CBP phosphorylation maintains intestinal homeostasis by supporting the stem cell niche through versican

Domain: biology
Published: 2026-03-28
Editorial outcome: major_revision

Abstract
CREB-binding protein (CBP) is a transcriptional coactivator, and we reported its phosphorylation regulates cell fate. Impaired CBP phosphorylation alters its binding preference for p53 in CBPAA mice, leading to distal colonic abnormalities. These changes are accompanied by reduced expression of versican, an extracellular matrix component that supports the intestinal stem cell niche, leading to impaired intestinal epithelial cell (IEC) proliferation and organoid formation. Deleting p53 in the IECs of CBPAA mice (CBPAAp53ΔIEC) restores versican expression and reverses the colonic phenotype. Versican replenishment and phosphomimetic CBP mutants rescue versican expression and IEC stemness in CBPAA organoids. Importantly, colonic tissues from ulcerative colitis patients exhibit impaired CBP phosphorylation, increased CBP–p53 binding, decreased versican expression and IEC proliferation, highlighting the clinical relevance of this phenomenon. In this work, we demonstrate CBP phosphorylation safeguards intestinal homeostasis by maintaining the stem cell niche through versican, highlighting versican as a potential therapeutic target for inflammatory bowel disease.

Introduction
Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract that includes ulcerative colitis (UC) and Crohn’s disease (CD). The quality of life of IBD patients is diminished, and the associated disability leads to substantial health and economic burdens worldwide1. Currently, IBD has no cure, which creates an unmet medical need. Therefore, elucidating the underlying mechanisms is essential for identifying potential therapeutic targets.

The pathogenesis of IBD remains incompletely understood. However, its etiology involves dynamic alterations in multiple cell types; including epithelial, stem, mesenchymal and immune cells2; driven by a complex interplay of genetic, environmental, immunologic and gut microbial triggers3. Colitis is induced by the dysregulation of the epithelial barrier, which consists of a single-layered epithelium3. The single-layer epithelium, comprising different specialized intestinal epithelial cells (IECs), is characterized by a high turnover rate that is sustained by intestinal stem cells (ISCs) at the crypt base to maintain intestinal homeostasis4, 5, 6. Moreover, ISC homeostasis is tightly orchestrated by the surrounding stem cell niche, a complex microenvironment in the lamina propria encompassing cellular and extracellular matrix (ECM) components to support intestinal stem cell maintenance, proliferation and differentiation7.

Recent studies have highlighted the role of posttranslational modifications (PTMs) such as protein neddylation and hydroxylation in IBD pathogenesis beyond direct transcriptional control and de novo protein synthesis8. CREB-binding protein (CBP) is a transcriptional coactivator that modulates DNA accessibility for transcription factors9. The competition between NF-κB and p53 for CBP has been reported to be a key determinant of whether a cell proliferates or undergoes apoptosis10, 11. We have previously shown that the phosphorylation of CBP at Ser1382/1386 by IKKα enhances NF-κB-mediated gene expression but suppresses p53-mediated gene expression, thereby promoting cell growth12. However, mutation of serines to alanines, which impairs CBP phosphorylation, increases its recruitment to p53-responsive promoters instead of NF-κB-responsive promoters12. These findings suggest that PTM of CBP may play an important role in regulating cell fate.

