and in response to radiation (Fig. 1E and F). Lgr5⁺ ISCs play a crucial role in maintaining intestinal homeostasis and promoting regeneration after IR-induced damage. We observed that the number of Lgr5⁺ ISCs per crypt was significantly lower in DAB2IP^−/− mice both in untreated conditions and after radiation exposure compared to the DAB2IP^+/+ and DAB2IP^+/− mice (Fig. 1G and H). To further assess the self-renewal capacity of ISCs, organoids formation assay was conducted ex vivo. The loss of DAB2IP resulted in markedly reduced efficiency and size of organoid formation, both under normal conditions and in response to radiation (Fig. S1D and E). Finally, attenuated intestinal regeneration following radiation-induced injury in DAB2IP^−/− mice correlated with decreased survival to TBI compared to DAB2IP^+/+ and DAB2IP^+/− mice (Fig. 1I). Collectively, these results indicate that DAB2IP play a crucial role in protecting the homeostasis of undifferentiated intestinal crypt cells under normal conditions and in response to cellular stress.

Loss of DAB2IP exacerbates DNA replication stress and genetic instability by prolonging DNA replication progression

Our data shows that loss of DAB2IP results in decreased proliferation and increased apoptosis in intestinal crypt cells, suggesting that DAB2IP protects against an intrinsic cellular stress. These observations drove us to delineate the mechanism by which DAB2IP regulates intestinal homeostasis and proliferation. To initiate this, we isolated mouse embryonic fibroblasts (MEFs) from DAB2IP^+/+ and DAB2IP^−/− mice. Consistent with DAB2IP modulating proliferation, we found that DAB2IP^−/− MEFs display a decrease in growth rate compared to DAB2IP^+/+ MEFs as assessed by proliferation and colony formation assays (Fig. 2A-C). To expand on this, we examined DNA synthesis by assessing the incorporation of 5-ethynyl-2′-deoxyuridine (EdU) in DAB2IP^+/+ and DAB2IP^−/− MEFs. We observed that DAB2IP^−/− MEFs exhibited reduced EdU incorporation compared to DAB2IP^+/+ MEFs, indicating limited DNA synthesis upon loss of DAB2IP (Fig. 2D and E). Moreover, this was supported by the observation that knock-down of DAB2IP in the cervical cancer line HeLa resulted in a decrease in EdU incorporation compared to the siRNA control treated cells (Fig. S2A and B). We then assessed if DAB2IP regulates DNA replication by utilizing DNA fiber assays by sequentially pulse-labeling cells with iododeoxyuridine (IdU) and chlorodeoxyuridine (CIdU). The results show that progression of ongoing DNA replication tracks is markedly attenuated in DAB2IP^−/− MEFs compared to DAB2IP^+/+ MEFs (Fig. 2F-H), suggesting that loss of DAB2IP attenuates normal DNA replication progression. As compromised DNA replication can lead to replication stress, we next monitored overall levels and chromatin binding of the ssDNA binding protein RPA in untreated cycling cells and after the induction of replication stress via treatment with hydroxyurea (HU) [25, 26]. In DAB2IP^−/− MEFs, the chromatin loading levels of RPA2 was higher in the absence and presence of HU compared to DAB2IP^+/+ MEFs (Fig. 2I-K). The increase in RPA2 intensity in the presence and absence of HU was also found in HeLa cells in which DAB2IP was knocked down compared to control cells (Fig. S2C and D). Additionally, higher ssDNA formation was observed in DAB2IP^−/− MEFs compared to DAB2IP^+/+ MEFs both under normal conditions and following HU treatment, indicating that loss of DAB2IP results in increased ssDNA formation and replication stress (Fig. S3E and F). As increased replication stress can lead to the induction of DNA double strand breaks (DSBs) and genomic instability, we subsequently assessed if these were increased in DAB2IP^−/− MEFs. We observed that DAB2IP^−/− MEFs display more intense staining for the DSBs marker γH2AX compared to DAB2IP^+/+ MEFs in the absence and presence of HU treatment (Fig. 3A and B). Further, immunoblotting showed that γH2AX levels were increased and this correlated with increased apoptosis as assessed by cleaved caspase 3 and PARP1 cleavage in DAB2IP^−/− MEFs compared to DAB2IP^+/+ MEFs in normal cycling cells and after induction of exogenously induced replication stress (Fig. 3C). DAB2IP^−/− MEFs also display a higher frequency of chromosomal aberrations and increased incidence of micronuclei in the absence and presence of HU in comparison to DAB2IP^+/+ MEFs (Fig. 3D and E). Finally, loss of DAB2IP in MEFs, HeLa, and HCT116 cells results in increased sensitivity to HU (Fig. 3F and Figs. S3A and B). Collectively, these observations illustrate that DAB2IP promotes DNA replication and genomic stability in response to replication stress.

