Dynamic role of CUL4B in radiation-induced intestinal injury-regeneration

CUL4B, a crucial scaffolding protein in the largest E3 ubiquitin ligase complex CRL4B, is involved in a broad range of physiological and pathological processes. While previous research has shown that CUL4B participates in maintaining intestinal homeostasis and function, its involvement in facilitating intestinal recovery following ionizing radiation (IR) damage has not been fully elucidated. Here, we utilized in vivo and in vitro models to decipher the role of CUL4B in intestinal repair after IR-injury. Our findings demonstrated that prior to radiation exposure, CUL4B inhibited the ubiquitination modification of PSME3, which led to the accumulation of PSME3 and subsequent negative regulation of p53-mediated apoptosis. In contrast, after radiation, CUL4B dissociated from PSME3 and translocated into the nucleus at phosphorylated histones H2A (γH2AX) foci, thereby impeding DNA damage repair and augmenting p53-mediated apoptosis through inhibition of BRCA1 phosphorylation and RAD51. Our study elucidated the dynamic role of CUL4B in the repair of radiation-induced intestinal damage and uncovered novel molecular mechanisms underlying the repair process, suggesting a potential therapeutic strategy of intestinal damage after radiation therapy for cancers.


CUL4B deficiency facilitates intestinal recovery from IR-induced damage via suppression of cell apoptosis
As mentioned above, the absence of CUL4B resulted in more severe intestinal damage after radiation exposure, but the intestine possessed greater resilience compared to Cul4b WT mice.To investigate the function of CUL4B in intestinal damage repair, we first examined the proliferative signals in regenerating intestines after IR injury.
We analyzed the number of Ki67 + proliferative cells and found that Ki67 expression was significantly elevated after intestinal injury induced repair in Cul4b WT mice, while there was no higher expression of Ki67 in Cul4b ΔIEC mice (Fig. 2A, B).Similarly, we did not detect more pronounced proliferation by EdU staining of shCul4b IEC-6 cells during regeneration (Fig. 2C, D).As shown in Fig. S2, the expression of inflammation factors such as Tnfα, Il6 and Il1β displayed unchanged or similar expression profiles in Cul4b deficiency mice before and after IR.Moreover, the expression of key proteins in cGAS/STING signaling, which bridges the IR-induced DNA damage with immune activation, was not changed.Subsequently, we further examined the alterations in apoptosis during intestinal recovery.Notably, CUL4B deletion led to an increase in Cleaved Caspase3/Caspase3 in intestinal cells in mice without radiation.Conversely, CUL4B deletion resulted in an attenuated apoptosis in mice during intestinal recovery, as evidenced by a decrease of Cleaved Caspase3/Caspase3 (Fig. 2E, F).The contrasting pattern of apoptosis under CUL4B deletion pre-and post-radiation was also verified in cell lines (Fig. 2E, F, Fig. S3).Above results suggest that CUL4B is involved in the intestinal recovery from radiation-induced damage by orchestrating cell apoptosis process.

CUL4B manipulates intestinal repair of injury through p53-mediated apoptosis
Given that the intestinal repair after IR injury proceeds via a p53-dependent pathway 20 , we evaluated the p53 expression in both CUL4B-deficient mice and cell lines to further investigate the involvement of CUL4B in the regulation of apoptosis through the p53 signaling pathway in intestinal injury repair.Surprisingly, CUL4B deficiency dramatically elevated the p53 protein expression in non-irradiated mice, but downregulated p53 protein levels during intestinal recovery process (Fig. 2E, G).Thus, we speculated that CUL4B could attenuate p53-mediated apoptosis in the absence of IR injury, whereas promote p53-mediated apoptosis during repair of intestinal injury.
To further verify this hypothesis, we detected the downstream target genes of p53, Puma (p53 upregulated modulator of apoptosis) and Bax (BCL2 associated X, apoptosis regulator), through RT-qPCR and Western blot.Accordingly, RT-qPCR results showed that, consistent with the pattern of p53 expression, Puma and Bax were significantly upregulated in CUL4B-deficient, non-IR induced mice and IEC-6 cells, but expressed less in CUL4B-deficient, IR induced injury models (Fig. 3A, B).Similar changes were observed at the protein level (Fig. 3C-F).In brief, the coordinated expression of CUL4B and p53 protein, together with the apparent variation in p53 downstream gene expression, demonstrate that CUL4B could orchestrate intestinal injury repair through p53-mediated apoptosis.