Building upon our molecular studies in vitro12, we generated CBP knock-in mice (CBPAA mice) by replacing Ser1383/1387 (corresponding to human CBP Ser1382/1386) with alanines (A). In contrast to the phosphorylation observed at these two serine residues in colonic crypt epithelial cells of CBPWT mice, CBPAA mice lacking CBP phosphorylation exhibit distal abnormalities despite comparable CBP expression. Analyses of colon tissues from UC patients, bone marrow chimera experiments, human cell-based sphere formation assays, colonic organoid models, and crypt RNA sequencing revealed that impaired CBP phosphorylation enhances CBP–p53 binding, leading to the downregulation of the expression of the ECM component versican (Vcan), which in turn reduces IEC stemness and causes colonic abnormalities in CBPAA mice. The specific deletion of p53 in IECs disrupts CBP–p53 binding in CBPAA mice and fully reverses these phenotypic changes. The replenishment of versican in the CBPAA organoids restores the proliferative capacity and organoid-forming efficiency. Furthermore, phosphomimetic CBP mutants (DD and EE) restore versican expression and stemness in CBPAA IECs. These results indicate that CBP phosphorylation plays a critical role in maintaining the stem cell niche and the proliferative potential of IECs by regulating versican expression and that its disruption impairs IEC self-renewal, contributing to colonic abnormalities. Importantly, the clinical relevance of these findings is supported by observations in colonic tissues from UC patients, which exhibit reduced IKKα expression and impaired CBP phosphorylation, increased CBP–p53 binding and decreased versican expression and IEC proliferation.

In the present study, we explore the role of the ECM component, versican, in maintaining IEC stemness. Our findings provide a basis for exploring potential therapeutic strategies for IBD and demonstrate how molecular-level insights may be translated into clinically relevant applications. Additionally, we characterize how defective PTMs of CBP contribute to the pathogenesis of IBD.

Results
Impaired CBP phosphorylation results in colonic abnormalities in CBPAA mice

The phosphorylation of CBP at Ser1383/1387 by IKKα is conserved across eukaryotic species (Supplementary Figure 1a). CBPAA mice were generated by replacing Ser1383/1387 with alanines (A) using a conventional gene targeting strategy in mouse embryonic stem cells, and the mutations were confirmed by PCR and DNA sequencing (Supplementary Figure 1b–d). The mice were born normally and fertile but did not exhibit the expected Mendelian ratio (Supplementary Table 1). The gross morphological and histopathological phenotypes of various organs were examined to determine whether impaired CBP phosphorylation is associated with pathology. CBP wild-type (CBPWT) and CBPAA mice displayed comparable body weights and overall appearance (Fig. 1a, b). However, CBPAA mice exhibited a reduction in colon length, along with increased occult blood in stool and stool consistency scores (Fig. 1c–e), as well as increased permeability of the colon (Fig. 1f, g). The histological evaluation of CBPAA mice revealed mild pathological changes in the distal colon, including localized low-grade immune cell infiltration in the mucosa and submucosa as well as mild crypt architectural irregularities (Fig. 1h, right panel, and Supplementary Figure 1e). Since these changes do not meet the criteria for colitis, we therefore described these observed lesions as colonic abnormalities. CBP phosphorylation was detected in the crypt epithelial cells of CBPWT mice, but this modification was absent in CBPAA mice although the levels of total CBP protein being comparable between the two genotypes (Fig. 1i). Importantly, CBP phosphorylation was detected in the colon tissue of healthy individuals but markedly reduced in that of UC patients, without changes in total CBP levels (Fig. 1j). These findings highlight the potential clinical relevance of impaired CBP phosphorylation in UC and underscore the role of CBP phosphorylation in maintaining intestinal homeostasis.