DAB2IP interacts with HBO1 and promotes PLK1-mediated phosphorylation of HBO1

Subsequently, we aimed to elucidate how DAB2IP regulates DNA replication and the response to replication stress. In our previous study, we reported that DAB2IP interacts with PLK1 to regulate mitosis [33]. PLK1, in addition to its role in mitosis, is an essential regulator of DNA replication and the response to replication stress via its ability to interact and phosphorylate HBO1 and facilitate the loading of the MCM complex onto chromatin [27–31]. This drove our hypothesis that DAB2IP regulates DNA replication and the response to replication stress by modulating the PLK1-HBO1 signaling axis. To examine this postulate, we initially examined if DAB2IP interacts with HBO1. Our findings demonstrated a reciprocal co-immunoprecipitated (co-IP) of DAB2IP and HBO1, indicating that proteins interact (Fig. 4A and B). Additionally, we observed that endogenous HBO1 interacts with transiently expressed FLAG-tagged DAB2IP (Fig. S4A). To identify the region of DAB2IP that mediates its interaction with HBO1, FLAG-tagged truncations of DAB2IP were transiently expressed in HeLa cells, immunoprecipitated using an anti-FLAG antibody, and the ability to interact with endogenous HBO1 was assessed. The results show that the GAP domain of DAB2IP preferentially interacts with endogenous HBO1 (Fig. 4C and D). It was previously reported that Cdk1 phosphorylates HBO1 at threonines 85 and 88, which are threonines followed by proline residues (pTP motif) [29]. These phosphorylated TP sites serve as a docking platform for the recruitment and activation of PLK1. Subsequently, activated PLK1 phosphorylates HBO1 at serine 57, also followed by a proline residue (pSP motif). This phosphorylation event plays a crucial role in facilitating the loading of the MCM complex onto chromatin and promoting DNA replication [29]. To investigate the influence of DAB2IP on phosphorylation of HBO1, we immunoprecipitated HBO1 from DAB2IP^−/− and DAB2IP^+/+ MEFs and observed a substantial reduction in HBO1 phosphorylation at SP site(s) in DAB2IP^−/− MEFs compared to DAB2IP^+/+ MEFs (Fig. 4E and F). Moreover, we did not observe HBO1-specific pTP immunostaining (Fig. S4B). This data indicates that DAB2IP influences PLK1-mediated, but not Cdk1, phosphorylation of HBO1. Additionally, knockdown of DAB2IP expression in HeLa cells led to a diminished phosphorylation of HBO1 at SP site(s) (Fig. 4G and H). Based on these findings, we aimed to investigate whether DAB2IP serves as a scaffolding protein, playing a role in stabilizing the interaction between HBO1 and PLK1. To test this hypothesis, we examined if DAB2IP regulates the interaction between FLAG-tagged PLK1 and endogenous HBO1. Our results demonstrated a significant reduction in the interaction between PLK1 and HBO1 in the DAB2IP knockdown cells (Fig. 4I and J). Taken together, these data suggest that DAB2IP interacts with both HBO1 and PLK1, contributing to the stabilization of the PLK1-HBO1 association and facilitating PLK1-mediated HBO1 phosphorylation.

The loss of DAB2IP leads to reduced H3K14 acetylation, impairment of MCM chromatin-binding, and decreased MCM2 phosphorylation

HBO1 is required for the acetylation of histone 3 on its K14 site (H3K14) [32]. As we found that DAB2IP modulates HBO1 phosphorylation, we next investigated if DAB2IP influences the activity of HBO1. We found that the acetylation of H3K14 is reduced in DAB2IP^−/− MEFs compared to DAB2IP^+/+ MEFs, as evidenced by immunoblotting and immunefluorescent staining (Fig. 5A-C). Furthermore, we observed that knockdown of DAB2IP in HeLa cells also resulted in reduced H3K14 acetylation and that this was rescued when expression of DAB2IP was restored (Fig. S4C and D). Considering the significant role of HBO1 in DNA replication licensing, we examined if the loss of DAB2IP affects this process by assessing the loading of the MCM complex onto chromatin in DAB2IP^−/− MEFs compared to DAB2IP^+/+ MEFs in absence and presence of HU. The loss of DAB2IP resulted in a significantly impairment of the chromatin loading of MCM2 and MCM7, both in the absence and presence of HU (Fig. 5D). Consistently, a reduction in chromatin-bound MCM complex was also observed in DAB2IP-depleted HeLa cells (Fig. S4E). Phosphorylation of MCM2 on serines 40 and 41 (S40/41) has been shown to play a dual role in the activation of pre-replication complexes and the facilitation of replication fork progression [33, 34]. With this in mind, we proceeded to investigate the influence of DAB2IP on MCM2 phosphorylation. Our analysis identified a clear reduction in MCM2 phosphorylation at the S40/41 sites in DAB2IP^−/− MEFs compared to DAB2IP^+/+ MEFs (Fig. 5E). The phosphorylation of MCM2 at the Ser40/41 sites was also examined in intestinal sections obtained from DAB2IP^+/+, DAB2IP^+/− and DAB2IP^−/− mice. We found that the activation of MCM2 primarily occurs in the transit-amplifying (TA) cells of the crypt and that loss of DAB2IP dramatically decreases the proportion of MCM2-pSer40/41⁺ cells within the crypts (Fig. 5F and G). Collectively, our results show that loss of DAB2IP leads to reduced H3K14 acetylation, impairment of MCM chromatin-binding, and decreased MCM2 phosphorylation.

ATR inhibition contributes to HBO1 phosphorylation by promoting Cdk1-dependent DAB2IP-PLK1 association, and subsequent activation of PLK1

Recent studies demonstrated the crucial involvement of the ATR signaling pathway in restricting Cdk1 activation and replication origin firing during normal DNA replication [35, 36]. To explore the impact of ATR on PLK1 activity toward HBO1 and the role of DAB2IP in this process, we treated DAB2IP-depleted and control HeLa cells with various combinations of inhibitors, including VE-821 (ATR inhibitor), VE-821 + RO3306 (combination of ATR and Cdk1 inhibitor), and VE-821 + BI2536 (combination of ATR and PLK1 inhibitor). HBO1 was immunoprecipitated from cells subjected to the various treatment and the phosphorylation of HBO1 at SP sites was detected. The data revealed that the ATR inhibitor notably enhanced the phosphorylation of HBO1 on SP sites. However, the addition of Cdk1 or PLK1 inhibitors completely abolished the SP phosphorylation of HBO1, even in response to ATR inhibition (Fig. 6A). Furthermore, in DAB2IP-depelated cells, ATR inhibition failed to enhance the SP phosphorylation of HBO1 (Fig. 6A). This finding suggests that DAB2IP plays an essential role in facilitating Cdk1-primed and PLK1-mediated HBO1 phosphorylation. Additionally, we observed that ATR promoted the interaction between PLK1 and HBO1 in control HeLa cells, whereas its impact was only marginal in DAB2IP-depleted HeLa cells (Fig. 6B).