PSME3 synergizes with CUL4B before IR to modulate p53-mediated apoptosis
To identify the key factor that incorporate CUL4B dynamically regulating p53-mediated apoptosis, mass spectrometry was conducted to analyze the proteins that interacted with CUL4B in both unirradiated and 3-day post-irradiation small intestine tissues of mice.A total of 173 CUL4B-binding proteins were detected in the absence of radiation, while 81 CUL4B-binding proteins were detected in 3-day post-irradiation samples (full lists in Table S1-S3).Among these, 61 proteins were found to consistently bind to CUL4B after irradiation, while 112 proteins initially bound to CUL4B, but dissociated after irradiation (Fig. 4A).We also comprehensively analyzed differentiated expressional proteins and ubiquitylated proteins in intestines with CUL4B deletion (MS data in PRIDE: PXD-021528).Notably, the PSME3 protein was uniquely observed as the candidate key regulator among these proteins (Fig. 4A, B).PSME3 is a member of proteasome activator complex that mediates protein degradation and essential for protein homeostasis 21 .It also indicated a decrease in PSME3 protein levels (0.285-fold) and an increase in its ubiquitination form (5.617-fold) in Cul4b ΔIEC mice 15 (Fig. 4C, full lists in Table S4-S6).As PSME3 can enhance MDM2-mediated ubiquitination and degradation of p53 22 , the protein level of p53 was measured in IEC-6 cells with Psme3 knockdown and overexpression, respectively.Western blot results revealed an increase in p53 levels with Psme3 knockdown, and a decrease with Psme3 overexpression (Fig. 4D, E).The negative correlation between PSME3 and p53 protein levels, giving rise to the speculation that PSME3 may play a critical role in CUL4B-regulated cell apoptosis.To further investigate the role of PSME3 in CUL4B-coordinated repair of IR-damage intestine, we initially evaluated the binding of CUL4B to PSME3 pre-and post-irradiation.Immunoprecipitation experiments on intestinal tissues of both unirradiated and irradiated mice revealed a reduction in the amount of PSME3 combined with CUL4B after radiation (Fig. 5A, B).Coordinately, proximity ligation assay (PLA) was conducted on tissue slices of unirradiated and irradiated mice small intestine to visualize the interaction between CUL4B and PSME3.Consistently, the fluorescent signal that indicating the binding of CUL4B and PSME3 protein were significantly decreased after IR exposure (Fig. 5C, D).Moreover, PSME3 expression was downregulated in CUL4B deletion mice and IEC-6 cells, confirming the interaction between CUL4B and PSME3 (Fig. 5E, F).
Given the important role of CUL4B in ubiquitination modifications, the ubiquitination modification of PSME3 was further examined.It was found that the level of ubiquitination of PSME3 was increased in shCul4b IEC-6 cells, resulting in enhanced protein instability and shortened half-life (Fig. 5G-I), suggesting that CUL4B could inhibit PSME3 ubiquitination and thus maintain the long-term presence of PSME3.As mentioned above, overexpression of Psme3 lead to the decrease of p53 expression.Therefore, we assume that CUL4B potentially regulating downstream p53 pathway activation through ubiquitination of PSME3.To verify this hypothesis, we detected whether the presence of PSME3 interfered with the effect of CUL4B on p53 expression.Addition of PSME3 in CUL4B deficiency cells significantly blocked the accumulation of p53 induced by CUL4B deletion (Fig. 5J, K), verifying that the presence of PSME3 inhibited the facilitation of p53 expression induced by CUL4B.
Taken together, PSME3 was a pivotal factor in the CUL4B modulation of p53-mediated apoptosis before IR, with CUL4B maintaining the existence of PSME3 by inhibiting its ubiquitination, and the persistent presence of PSME3 inhibiting the expression of p53, thereby suppressing the occurrence of apoptosis.