Nonhematopoietic cell-driven colonic abnormalities in CBPAA mice

The function of the intestinal barrier is maintained by nonhematopoietic cells, such as epithelial cells, and/or by hematopoietic cells, such as immune cells, in the lamina propria13. Bone marrow chimera experiments were performed to identify the cellular component responsible for the colonic abnormalities observed in CBPAA mice. CD45.1 and CD45.2 mouse strains are widely used to track immune cell development in vivo14. CBPAA mice were generated in the C57BL/6J (B6) strain and express a common CD45.2 allele, whereas B6.SJL-Ptprcα pepcβ/BoyJ mice expressing the CD45.1 allele were used as control (WT) mice (Fig. 2a). More than 80% of the donor bone marrow cells were reconstituted in chimeric recipient mice (Fig. 2b). Chimeric CBPAA mice (WT→AA) presented with elevated fecal occult blood, increased stool consistency scores, and reduced colon length (Fig. 2c–e). In addition, chimeric CBPAA mice exhibited substantial immune cell infiltration in all layers of the bowel wall, severe crypt distortion with the loss of entire crypts, and moderate mucosal hyperplasia, accompanied by increased histological scores and elevated mesenteric lymph node (mLN) cellularity (Fig. 2f, g, h). These mice also showed greater infiltration of macrophages (F4/80+) and neutrophils (Ly6G+) than chimeric WT mice did (AA→WT) (Fig. 2i, j), in which minimal levels of infiltration markers were observed, likely because of irradiation prior to the chimera experiments. These results indicate that chimeric CBPAA mice exhibit colonic inflammation driven by nonhematopoietic cells, such as epithelial cells. Since the intestinal barrier is composed of a single layer of IECs15, 16, 17, disrupted CBP phosphorylation within IECs may lead to impaired colonic barrier integrity.

The differentiation potential of naïve T cells and T subsets was evaluated to further confirm that colonic abnormalities in CBPAA mice are driven by nonhematopoietic cells. The total cell numbers in the spleen or peripheral lymph nodes (pLNs) were comparable between CBPWT and CBPAA mice (Supplementary Figure 2a, b). However, the numbers of CD4+ and CD8+ T cells in the mLNs, the key local sites of gut inflammation18, were increased in CBPAA mice (Supplementary Figure 2c–f). In the spleen, a decrease in the number of naïve CD4+ and CD8+ T cells but an increase in the number of effector or memory T cells were observed in CBPAA mice (Supplementary Figure 2g, h), suggesting alterations in T-cell properties despite unchanged total cell numbers. In addition, the production of cytokines and mRNA expression levels of T-specific transcription factors in naïve CD4+ T cells were comparable between CBPAA and CBPWT mice under both neutral and T-skewed conditions (Supplementary Figure 2i–n and 3). Although increased expression of the T2-associated transcription factors GATA-3 and c-Maf was observed in CBPAA mice, neither mRNA expression nor secretion of IL-4 differed (Supplementary Figure 3b). These findings further indicate that the colonic abnormalities in CBPAA mice are attributed to impaired CBP phosphorylation in IECs per se, rather than to the regulation of baseline immune homeostasis.

Impaired CBP phosphorylation hinders the renewal of colonic epithelial cells and the mutant protein exhibits a binding preference for p53 in CBPAA mice

Because impaired CBP phosphorylation in IECs contributes to colonic abnormalities in CBPAA mice, the status of epithelial cells was examined by staining for Ki-67, a widely used marker of cell proliferation. A reduction in the number of proliferating IECs was observed in CBPAA mice compared with control mice (Fig. 3a).

The integrity of the colonic epithelial barrier is challenged by the high turnover rate of IECs16. BrdU pulse-chase experiments were performed to assess IEC turnover in vivo. Two hours after the BrdU injection, BrdU-labeled cells were restricted to the crypt base, reflecting the baseline proliferation of IECs19. The subsequent migration of BrdU+ cells from the crypt base toward the lumen was examined by performing immunohistochemical (IHC) staining at 24 h, 48 h and 96 h after the BrdU injection. The BrdU+ cells in CBPWT mice had already reached the luminal surface by 96 hours, whereas those in CBPAA mice were still located within the crypt region and had not yet migrated to the lumen (Fig. 3b), suggesting a potential delay in IEC turnover.