In our previous study, we demonstrated that Cdk1 phosphorylates DAB2IP on its Thr-531 and Thr-546 residues; therefore, we investigated if Cdk1-mediated phosphorylation of DAB2IP also primes PLK1 and activates PLK1’s activity toward HBO1 [10]. To assess this, FLAG-tagged wild-type (WT) DAB2IP and the DAB2IP-Thr531Ala/Thr-546Ala double site mutants (DAB2IP-2A) were expressed in HeLa cells. We then synchronized HeLa cells at the G1/S boundary using double thymidine blockage. After release from the block, we immunoprecipitated the FLAG-tagged DAB2IP from cells at various cell cycle phases, including G1/S, S, and mitosis. Our findings revealed that DAB2IP, but not DAB2IP-2A mutants, is phosphorylated on TP sites in each phase of the cell cycle (Fig. S5A). Next, we examined this using our phospho-Thr531 antibody. We observed that treatment with the ATRi increased phosphorylation at T531 and this was attenuated with co-treatment with Cdk1i (Fig. 6C). Subsequently, we examined if modulation of DAB2IP phosphorylation by ATR and/or Cdk1 affects the interaction between DAB2IP and PLK1 as well as the activation of PLK1. We found that pretreatment with the ATRi significantly increased the interaction between PLK1 and DAB2IP (Fig. 6D). Conversely, co-treatment of Cdk1i + ATRi eliminated the ATR inhibitor-mediated enhancement of DAB2IP-PLK1 association (Fig. 6D). We then determined if DAB2IP phosphorylation affects HBO1 phosphorylation and the HBO1-PLK1 interaction. Transient expression of WT and DAB2IP phosphorylation mimic mutants (DAB2IP-2D) rescued HBO1 phosphorylation and promoted increased interaction between HBO1 and PLK1, which was not observed in DAB2IP knockout cells that were rescued with the DAB2IP 2A mutant (Fig. 6E-G). The mutation of DAB2IP on its Thr-531 and Thr-546 sites does not appear to have any influence on the interaction between DAB2IP and HBO1 (Fig. S5B). To further validate the role of phosphorylated DAB2IP in PLK1-mediated HBO1 phosphorylation, we purified FLAG-tagged WT DAB2IP, DAB2IP-2A, and DAB2IP-2D, as well as FLAG-tagged PLK1 and FLAG-HBO1, from HeLa cells treated with ATRi (Fig. S5C and D). Subsequently, FLAG-tagged PLK1 and HBO1 proteins were treated with λPP to remove endogenous phosphorylation signals and then these proteins were mixed together for an in vitro kinase assay. We found that WT FLAG-tagged DAB2IP and DAB2IP-2D proteins, but not the DAB2IP-2A mutant, significantly enhanced PLK1-mediated phosphorylation of HBO1 on its SP sites (Fig. 6H). Together, these data indicate that Cdk1-mediated phosphorylation of DAB2IP generates a docking site for PLK1 to facilitate the DAB2IP-PLK1 interaction and that this promotes PLK1-mediated phosphorylation of HBO1.

Phosphorylation of DAB2IP is essential for maintaining genomic stability

To gain further insight into the role of phosphorylated DAB2IP in the regulation of DNA replication and genome maintenance, siRNA-resistant wild-type DAB2IP, DAB2IP-2A, and DAB2IP-2D were reintroduced into DAB2IP-depleted HeLa cells. The expression of WT DAB2IP and the DAB2IP-2D mutant, but not DAB2IP-2A, led to the restoration of acetylated H3 on K14 and the phosphorylation of MCM2 on S40/41, suggesting that phosphorylation of DAB2IP promotes HBO1-directed activities (Fig. 7A). Dysregulated DNA replication rate can cause replication stress, leading to accumulation of DNA damage during mitosis, and ultimately resulting in the formation of ultrafine chromatin bridges (UFBs) during anaphase [37, 38]. We postulated that replication stress associated with DAB2IP loss would lead to these events. Consistent with this, loss of DAB2IP resulted in marked increase in the number of mitotic DSBs as assessed by γH2AX foci per prometaphase cell (Fig. 7B and C). Moreover, this correlated with increased formation of anaphase bridges and lagging chromosomes, and subsequent formation of 53BP1 bodies in G1 phase of the cell cycle (Fig. 7D-G). In contrast, the heightened genomic instability observed upon loss of DAB2IP was effectively reversed by expressing wild-type DAB2IP and DAB2IP-2D proteins (Fig. 7B-G). However, the expression of the siRNA-resistant DAB2IP-2A mutant did not exhibit the same rescue effect. Additionally, our findings indicate that phosphorylated DAB2IP plays crucial role in maintaining resistance to HU treatment (Fig. 7H). These results show that phosphorylation of DAB2IP at the Thr531 and Thr546 sites is necessary for proper DNA replication and the maintenance of genomic stability. Collectively, our study indicates the DAB2IP-Cdk1-PLK1-HBO1 axis regulates DNA replication by promoting HBO1-mediated acetylation of histone H3, leading to MCM loading, and onset of DNA replication, and if this process is dysregulated it leads to DNA replication stress and genomic instability (Fig. S6). We postulate that this process is vital for intestinal homeostasis in normal cycling cells and in response to cellular stress.