CUL4B dissociated from PSME3 and bound to chromatin after IR damage discouraging the repair process
We have shown that CUL4B inhibits ubiquitination of PSME3 and thus impedes p53 protein expression in unirradiated mice.However, following radiation treatment, CUL4B no longer bound to PSME3 and deficiency of CUL4B not hinders but assists with intestinal recovery from radiation-induced damage, suggesting that CUL4B exerts a different mechanism during damage repair.It has been reported that nuclear localization of CUL4B is critical for its regulation of cell proliferation, so we speculate that CUL4B plays a different functional role after experiencing damage possibly due to its translocation in cells.Therefore, we detected the subcellular localization of CUL4B protein through immunofluorescence and nucleoplasm separation.At 3 days post-irradiation, a markedly increased percentage of nuclear importation of CUL4B was detected (Fig. 6A, B).More importantly, by measuring CUL4B in separated nucleoplasm, we found that CUL4B protein bound to chromatin was significantly elevated after irradiation (Fig. 6C, D), suggesting that CUL4B might enter the nucleus and positioned at the site of DNA damage after irradiation.
γH2AX, an indicator of DNA damage induced by radiation and the loss rate of which correlated with the rate of DBS repair 23 .To confirm whether CUL4B interacts with γH2AX and drives DNA damage, we carried out the immunoprecipitation and detected the clear combination between CUL4B and γH2AX (Fig. 6E, F).The colocalization of them in nucleus was evidenced in Fig. S4.Meanwhile, CUL4B deletion decreased γH2AX expression in intestine of mice and IEC-6 cells after irradiation treatment, confirming the correlation of CUL4B and γH2AX (Fig. 6G, H, Fig. S5A-S5B, Fig. S5G-S5H).Moreover, BRCA1 (p-BRCA1) and RAD51, DDR-related proteins recruited by γH2AX at damage lesions, were assayed by Western blot and immunofluorescence.We found that absence of CUL4B resulted in significantly elevated in p-BRCA1 expression and obviously increased RAD51 foci (Fig. 6I-L, Fig. S5C-S5H), further validating loss of CUL4B alleviates DNA damage.
Taken together, our findings suggest that while CUL4B can inhibit p53-mediated apoptosis by coordinating and regulating PSME3 before irradiation.After irradiation, CUL4B dissociated from PSME3 and located at γH2AX foci in nuclear.On one hand, CUL4B deficiency diminished PSME3 expression and upregulated p53-mediated apoptosis upon damage.On the other hand, loss of nuclear CUL4B ultimately accelerated the cellular repair of DNA damage (Fig. 7).