Since the impairment in CBP phosphorylation induced by substituting serines with alanines increased its recruitment to p53-responsive promoters instead of to NF-κB-responsive promoters at the molecular level12, we next sought to determine whether this event occurred in CBPAA mice. A proximity ligation assay (PLA, Duolink®) evaluating protein–protein interactions in colonic IECs based on antibody detection was employed to examine CBP binding to p53 in CBPAA and CBPWT mice. CBP in the IECs of CBPAA mice preferentially bound to p53 and the expression of the p53-mediated gene p21 increased (Fig. 3c, d). The expression levels of p53 (Fig. 3e) and CBP (Fig. 1i, lower panel) were comparable between CBPWT and CBPAA mice, indicating that the binding preference of CBP for p53 in CBPAA mice is not due to alterations in p53 and CBP levels. CUT&RUN assays revealed that both CBP- and p53-precipitated chromatin were enriched at the p21 promoter (Fig. 3f, g), supporting that enhanced recruitment of CBP by p53 regulates p21 expression in CBPAA mice.

Impaired CBP phosphorylation reduces spheroid formation by HCT116 cells

The constantly self-renewing IECs are replenished by ISCs at the crypt base to maintain intestinal barrier integrity4. Dysregulation of ISCs results in delayed IEC turnover20, leading to epithelial barrier dysfunction and colitis21. Since impaired CBP phosphorylation in the colonic IECs of CBPAA mice resulted in a binding preference of mutant CBP for p53 that paralleled the reduced IEC proliferation and delayed epithelial turnover (Fig. 3), WT and p53-/- human HCT116 cells22, 23, 24 were first utilized to examine whether and how CBP–p53 binding dysregulates stemness. Spheroids are highly enriched in stem cell populations25, and the functions of normal stem cells and cancer stem cells are conceptually similar26.

An IKK inhibitor, CYL-19s, has been shown to abolish TNF-α-induced CBP phosphorylation in HeLa cells and increase CBP binding to p53-responsive promoters instead of NF-κB-responsive promoters12, 27. Similar results were also observed in WT HCT116 cells in the present study (Supplementary Figure 4a–c). The inhibition of basal CBP phosphorylation by CYL-19s led to increased CBP–p53 binding without affecting p53 levels, along with increased expression of the p53-responsive gene p21 (Supplementary Figure 4a–e). In addition, the viability of WT HCT116 cells decreased in response to CYL-19s treatment (Supplementary Figure 4f). None of these changes were observed in p53-/- HCT116 cells (Supplementary Figure 4b, c, d, f).

We determined whether and how increased CBP–p53 binding weakens HCT116 stemness by examining the effects of spheroid formation by WT and p53-/- HCT116 cells in the presence of CYL-19s. CYL-19s treatment significantly inhibited sphere formation by WT HCT116 cells (Supplementary Figure 4g) and reduced the expression of the stemness marker ALDH1A1 (Supplementary Figure 4d), suggesting that increased CBP–p53 binding suppresses spheroid formation (Supplementary Figure 4b, c, g).

The spheres formed by p53-/- HCT116 cells were larger than those formed by WT HCT116 cells (Supplementary Figure 4g), and ALDH1A1 expression was increased (Supplementary Figure 4d), indicating that increased CBP–p53 binding played a negative role in regulating HCT116 cell stemness since this event was relieved in p53-/- HCT116 cells (Supplementary Figure 4b). Increased ALDH1A1 expression and larger spheres were sustained in p53-/- HCT116 cells in the presence of CYL-19s (Supplementary Figure 4d, g), further indicating that increased CBP–p53 binding decreases HCT116 cell stemness.

Impaired CBP phosphorylation hinders the formation of organoids from CBPAA mice

Since increased CBP–p53 binding suppressed sphere formation and reduced the expression of stemness marker in HCT116 cells, confirming this event in colonic IECs by using ex vivo primary 3D colonic organoids is essential. We established a near-physiological primary epithelial 3D organoid culture system from colonic crypts. Crypt-derived organoids consisting of various differentiated epithelial lineages were generated from single Lgr5+ (Leu-rich repeat-containing G protein-coupled receptor 5-expressing) stem cells28. CBP phosphorylation was detected in CBPWT organoids using immunofluorescence staining, but this PTM was absent in CBPAA organoids, despite the comparable total CBP levels between the two groups (Fig. 4a). Similar to colon tissues, CBPAA organoids exhibited a reduced number of Ki-67+ cells and decreased cell viability (Fig. 4b, c). Additionally, both the formation efficiency and the diameter of CBPAA organoids were reduced (Fig. 4d). These findings indicate that the loss of CBP phosphorylation in CBPAA mice is associated with impaired IEC proliferation and reduced stemness, likely through its preferential binding to p53, and contributes to colonic abnormalities in CBPAA mice.