In this study, we have uncovered a previously unknown function of DAB2IP in small intestine homeostasis maintenance and regeneration following radiation-induced intestinal injury. The preservation of intestinal epithelium maintenance depends on a delicate balance between the continuous proliferation of undifferentiated cells in the crypt and apoptosis of differentiated epithelial cells [20]. Our data revealed a significant reduction in the proliferative activity of the crypt in DAB2IP^−/− mice compared to DAB2IP^+/+ mice. Additionally, the loss of DAB2IP resulted in notable changes in the architecture of crypt and villus, including shorter villi and reduced crypt depth, even under normal growth conditions. In addition to the changes observed in vivo, we also observed a reduction in organoid formation and budding efficiency in ex vivo-cultured crypts derived from DAB2IP^−/− mice compared to DAB2IP^+/+ mice. Intriguingly, we observed the accumulation of DNA damage and subsequent chromosomal abnormalities in DAB2IP^−/− cells, particularly under HU exposure. These findings further support the role of DAB2IP is maintaining genomic stability and preventing the detrimental effects of DNA damage. Our study demonstrated that DAB2IP deficiency perturbs normal progression of DNA synthesis, resulting in the accumulation of extended ssDNA. Following exposure to IR, the intestinal homeostasis is disrupted, and to regenerate epithelium after injury, the proliferative activity of ISCs and progenitor cells in the crypt is typically enhanced [39]. However, in the context of DAB2IP deficiency, we propose that the decreased proliferative capacity of undifferentiated cells in the crypt, resulting from DNA replication stress, exacerbates IR-induced intestinal injury. Moreover, a recent study have revealed that while direct DSBs induced by IR can be efficiently repaired, the secondary replication stress associated DSBs are not easily repairable and can lead to genomic instability [40]. We propose that the sustained replication stress, in normal cycling cells and from induced by DNA damaging agents, leads to increased DSB formation and eventually causes apoptosis. In line with this hypothesis, our study showed a more severe loss of Lgr5⁺ ISCs and increased apoptosis of undifferentiated crypt cells in DAB2IP^−/− mice.

We previously established that DAB2IP is a novel regulator of PLK1 in the context of mitotic progression [9, 10]. PLK1 is a critical kinase involved in orchestrating various aspects of mitotic progression. Loss of PLK1 expression or its inactivation results in severe mitotic aberrations and subsequent cell death. Additionally, PLK1 also plays a role in DNA replication progression, in cycling cells and in response to stress exposure [28, 29]. Indeed, PLK1 has been shown to directly interact with members MCM complex, including MCM2, MCM3, and MCM7 [41]. Depletion of PLK1 disrupts the proper chromatin loading of the MCM complex and subsequently reduces DNA synthesis during the S phase of the first cell cycle following depletion [30]. This observation is supported by another study demonstrating that PLK1 phosphorylates the Ser-57 site of HBO1, which is crucial for MCM complex assembly [44]. The polo-box domain (PBD) of PLK1 has propensity to bind to proteins that are primed-phosphorylated by CDKs, with a consensus sequence typically represented by S-pS/pT-P/X [42]. The binding of the PBD domain to these primed-phosphorylated proteins triggers the release of PLK1’s N-terminus kinase domain, leading to the activation of PLK1. It was demonstrated that Cdk1-mediated priming phosphorylation of HBO1 plays a crucial role in facilitating the interaction between PLK1 and HBO1 [29]. This priming phosphorylation event by Cdk1 primes HBO1 for subsequent phosphorylation by PLK1, further regulating HBO1’s functions towards MCM chromatin loading. Our data provided evidence for the interaction of DAB2IP with both HBO1 and PLK1 through adjacent motifs. This suggests a novel role for DAB2IP as a scaffolding protein that can simultaneously recruit HBO1 and PLK1, leading to the specific activation of PLK1 on its target HBO1. Supporting this concept is the observation that although the phosphorylation-deficient mutant of DAB2IP (Thr531Ala and Thr546Ala) does not affect the interaction between DAB2IP and HBO1, it significantly impairs the effect of DAB2IP on the association between PLK1 and HBO1, as well as the PLK1-mediated phosphorylation of HBO1.

The ATR-Chk1 signal pathway plays crucial role in the cell cycle checkpoint response to genotoxic stress. It functions by inhibiting Cdks activity, allowing cells sufficient time to resolve the stress and maintain genomic integrity. In normal DNA replication, ATR is activated at relatively low levels and serves to regulate the S/G2 cell cycle transition, ensuring that it proceeds only after DNA replication has been completed [43]. The ATR-Chk1 signaling pathway also inhibits the Cdk1-mediated phosphorylation of Rif1 on its Ser2205 residue in mammalian cells [36]. Rif1 is known to interact with protein phosphatase 1 (PP1) and plays a critical role in recruiting PP1 to chromatin. This recruitment enables PP1 to counteract the phosphorylation of MCM proteins induced by CDC7 and CDK2, thus regulating the initiation of DNA replication [44]. Thus, the inhibition of ATR-Chk1 signaling has been shown to increase the Cdk1-mediated phosphorylation of Rif1. This enhanced phosphorylation disrupts the interaction between Rif1 and PP1, resulting in the activation of replication origin firing. A recent study using Xenopus as a model system revealed that PLK1 phosphorylates the same conserved site of Rif1, leading to the disruption of the Rif1-PP1 interaction [31]. Although it remains unknown whether ATR directly suppresses the activity of PLK1 on Rif1, our study provides evidence that ATR regulates PLK1-mediated phosphorylation of HBO1. Furthermore, our finding suggest that this regulation by ATR relies on Cdk1-primed phosphorylation. Direct evidence comes from the observation that the inhibition of ATR enhances HBO1 phosphorylation, and this effect can be effectively suppressed by PLK1 and Cdk1 inhibitors. Moreover, our study unveils the role of DAB2IP in supporting the activation of Cdk1 and PLK1 on their targets after ATR inhibition. In the context of DAB2IP depletion, the ATR inhibitor only slightly enhances the interaction between HBO1 and PLK1, as well as the PLK1-mediated phosphorylation of HBO1 (Fig. 7). Further investigation is warranted to fully elucidate the specific contribution of DAB2IP in ATR-suppression-related molecular events during replication regulation, such as the disassociation of Rif1-PP1 and the activation of MCM complex. Our data showing that DAB2IP promotes MCM2 phosphorylation in both in vivo and in vitro provide supportive evidence for this hypothesis.