Discussion
The dependence of p53 signals characterizes IR-induced damage to the epithelium of small intestine 24,25 , while more regulators not fully understood.In this study, CUL4B was identified to play a promising role in the context of radiation-induced intestinal damage and recovery depending on p53 regulation, strengthening the comprehension of CUL4B in modulating impaired intestinal functions.Here we demonstrated the flexible and reversed function of CUL4B delimited by IR exposure.Prior to IR, CUL4B encouraged the expression of PSME3 through binding with PSME3 and inhibiting its degradation after ubiquitination.p53 was subsequently repressed by CUL4B-mediated PSME3.Post IR, CUL4B dissociated from PSME3, translocated into nucleus and colocalized at DNA damage sites, impeding DNA repair by down-regulating p-BRCA1 and RAD51.Our findings suggest a novel function of CUL4B in repair IR-induced damage in intestinal tissues.
It has been reported that CUL4B-DDB1 (DNA damage-binding protein 1) targeted to UV-damage chromatin and initiated efficient nucleotide excision repair (NER) 17 .Here we examined γH2AX as recognition substrate of CUL4B at the site of radiation lesions.The fact of high efficiency of monoubiquitinated histone H2A by CUL4B provides evidence for the above result 17,26 .The identification of subcellular localization of CUL4B is based on the its specific cytoplasm location in intestine cells 15 .CUL4B can be transported into the nucleus after radiation treatment to participate the DDR, due to its nuclear localization signal (NLS) 27 .This abrupt change indicates CUL4B will certainly play assignable role, and other related functions need to be further detected apart from DDR.
The occurrence of cell apoptosis is the consequence of redundancy DNA damage after radiation, in which p53 is a key factor and affected by multiple upstream signals.Nevertheless, it is worth noting the controversial role of p53 in cell specificity.p53 deficiency blocks apoptosis of most intestine epithelial cells and crypt cells rather than endothelial cells 24,[28][29][30] .Undeniably, all findings in this study based on the comparison with and without CUL4B deletion in the intestinal epithelium, revealing CUL4B as a novel regulator of p53 in IR-induced apoptosis.In addition, a contrasting perspective has emerged in the function of p53, which prevents genotoxicity caused by IR through inducing revival and decreasing apoptosis of stem cells to reinstate intestine integrity 31,32 .Whether the protective property of p53 is related to CUL4B and various effects of downstream molecules of p53 seem to be further detected.Therefore, the alternate exact mechanism by which CUL4B regulates p53 needs further verification.
PSME3 is a member of the 11S proteasome activator, which binds and activates the 20S proteasome in a ubiquitin and ATP-independent manner 21 .Not only that, PSME3 is required in DDR and maintenance of centrosome and chromosomal stability 33,34 .Its exhaustion leads to cell radiosensitivity and significant delay in DSB repair 34 .Moreover, PSME3 is implicated in the existence of p53 in multiple levels.PSME3 facilitates the physical interaction of MDM2-p53 in the form of polymers, promoting the polyubiquitination of p53, subsequent nuclear export and MDM2-dependent proteasomal degradation to make cells resistant to apoptosis 35,36 .In turn, p53 protein promoted PSME3 transcription, which can be inhibited by PSME3 itself thus forming a negative feedback loop 22 .We found that PSME3 acts as a dynamic binding substrate of CUL4B, providing strong support for the change in DNA damage and cell apoptosis with or without radiation.Before irradiation, the combination of CUL4B between PSME3 resulted in a decrease of its ubiquitination level and subsequent increased of its accumulation, reduced DNA damage and p53-induced cell apoptosis.After irradiation, CUL4B dissociated from PSME3 and entered nucleus.p53-induced cell apoptosis exhibited the opposite effect.However, in terms of DDR, sustained higher expression of γH2AX seems to play a dominant role instead of PSME3 controlled by CUL4B, suggesting that the impact on cellular and molecular effects still needs further investigation.
In conclusion, our findings demonstrate an inventive function of CUL4B during the intestine damage and recovery stage caused by radiation.CUL4B binds to PSME3 to inhibit the expression of p53 without any stimulation but abandons PSME3 to bound with γH2AX at the DNA damage sites on chromatin and down-regulates phosphorylation of the DNA repair protein BRCA1 and RAD51 after radiation.In addition, CUL4B has been reported to be overexpressed in a range of cancers and promotes carcinogenesis through regulating multiple cellular malignant behaviors [37][38][39][40][41][42] , suggesting that CUL4B can serve as a potential therapeutic target simultaneously for cancers and intestinal injury caused by radiotherapy of cancers.

Flow cytometry analysis
For analysis of apoptosis, IEC-6 cells were fixed and stained with PE Annexin V Apoptosis Detection kit with 7-AAD (Vazyme, A213-01) according to the manufacturer's instructions.The proportion of PI-positive and PE-positive cells were recorded.Data acquisition was performed on Accuri C6 Plus and analyzed via FlowJo (RRID:SCR_008520).