Targeted deletion of p53 in IECs diminishes colonic abnormalities and restores epithelial stemness in CBPAA mice

Because increased CBP–p53 binding, which decreases IEC stemness, was observed in vitro and ex vivo, CBPAA mice with an IEC-specific p53 deletion (CBPAAp53ΔIEC mice) were generated to further examine whether the abnormal colonic phenotypes would be alleviated and colonic IEC stemness would be restored in the absence of CBP–p53 binding.

p53 was specifically knocked out in the IECs of CBPAA mice by crossbreeding CBPAA mice with Villin-Cre; p53f/f mice. qPCR and Western blotting confirmed the effective deletion of p53 in the colonic epithelial cells of CBPAAp53ΔIEC mice (Fig. 5a, b). CBPAAp53ΔIEC mice exhibited significant improvements in the abnormal colonic phenotypes caused by the CBPAA mutation, as evidenced by the restored colon length, reduced occult blood in stool and stool consistency scores, and decreased intestinal permeability (Fig. 5c–f), along with improvements in the histological abnormalities (Fig. 5g).

The preferential CBP–p53 binding observed in CBPAAp53-floxed mice was comparable to that in CBPAA mice (compare Fig. 5h, left two panels, and Fig. 3c). In contrast, CBP–p53 binding was lost in CBPAAp53ΔIEC mice (Fig. 5h, right two panels), which paralleled the restoration of the reduced numbers of Ki-67+ cells, organoid diameter and organoid-forming efficiency (Fig. 5i, j). These findings support that increased CBP–p53 binding impairs colonic IEC stemness and drives colonic abnormalities in CBPAA mice, whereas the loss of this interaction restores stemness and prevents these abnormalities in CBPAAp53ΔIEC mice.

Enrichments of both CBP- and p53-precipitated chromatin at the p21 gene promoter were observed in CBPAA mice (Fig. 3f, g). However, CBP enrichment at the p21 promoter was absent in CBPAAp53ΔIEC mice, accompanying a reduction in p21 expression (Supplementary Figure 5), in which CBP–p53 binding was lost (Fig. 5h, right two panels). These results confirm that direct regulation of CBP on p53 is enhanced at the chromatin level by impaired CBP phosphorylation in CBPAA mice, providing further evidence that differential phosphorylation of CBP regulates binding to the p53 locus and subsequent p21 expression.

Versican expression is downregulated in CBPAAp53-floxed mice and restored in CBPAAp53ΔIEC mice

RNA-seq was performed to profile the transcriptome of colonic crypt-enriched epithelial cells to investigate how impaired CBP phosphorylation results in decreased IEC stemness. The differentially expressed genes (DEGs) are depicted in heatmaps that show a distinct expression pattern in crypts between CBPWTp53-floxed and CBPAAp53-floxed mice (Fig. 6a), and a volcano plot shows that versican (Vcan), a proteoglycan in the ECM, was significantly downregulated in CBPAAp53-floxed mice (Fig. 6b). A Venn diagram depicts the overlapping DEGs between the two experimental groups. Versican was also the only gene overlapping between the comparisons of the CBPAAp53-floxed vs. CBPWTp53-floxed and CBPAAp53ΔIEC vs. CBPAAp53-floxed groups but was excluded from the comparisons of the CBPAAp53ΔIEC vs. CBPWTp53ΔIEC and CBPWTp53ΔIEC vs. CBPWTp53-floxed groups (Fig. 6c). RNA-seq revealed that versican expression was downregulated in CBPAAp53-floxed mice but restored in CBPAAp53ΔIEC mice to levels comparable to those in CBPWTp53ΔIEC and CBPWTp53-floxed mice (Fig. 6d), which was further verified by qPCR (Fig. 6e). Immunofluorescence staining showed reduced versican expression in the mucosal stroma of CBPAAp53-floxed mice, which was restored in CBPAAp53ΔIEC mice to levels comparable to those in CBPWTp53ΔIEC mice (Fig. 6f, g).