PLK1 is essential for embryonic development. PLK1 knockout mice are known to be embryonic lethal, with early embryos failing to survive beyond the eight-cell stage [45]. In study utilizing inducible knockdown mouse model, it has been observed that loss of PLK1 leads to a reduction in the number of Ki67⁺ cells in large intestine crypts [46]. Additionally, the studies demonstrating the involvement of PLK1 in maintaining intestinal homeostasis in sepsis provide further evidence for the potential role of PLK1 in the regulation of intestinal integrity and repair processes [47]. Here, our study, which reveals the role of PLK1 in intestinal regeneration after IR-induced intestinal injury, adds to the growing body of evidence supporting the importance of PLK1 in intestinal homeostasis maintenance. The phosphorylation of DAB2IP on the Thr531 and Thr546 sites has been shown to play a crucial role in the activation of PLK1 and its subsequent signaling during mitosis [34] and DNA replication regulation. There will be considerable interest in generating DAB2IP Thr531Ala and Thr546Ala knock-in mice to better understand the specific DAB2IP-PLK1 signal pathway in intestinal homeostasis maintenance in unperturbed condition and intestinal regeneration following damage by radiation/chemotherapy or under pathophysiological conditions.

PLK1 is commonly known as an oncogenic protein due to its essential role in driving cell cycle progression in tumor cells. However, emerging evidence suggests that PLK1 can also have tumor-suppressive functions in specific contexts. PLK1 overexpression results in abnormal chromosome segregation and cytokinesis failure to generate polyploid cells with reduced proliferative activity [48]. The in vivo study showed that PLK1-overexpression delays Kras- and Her2-induced mammary gland tumor development [48]. In contrast, PLK1 inhibition increased intestinal tumors in two independent Apc^Min/+ mouse models [49]. PLK1 suppresses genomic instability, a major driving force of tumor development, by not only guarding the mitosis progression but also preventing incomplete DNA synthesis and loss of DNA integrity [30]. The prolonged DNA synthesis leads to incomplete replication of genomic DNA, especially at the difficult-to-replicate regions such as common fragile sites (CFS), followed by the accumulation of mitotic UFBs [50]. The resolve of UFBs is commonly accompanied by the generation of micronuclei and G1-phase 53BP1 nuclear bodies, providing fuel for the selection of cancer evolution. The increased levels of UFBs during anaphase and G1-phase 53BP1 nuclear bodies in DAB2IP-deficient or non-phosphorylatable DAB2IP cells in response to replication stress, suggesting the involvement of DAB2IP in genomic stability maintenance and tumor development suppression, at least in part, through PLK1-dependent pathway.

Generation of DAB2IP knockout mice

DAB2IP knockout mice were obtained from Cyagen Biotechnology Co. Ltd (Guangzhou, Guangdong, China). The DAB2IP knockout C57BL/6 mice were generated by CRISPER/Cas9-mediated genome engineering. The mouse DAB2IP gene (GenBank accession number: NM_001114124.2) is located on mouse chromosome 2. The gRNA was designed to remove exon 4–6, and sequence are as followed: gRNA1: 5’-CTTGCAACAGACAAGTCCGGAGG-3’; gRNA2: 5’-CCAGAACTAGTAGCGCTGAGTGG-3’. Cas9 mRNA and gRNA were added into fertilized eggs for the production of DAB2IP knockout mice. The mice were backcrossed four times with wild-type C57BL/6 mice in order to limit potential off-target mutations generated by CRISPR-Cas9.

Mouse irradiation protocol

The DAB2IP^+/+, DAB2IP^+/−, and DAB2IP^−/− mice were exposed to 9 Gy of total body irradiation (TBI) which was delivered using a ⁶⁰Co γ source at a dose rate of 2 Gy/min. For the survival experiments, the weight and activity were monitored until 30 days post-irradiation. The mice were sacked when they lost more than 20% of their initial body weight; their Body Conditioning Score (BCS) fell below 2%, or at the date of experimental termination. For short-term experiments, mice were euthanized 6 h or 3 days post-irradiation.

Histological analysis

The histological analysis was performed as previously described [51, 52]. Small intestine tissue from DAB2IP^+/+, DAB2IP^+/−, and DAB2IP^−/− mice was isolated at the indicated time after TBI. Tissues were fixed overnight in 10% neutral formalin and were embedded in paraffin. Three mm-thick sections were cut and stained with hematoxylin, eosin (H&E), or other staining reagents under standard protocols and observed under a light microscope (Olympus Corp., Tokyo, Japan). Villus height and crypt depth were determined from H&E-stained sections using Image J 1.43 software. At least 30 well-oriented, full-length crypt-villus units per mouse were measured in pixels and converted into micrometer units by dividing with the conversion factor of 1.46.

Immunohistochemistry (IHC)

The IHC staining was performed as previously described [51, 52]. Intestine tissue sections from DAB2IP^+/+, DAB2IP^+/−, and DAB2IP^−/− mice were deparaffinized and rehydrated. The endogenous peroxidase was quenched after incubation with 3% hydrogen peroxide for 15 min at room temperature. Following the antigen retrieval in sodium citrate buffer (pH 6) for 3 min in a pressure cooker, tissues were incubated with 5% bovine serum albumin (BSA) for 30 min at room temperature to block the non-specific signal. The intestinal sections were exposed to the indicated antibody overnight, including antibodies against Lgr5 (1:200; Abbiotec, San Diego, CA, USA), Ki-67 (1: 100; Abcam, Cambridge, MA, USA), MCM2-pS40/41 (1:100; Bethyl Laboratories, Montgomery, TX, USA) and cleaved caspase 3 (1:50; Cell Signaling Technology, Beverly, MA, USA) at 4°C. Followed incubation with a pre-diluted secondary antibody (Zhongshan Golden Bridge Biotechnology, Beijing, China) at 37°C for 1 hour, color was developed using 3, 3N-diaminobenzidine tetrahydrochloride (DAB), and the sections were counterstained with hematoxylin.