RNA isolation and RT-qPCR
For RNA isolation, intestine tissues were lysed in Trizol (Invitrogen, 15596018) and reversely transcribed with HiScript III RT SuperMix for qPCR (Vazyme, R323).RT-qPCR analysis was performed with SYBR Green Mixture (Roche, 4729692001) in qTOWER3G.The primers for RT-qPCR were designed using Primer3 (RRID:SCR_003139) and BLAST through NCBI and listed in Table 2. Gene expression was normalized to the referee Gapdh or Rps18 to control.

Immunoprecipitation
Protein supernatants were incubated with indicated antibodies and protein A/G PLUS-Agarose (Santa Cruz, sc-2003) for 2 h at 4 °C.Immunoprecipitates were boiled in sample loading dye at 95 °C for 5 min, followed by Western blotting.

EdU assay
EdU staining of IEC-6 was performed with Cell-LightTM EdU Apollo 567 In Vitro Kit (RiboBio, C10310-1) following the manufacturer's instructions.EdU medium (1:1000) was added when IEC-6 cells were passaged and incubated for 2 h at 37 °C in a humidified chamber with 5% CO 2 .Reaction mix was added to label EdU after cells were fixed and permeabilizated, and the images were captured with Olympus BX51.

Figure 1 .
Figure 1.CUL4B disrupted IR induced intestinal damage and recovery.(A) Schematic overview of the mice (Cul4b WT and Cul4b ΔIEC ) model and the IEC-6 cells (shCul4b and shNC).Mice were sacrificed at 0, 1, 3, 5 and 7 days after 12 Gy whole-body irradiation and cells were collected at 0, 12, 24, 36 and 48 h after 3 Gy irradiation.(B, C) Western blot analysis of CUL4B expression in intestine of Cul4b WT mice (n = 3) after radiation at 0, 1, 3, 5 and 7 days and in IEC-6 cells after radiation at 0, 12, 24, 36 and 48 h (B) and statistical analysis (C).Three independent experiments were performed.Error bars represent SD. (D) Overall survival analysis of the Cul4b WT and Cul4b ΔIEC mice post-irradiation.ns not significant, based on Log-rank test.(E) Statistical analysis of the body weight of the Cul4b WT and Cul4b ΔIEC mice post-irradiation relative to day 0. (F, G) Representative H&E images of Cul4b WT and Cul4b ΔIEC mice after radiation and the quantification of the length of crypt-villi (3 vs. 3) (F) and statistical analysis of villi length (G).Scale Bar = 50 μm.Error bars represent SD, *P < 0.05; **P < 0.01; ns not significant, based on Student's t test.The original western blots are presented in Fig. S6.

Figure 3 .
Figure 3. Apoptosis associated p53 downstream genes were regulated by CUL4B.(A) RT-qPCR analysis of Puma and Bax expression in Cul4b WT and Cul4b ΔIEC mice before and after radiation at 3 days.Error bars represent SD, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, based on Student's t test.(B) RT-qPCR analysis of Puma and Bax expression in shCul4b and shNC IEC-6 cells before and after radiation at 24 h.Error bars represent SD, *P < 0.05; **P < 0.01; ****P < 0.0001, based on Student's t test.(C, D) Western blot and statistical analysis of CUL4B and BAX expression in intestine from Cul4b WT and Cul4b ΔIEC mice before and after radiation at 3 days.Error bars represent SD, ***P < 0.001; ****P < 0.0001, based on Student's t test.(E, F) Western blot and statistical analysis of CUL4B and BAX expression in shCul4b and shNC IEC-6 cells before and after radiation at 24 h.Error bars represent SD, *P < 0.05; **P < 0.01, based on Student's t test.The original western blots are presented in Fig. S6.

Figure 4 .
Figure 4. PSME3 bridged CUL4B and its regulation of p53.(A) Venn diagram of CUL4B binding-protein using MS in intestine of Cul4b WT and Cul4b ΔIEC mice pre-IR and post-IR at 3 days.(B) Venn diagram of the overlap of up-regulated ubiquitylated proteins and down-regulated proteins identified in Cul4b ΔIEC mice compared with Cul4b WT mice.(C) Changing folds and detail modification site of PSME3.(D, E) Western blot and statistical analysis of the expression of p53 in Psme3-knockdown (siPsme3) and PSME3-overexpressed (OE PSME3) IEC-6 cells relative to control (siNC and OE NC).Error bars represent SD, **P < 0.01; ***P < 0.001; ****P < 0.0001, based on Student's t test.The original western blots are presented in Fig. S6.