Versican mediates the mesenchymal–epithelial transition by promoting the switch from N-cadherin expression to E-cadherin expression, thereby facilitating the formation of epithelial-specific adhesion junctions29. E-cadherin is required for maintaining epithelial integrity, but defects in E-cadherin expression are associated with IBD30, 31. Notably, the expression levels of E-cadherin paralleled those of versican (Fig. 6h, i vs. 6f, g), suggesting that versican expression may influence cell–cell adhesion and epithelial barrier integrity.

The recombinant versican protein was added to the culture medium of CBPAAp53-floxed organoids (AA + Vcan) to examine the role of versican in IEC stemness. The organoid diameter, organoid-forming efficiency, number of Ki-67+ cells and E-cadherin expression were restored to levels comparable to those in CBPWTp53-floxed organoids (Fig. 6j–p), suggesting that versican contributes to the maintenance of IEC stemness and the regulation of intestinal epithelial cell behavior. Versican contains two EGF-like repeats in its G3 domain, and upon cleavage, EGF may be released to promote proliferation32, 33. The restorative effect of recombinant versican was abolished by gefitinib (Fig. 6j–p), suggesting the involvement of the activated EGFR pathway in its function.

Versican is produced by various cell types, including epithelial, endothelial, and stromal cells, as well as leukocytes34. In addition to the RNA-seq and qPCR results (Fig. 6a, d, e), the epithelial-intrinsic defect and reduced versican expression observed in CBPAA mice were reversed by epithelial-specific p53 deletion (Fig. 6f), suggesting that versican is derived primarily from epithelial cells. Consistent with these tissue-level changes (Fig. 6f), versican expression was also reduced in organoids derived from the crypts of CBPAAp53-floxed mice and restored in those derived from CBPAAp53ΔIEC mice (Fig. 6q, r), further confirming its epithelial origin.

Overexpression of phosphomimetic CBP mutants restore versican expression and rescue the phenotype of CBPAAp53f/f organoids

As mentioned, CBP phosphorylation is important for maintaining intestinal homeostasis, and versican contributes to the maintenance of IEC stemness and epithelial cell behavior. We performed gain-of-function experiments using phosphomimetic CBP mutants to further determine whether CBP phosphorylation affects versican levels. Single cells dissociated from crypt-derived CBPAAp53f/f (AA) organoids were transfected with either a control vector expressing EGFP alone (pCMV-puro-IRES2-EGFP) or a construct encoding HA-tagged CBP phosphomimetic mutant coexpressing EGFP (DD: pCMV-puro-IRES2-EGFP mCBPS1383D/S1387D-HA; EE: pCMV-puro-IRES2-EGFP mCBPS1383E/S1387E-HA) using Lipofectamine™ Stem Transfection Reagent. Transfected cells were identified by EGFP fluorescence. Successful delivery of HA-tagged CBP mutants into AA organoids was confirmed by HA-CBP expression detected by Western blotting (Supplementary Figure 6). Compared with the vector control, AA organoids overexpressing DD or EE showed restored versican expression and a reversal of AA phenotype, as evidenced by the rescued organoid-forming efficiency and organoid diameter (Fig. 7a–b). Additionally, these organoids exhibited increased IEC proliferation and E-cadherin expression (Fig. 7c–d). These findings indicate that CBP phosphorylation plays important roles in maintaining versican expression and supporting the intestinal stem cell niche, thereby contributing to the preservation of epithelial homeostasis in the colon. Despite the low number of EGFP-positive cells, overexpression of DD or EE in AA organoids upregulated versican expression (Fig. 7a, e). This finding parallels the concept that localized alterations within the stem cell niche can have broad effects on epithelial structure and function35.