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay

The TUNEL assay was performed using an in situ Cell Death Detection Kit (Roche Diagnostic, Mannheim, Germany) according to the manufacturer's instructions. Briefly, paraffin intestine sections were dewaxed, rehydrated, and incubated with the TUNEL reaction mixture at 37°C for 1 h. The nuclei were visualized using staining of DAPI, and then intestine sections were analyzed under a confocal laser scanning microscope (Olympus Corp., Tokyo, Japan). TUNEL-positive cells exhibiting green fluorescence were counted in at least 30 well-oriented crypts per group.

Crypt isolation and ex vivo culture

The ex vivo organoid cultures were generated as previously described [51]. Approximately 20-cm small intestine segments from DAB2IP^+/+, DAB2IP^+/−, and DAB2IP^−/− mice were harvested. The intestinal segments were placed into 100-mm dishes containing 5 mL of cold PBS, and the segments were flushed gently with cold PBS. The intestinal sections were transferred to clean dishes with 15 mL of cold PBS, cut into 2 mm pieces, and washed with fresh PBS 25–30 times. Intestinal pieces were then incubated with 25 mL Gentle Cell Dissociation Reagent (Stem Cell Technologies, Vancouver, BC, Canada) for 15 min on a rocking platform at 20 rpm at room temperature (15–25°C). The supernatants were discarded, and 10 mL of cold PBS with 0.1% BSA was added. The suspension was allowed to settle and then filtered through a 70 µm cell strainer (BD Biosciences, San Diego, CA, USA) to remove the residual villous material. The filtrate was centrifuged at 290 x g for 5 min at 4°C. The pelleted crypts were re-suspended in complete IntestiCult™ Organoid Growth Medium (Stem Cell Technologies) at the density of 500 crypts per 50 µl. The cell suspension was then mixed with 50 µl undiluted Matrigel® (BD Biosciences) and seeded in pre-warmed 24-well plates. The polymerized Matrigel was layered with 750 µl complete IntestiCultTM. For the irradiation groups, the intestinal crypts were subjected to 9 Gy γ-ray irradiation and cultured continuously at 37°C in 5% CO². Images were taken 1, 2, 3, 4, and 5 days post-irradiation.

Cell culture, siRNA transfection, plasmids, and treatment

Human cervical cancer HeLa cells, human embryonic kidney 293T cells, human colon cancer HCT116 cells, and mouse embryonic fibroblasts (MEF) were maintained in α-minimum essential medium (α-MEF, HyClone, Hudson, NH, USA), supplemented with 10% fetal bovine serum (FBS, HyClone, Hudson, NH, USA), 10 mM HEPES, 1 mM sodium bicarbonate, 100 U/ml penicillin, and 100 µg/ml streptomycin, 10 mM HEPES (HyClone, Hudson, NH, USA) in a humidified incubator at 37°C with 5% CO₂. All cell lines were authenticated using short tandem repeat (STR) profiling detection by the Cell Bank of Typical Culture Preservation Committee of the Chinese Academy of Sciences. Cell lines were routinely tested using Mycoplasma-free Mycoplasma Detection Kit (Beyotime, Shanghai, China).

The primary MEFs were kindly provided by Pro. Jer-Tsong Hsieh at UT Southwestern Medical Center [33]. The MEFs used in this study were immortalized by transfecting with SV-40. HeLa cells were transfected with pGIPZDAB2IP-lentiviral-shRNAmir and pGIPZ-non-silencing-lentiviral shRNAmir [4] according to the manufacturer’s protocol. The cells were then selected by puromycin (ThermoFisher Scientific, Carlsbad, CA, USA) at a concentration of 0.5 g/ml for 3–4 weeks. Duplex siRNAs were synthesized (ThermoFisher Scientific, Carlsbad, CA, USA) based on experimentally validated target sequences for DAB2IP siRNA (5’-GGAGCGCAACAG UUACCUGTT-3’), siRNA2 (5’-GGUGAAGGACUUCCUGACATT-3’) or control siRNA (5’-CTGGACTTCC AGAAGAACA-3’). siRNA was transfected into HeLa cells using Lipofectamine 3000 (ThermoFisher Scientific, Carlsbad, CA, USA).

The DAB2IP full-length expressing plasmids were cloned from human DAB2IP isoform 2 cDNA (1065a.a.). The DAB2IP siRNA-resistant expression plasmid and various DAB2IP full-length and truncated expression plasmids were described previously [4, 9]. A sequence-verified open reading frame clone of PLK1 was cloned into pcDNA3.1 3xFlag, pCMV-HA, pcDNA5/FRT/TO V5 expression plasmids. A sequence-verified open reading frame clone of human HBO1 was cloned into pcDNA3.1 3xFlag expression plasmid. Point mutations for DAB2IP were generated by using a QuikChange II site-directed mutagenesis kit (Agilent, Santa Clara, CA, USA).

Cells were treated with 0.5 mM (MEFs) or 2 mM (HeLa cells) HU (Sigma, St. Louis, MO, USA), 5 µM Cdk1 inhibitor RO-3306 (Selleckchem, Houston, TX, USA), 10 nM PLK1 inhibitor BI2536 (Sigma, St. Louis, MO, USA) or 5 µM ATR inhibitor VE-821 (Selleckchem, Houston, TX, USA) for indicated times. For the colonies survival experiment, MEFs were treated with 5, 10, 25 and 50 µM HU, HeLa cells were treated with 100 and 200 µM HU.