Figure 6 .
Figure 6.CUL4B converted subcellular localization and repressed DDR.(A, B) Representative images of immunofluorescent staining of CUL4B + cells and statistical analysis of the rate of nuclear import of CUL4B pre-IR and post-IR at 3 days in intestine from Cul4b WT mice.Scale Bar = 50 μm.Error bars represent SD, **P < 0.01, based on Student's t test.(C, D) Western blot and statistical analysis of the CUL4B expression of cytoplasmic (HSP90 as control), nuclear (HDAC2 as control) and chromatin-bound (H3 as control) in intestine from Cul4b WT mice pre-IR and post-IR at 3 days.Error bars represent SD, **P < 0.01; ***P < 0.001; ns not significant, based on Student's t test.(E, F) Immunoprecipitation analysis of CUL4B and γH2AX in intestine from Cul4b WT mice pre-IR and post-IR at 3 days.Error bars represent SD, ***P < 0.001, based on Student's t test.(G, H) Western blot and statistical analysis of CUL4B and γH2AX expression in intestine after radiation at 3, 5 and 7 days.Error bars represent SD, *P < 0.05; **P < 0.01; ****P < 0.000, based on Student's t test.(I, J) Western blot and statistical analysis of CUL4B, BRCA1 and p-BRCA1 expression in intestine after radiation at 3, 5 and 7 days.Error bars represent SD, **P < 0.01; ****P < 0.0001, based on Student's t test.(K, L) Western blot and statistical analysis of CUL4B and RAD51 expression in intestine at 3 days post IR.Error bars represent SD, **P < 0.01; ****P < 0.0001, based on Student's t test.The original western blots are presented in Fig. S6.

Figure 7 .
Figure 7. CUL4B plays a dynamic role in p53-mediated cell apoptosis during IR-induced intestine damage repair.
forward AGG TCG GTG TGA ACG GAT TTG m-Gapdh reverse TGT AGA CCA TGT AGT TGA GGTCA m-Puma forward ATG AGC CAA ACC TGA CCA CT m-Puma reverse TGA GAT GGA TGG GGA TTG GG m-Bax forward AGA CAG GGG CCT TTT TGC TAC m-Bax reverse AAT TCG CCG GAG ACA CTC G m-Cul4b forward TGG AAG TTC ATT TAC CAC CAG AGA TG m-Cul4b reverse TTC TGC TTT TAA CAC ACA GTG TCC TA m-Il6 forward CCA AGA GGT GAG TGC TTC CC m-Il6 reverse CTG TTG TTC AGA CTC TCT CCCT m-Ilβ forward GCC CAT CCT CTG TGA CTC AT m-Ilβ reverse AGG CCA CAG GTA TTT TGT CG m-Tnfα forward GAC GTG GAA CTG GCA GAA GAG m-Tnfα reverse TTG GTG GTT TGT GAG TGT GAG m-Ifnβ forward CAG CTC CAA GAA AGG ACG AAC m-Ifnβ reverse GGC AGT GTA ACT CTT CTG CAT Rat-Rps18 forward ATC CCC GAG AAG TTT CAG CA Rat-Rps18 reverse ATT GTC GTG GGT TCT GCA TG Rat-Puma forward AAA CCT GAC CAC TAG CCT CC Rat-Puma reverse AAT GGG ATG GAT GGG GAC TG Rat-Bax forward GAG ACA CCT GAG CTG ACC TT Rat-Bax reverse CGT CTG CAA ACA TGT CAG CT Rat-Cul4b forward TTA AGG AAC GGG TGG CTG AT Rat-Cul4b reverse AAT CTC TGG GTT GTG GAG GG

Table 1 .
Primer sequence for genotyping.