Binding preference of CBP for p53 and reduced versican expression in colon tissues from UC patients

The observation of impaired CBP phosphorylation without changes in total CBP levels in UC patients underscores its clinical relevance (Fig. 1j). Consistently, increased CBP–p53 binding, reduced versican levels, and fewer Ki-67+ proliferative IECs were observed in UC patients (Fig. 8a–c), along with decreased IKKα expression (Fig. 8d). These findings strengthen the hypothesis that impaired CBP phosphorylation enhances CBP–p53 binding, which in turn suppresses versican expression and may ultimately contribute to IBD pathogenesis in humans.

Moreover, healthy individuals presented preserved IKKα expression, intact CBP phosphorylation, maintained versican levels, and abundant Ki-67+ IECs (Fig. 8b–d and Fig. 1j). Together, these results underscore the essential role of CBP phosphorylation in maintaining versican expression and the epithelial stem cell niche, thereby sustaining intestinal homeostasis and preventing IBD.

Methods
Mice
CBP mutation “knock-in” mice (CBPAA mice) were generated using traditional embryonic stem cell technology. These mice were generated via the targeted insertion of the mutated allele (S1383/1387A) at exon 25 with a neomycin selection cassette into mouse embryonic stem cells on a 129 background, followed by subsequent backcrossing to C57BL/6 mice over 10 generations. CBPWT mice were used as littermate controls. The mice were designed and generated by the Transgenic Mouse Models Core of the National Taiwan University Center of Genomic and Precision Medicine. Lgr5-eGFP-creERT2+/Tg; Villin-Cre mice and p53f/+ mice were gifts from Dr. Jr-Wen Shui and Dr. Kai-Chien Yang, respectively. p53f/+ mice were intercrossed to generate p53f/f mice. These mice were then bred to CBPAA mice to generate heterozygous CBP–p53f/+ mice. Heterozygous CBP–p53f/+ mice were intercrossed to generate CBPWTp53f/f and CBPAAp53f/f mice. CBPWTp53f/f mice were used as littermate controls. These mice were then bred to Villin-Cre mice to generate IEC-specific p53-knockout mice (CBPAAp53ΔIEC and CBPWTp53ΔIEC) for our experiments. CBPWTp53ΔIEC mice were used as littermate controls. The p53ΔIEC mice were used to verify whether the colitis phenotype was diminished and whether the decreased stemness of colonic IECs was recovered. The mouse genotypes were determined by the extraction and amplification of DNA from mouse toe tissues (KAPA Mouse Genotyping Kit; KAPA Biosystems, Cape Town, South Africa; KK7352). All three mouse strains were on a C57BL/6 background and were raised and bred under specific pathogen-free conditions at the Laboratory Animal Center, National Taiwan University College of Medicine. Female mice at 8 weeks of age were used in all experiments, unless indicated otherwise. All the mice were housed in isolated ventilated cages (maximum of five mice per cage) in a barrier facility at the Laboratory Animal Center, National Taiwan University College of Medicine. The mice were maintained on a 12-h light–dark cycle at 22–26 degrees Celsius, with sterile pellet food and water available ad libitum. All the mice were sacrificed between 10 am and 3 pm. All animal experiments were approved by the National Taiwan University College of Medicine, Institutional Animal Care and Use Committee.