Antibodies, immunoprecipitation, and immunofluorescence

The following primary antibodies were used in the present study: anti-DAB2IP, anti-MCM2, anti-MCM2-pSer40/41, anti-cyclin A (Bethyl Laboratories, Montgomery, TX, USA); anti-HBO1, anti-PARP, anti-cleaved caspase 3, anti-H3, anti-H3K14Ac, anti-p-SP motif, anti-p-TP motif, anti-53BP1, anti-Rif1 (Cell Signaling Technology Danvers, MA, USA); anti-HA, control mouse IgG and rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA); anti-α-tubulin, anti-V5, anti-Flag (M2), anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA); anti-γH2AX, anti-BrdU/IdU (BD Biosciences, San Jose, CA, USA); anti-FANCD2 (Novus Biologicals, Centennial, CO, USA); anti-RPA2 (EMD Millipore, Temecula, CA, USA); anti-BrdU/CldU (Bi-Rad, Hercules, CA, USA). The secondary antibodies used for immunofluorescence were Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 488 goat anti-human IgG, Alexa Fluor 568 goat anti-mouse IgG, Texas red goat anti-rat IgG and Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen Carlsbad, CA, USA).

HeLa cell lysates were incubated overnight with anti-DAB2IP, anti-HBO1, or anti-Flag (M2) antibodies and protein A/G sepharose. The sepharose beads were washed with lysate buffer three times and resuspended in a sodium dodecyl sulfate (SDS)-PAGE loading buffer for immunoblotting analysis using indicated antibodies.

For the immunofluorescent staining experiment, cells with the different treatment regimens were plated on 35 mm dishes with coverslips, fixed in 4% paraformaldehyde/PBS (phosphate-buffered saline) for 30 min, permeabilized in 0.5% Triton X-100/PBS for 15 min, and blocked in 5% bovine serum albumin (BSA)/PBS for 30 min. The samples were incubated with anti-α-γH2AX (1:1000), anti-53BP1 (1:1000), anti-RPA2 (1:500), anti-cyclin A (1:500), anti-Rif1 (1:200) and anti-H3K14Ac (1:200) antibodies for 3 h at room temperature, washed three times in PBS (5 min each), and incubated with Alexa Fluor 488 and 568 secondary antibodies (1:1000) for 1 h. The cells were washed in PBS three times (5 min each) and mounted using VECTASHIELD with 4', 6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). Images were taken under a fluorescence microscope (Axio Imager M2, Carl Zeiss, Thornwood, NY, USA) and were recorded with AxioVision SE64 Rel.4.8 software (Carl Zeiss, Thornwood, NY, USA). For chromatin-bound RPA2 staining, cells were pre-extracted with CSK buffer (10 mM Pipes, pH 7.0, 100 mM NaCl, 300 mM sucrose, and 3 mM MgCl₂, containing 0.5% Triton X-100) for 5 min before fixation.

Clonogenic survival and cell proliferation assays

For clonogenic survival, 400 or 800 DAB2IP^+/+ and DAB2IP^−/− MEFs were plated into 60-mm dishes. Cells were grown for 10 days. The colonies were fixed with 100% ethanol and stained with 0.1% crystal violet. A colony with more than 50 cells was counted as a survivor.

DAB2IP ^+/+ and DAB2IP^−/− MEFs were plated into 60-mm dishes and exposed to the indicated dose of γ-ray with a J. L. Shepherd and Associates irradiator that emitted ¹³⁷Cs γ-rays at a dose rate of 3.44 Gy/min to the cells’ positions. Cells were cultured for 10 days. For HU treatment, HeLa cells were transfected with siRNA to suppress DAB2IP expression or were co-transfected with siRNA and siRNA-resistant DAB2IP (rWT), siRNA-resistant DAB2IP 2A (r2A), and siRNA-resistant DAB2IP 2D (r2D) constructs, or DAB2IP^+/+ or DAB2IP^−/− MEFs, were plated in 60-mm dishes. Cells were treated with the indicated dose of HU for 10 days. The colonies were fixed with 100% ethanol and stained with 0.1% crystal violet. A colony with more than 50 cells was counted as a survivor.

For cell proliferation/viability assays, 1000 cells were plated in 96-well microplates. Cell numbers were analyzed by using Cytation 5 Multi-Mode Reader (BioTek, Winooski, Vermont) at indicated days after plating.

FLAG-tagged protein purification

For purification of 3×FLAG-tagged DAB2IP, DAB2IP-2A, DAB2IP-2D, HBO1, and PLK1 from HeLa cells, the plasmids were transfected into HeLa cells. The cells expressing FLAG-DAB2IP, FLAG-DAB2IP-2A, and FLAG-DAB2IP-2D were treated with 5 µM VE-821 for 2 h before harvest. FLAG-tagged protein purification was performed as described [53]. Cells were suspended briefly in lysis buffer 3 (50 mM HEPES 7.4, 500 mM NaCl, 0.5% NP-40, 10% Glycerol, 1× phosphatase inhibitor cocktail, 1× protease inhibitor cocktail) and sonicated on ice. Cell lysates were incubated with M2-FLAG beads (Sigma, St. Louis, MO, USA). Flag-tagged HBO1 and PLK1 were treated with lambda phosphatase (γPP) (Sigma, St. Louis, MO, USA). Protein-bound M2-Flag beads were washed with washing buffer 3 (50 mM HEPES pH 7.4, 1 M NaCl, 0.5% NP-40, 10% Glycerol, 1× phosphatase inhibitor cocktail, 1× protease inhibitor cocktail), lysis buffer 3, and washing buffer 4 (50 mM HEPES 7.4, 150 mM NaCl, 0.5% NP-40, 10% Glycerol), washing buffer 5 (50 mM HEPES 7.4, 150 mM NaCl, 10% Glycerol), and washing buffer 6 (50 mM HEPES 7.4, 80 mM KCl, 10% Glycerol). Finally, proteins were eluted with washing buffer 6 containing 250 µg mL^− 1 3×FLAG peptides. Protein purity was examined by Coomassie blue gel staining after SDS-PAGE, and the concentration was measured with Bio-Rad Protein Assay Kit 1.

In vitro kinase assay

For the in vitro PLK1 kinase assay, purified Flag-PLK1 was incubated with Flag-DAB2I, Flag-DAB2IP-2A, or Flag-DAB2IP-2D in PLK1 kinase reaction buffer [20 mM HEPES (pH 7.4), 100 mM KCl, 10 mM MgCl2, 1 mM EDTA, 0.5 mM DTT, 5% glycerol, 10 M ATP and 0.17 µM γ-32P ATP] with Flag-HBO1 as substrates. The reaction was incubated for 30 min at 30◦C and stopped by adding sodium dodecyl sulfate (SDS) sample buffer. After the kinase reaction, samples were subject to immunoblotting analysis using an anti-p-S-P antibody.

Subcellular fractionation

The association of the MCM complex and RPA2 with chromatin in DAB2IP-proficient or -deficient cells (DAB2IP^+/+ and DAB2IP^−/− MEFs, or control and siRNA-mediated knockdown HeLa cells) was examined using the Thermo Fisher Subcellular Protein Fractionation Kit as described [54]. Briefly, the DAB2IP^+/+, DAB2IP^−/− MEFs, or control or siRNA-mediated knockdown HeLa cells were mock-treated or treated with the indicated dose of HU for 2 h. Next, the cells were harvested after trypsinization and processed with the Thermo Fisher Subcellular Protein Fractionation Kit according to the manufacturer's instructions. The protein concentration of each sample was measured using a Pierce BCA Protein Assay kit (Thermo Fisher), and 30 µg protein of each sample was separated via SDS-PAGE. The indicated protein was determined by immunoblotting analysis.

Chromosome spread assays

MEFs were irradiated with 1 Gy γ-ray or treated with 0,5 mM HU for 2 h. After 6 h of treatment with 100 ng/ml colcemid (Irvine Scientific, Santa Ana, CA, USA), cells were collected and hypotonically swollen in pre-warmed 75 mM KCl for 13 min at 37◦C. Cells were fixed in freshly made Carnoys fixative solution (methanol: acetic acid 3:1) for 2–3 times of the fixative. Cells were dropped onto warmed glass slides and dried overnight. Slides were stained with 5% Giemsa (Sigma, St. Louis, MO, USA) for 10 min at room temperature, gently rinsed with running water, air dried, and mounted. Slides were visualized using a microscope (Axio Imager M2, Carl Zeiss, Thornwood, NY, USA) and were recorded with AxioVision SE64 Rel.4.8 software (Carl Zeiss, Thornwood, NY, USA).

DNA fiber assay

The DNA replication progression was examined using the DNA fiber assay as described previously [26]. Cells were labeled sequentially with 100 µM iododeoxyuridine (IdU) for 10 min and chlorodeoxyuridine (CldU) for 20 min. DNA fibers were spread as described [55] and stained with primary antibodies (mouse anti-BrdU/IdU and rat anti-BrdU/CldU) and fluorescence conjugated secondary antibodies (Alexa Fluor 488 anti-rat and Texas Red anti-mouse). Fibers were imaged using the Zeiss AxioImager M2 and measured using the AxioVision software (x64 version 4.9.1).

Statistical analysis

Student’s t-test (two-sided) was performed to assess statistical significance. All statistical analyses were performed using the Prism GraphPad software. Statistical significance was defined as ^*P < 0.05, and values of ^**P < 0.01, ^***P < 0.001 were also shown in different experiments. The intensity of H3K14Ac, EdU, RPA2, and γH2AX, the aberrant chromosome number, the γH2AX foci number in mitosis cells and 53BP1 nuclear bodies number, and the length CldU tract were analyzed by the significance using Welch’s t-test.

Data availability

All data generated during the current study are available upon request.

Acknowledgements

We thank Dr. Jer-Tsong Hsieh of UT Southwestern Medical Center for kindly providing plasmids and cell lines; We thank Dr. Tom K. Hei of the Columbia University and Dr. Guangming Zhou of Soochow University for their valuable suggestions and for Dr. Huiming Lu for or critical revision of the manuscript.

Funding

National Institutes of Health [CA175879] to DS; National Institute of Health [CA162804 and GM04725] to AJD; National Institutes of Health [CA233594] to BPC; National Natural Science Foundation of China [#81872546]

Author information

These authors contributed equally: Zeng-Fu Shang, Lan Yu, Yue Lang, Ming Li.

These authors jointly supervised this work: Zeng-Fu Shang, Anthony J. Davis, and Debabrata Saha.

Contributions

Conception and design: ZFS, AJD, DS, BC and MS. Development of methodology: LY, YL, MMG and ML. Analysis and interpretation of data: LY, YL, ML, WMC, CN and ZFS. Writing, review, and/or revision of the manuscript: ZFS, BC, DS, MS, and AJD. Administrative, technical, or material support: LY and ZFS.

Ethics declarations

Competing interests

The authors declare no competing interests.

Consent for publication

We have obtained consents to publish this paper from all the participants.

Ethics approval

The mouse study was passed through the laboratory animal welfare ethics review of Soochow University (201807A303).

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DAB2IP maintains intestinal epithelial homeostasis by modulating the DNA replication stress response

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Abstract

Figures

INTRODUCTION

RESULTS

DAB2IP promotes intestinal homeostasis and regeneration after IR-induced intestinal injury

Loss of DAB2IP exacerbates DNA replication stress and genetic instability by prolonging DNA replication progression

DAB2IP interacts with HBO1 and promotes PLK1-mediated phosphorylation of HBO1

Phosphorylation of DAB2IP is essential for maintaining genomic stability

DISCUSSION

MATERIALS AND METHODS

Mouse irradiation protocol

Histological analysis

Immunohistochemistry (IHC)

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay

Cell culture, siRNA transfection, plasmids, and treatment

Antibodies, immunoprecipitation, and immunofluorescence

Clonogenic survival and cell proliferation assays

FLAG-tagged protein purification

Subcellular fractionation

Chromosome spread assays

DNA fiber assay

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

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