Cell lines and reagents
The cell lines were cultured at 37 °C with 5% CO2. WT and p53-/- HCT116 cells were obtained from Prof. Chang ZF and grown in DMEM supplemented with 10% FBS with pen/strep. CYL-19s was synthesized by our collaborator, Prof. Cherng-Chyi Tzeng, a medicinal chemist27, and dissolved in DMSO to prepare a stock solution (50 mM). The recombinant human TNF-α protein was purchased from Peprotech (#300-01A) and dissolved in ddH2O. The recombinant mouse TNF-α protein was purchased from Peprotech (#315-01A) and dissolved in 3% BSA/PBS. The recombinant mouse versican protein (Abbexa, abx069663), produced in a bacterial (E. coli) expression system, was dissolved in ddH2O. The final working concentration of 100 ng/mL was selected based on a recent study49 and further supported by our preliminary dose–response tests (50, 100, and 200 ng/mL), in which 100 ng/mL elicited a robust biological effect with no additional increase observed at 200 ng/mL. The recombinant mouse epidermal growth factor (mEGF) protein was purchased from Peprotech (#315-09) and dissolved in 0.1% BSA/PBS. Gefitinib was purchased from LC Laboratories (G-4408) and dissolved in DMSO.

Construction of the targeting vector and genotyping of mice expressing the mutant CBP allele
A construct containing the mutated exon 25 was used to target the mouse CBP gene. A targeting vector was generated by the recombineering method to achieve the knock-in of this allele50, 51. Briefly, a 9.6 kb fragment of mouse genomic DNA containing the region from exon 25 of the mouse CBP gene was retrieved from a B6 strain-derived bacterial artificial chromosome clone (bMQ451N12, Children’s Hospital Oakland Research Institute). The fragment was inserted at the NotI–SpeI sites of pL253, in which the MC1-TK (thymidine kinase) gene served as a negative selection marker. The resulting construct was used as a backbone for CBP and a neo cassette (5′–loxP–PGK–NeobpA–loxP-3′) from pL451 downstream of exon 25. The final targeting construct contained a homologous 5′ arm of 4.7 kb and a 3′ arm of 4.9 kb. The primer sequences used for PCR to generate the 300-bp homologous arms for the retrieval of exon 25 of mouse CBP genomic DNA (AU, BD, YU and ZD) with the insertion of loxP and FRT sites (CU, DD, EU and FD) and the neo cassette were as follows: CBP-KI AU 114200, 5′-ATAGCGGCCGCAGTAACTTGAAGTGACTAATATC-3′; CBP-KI BD 114500, 5′-ATAAAGCTTATACTTAAGAGAACAAAGTAATTACTGCTAG-3′; CBP-KI CU 118130, 5′-ATAGTCGACTCCATTTTAAGTGAGCCTAGG-3′; CBP-KI DD 118890, 5′-ATATCTAGAAAAGAAAGTCACATGCTAGC-3′; CBP-KI EU 119300, 5′-ATAGGGCCCTAAGCTGTATATTTTGGTGCTAG-3′; CBP-KI FD 120000, 5′-ATAGCGGCCGCAAAAGCAGAGTAGACACTGTG-3′; CBP-KI YU 123900, 5′-ATAAAGCTTTAGAACATTGATGGTGAAG-3′; and CBP-KI ZD 124200, 5′-ATAACTAGTGTACTACAGCGCCCATACAG-3′. The knock-in targeting vector was confirmed by sequencing, linearized by DNA digestion at the unique NotI site and electroporated into C57BL/6N ES cells. Correctly targeted ES cell clones were selected by culture with G418 (240 mg/mL) and ganciclovir (2 mM). Correctly targeted clones were identified by Southern blotting to confirm homologous recombination at both 5' and 3' arms. Validated ES cells were subsequently injected into blastocytes of C57BL/6J origin (National Laboratory Animal Breeding and Research Center, Taiwan) to establish knock-in mice.

The mouse genotypes were determined by the extraction and amplification of DNA from mouse toe tissues (KAPA Mouse Genotyping Kit; KAPA Biosystems). Routine genotyping was performed by PCR with the primer 5′-CCCTTCCCTAAGAAACAGGACT-3′ (FokI-F) paired with 5′-CCCTTCCTCCCCTAAGAGTTTA-3′ (FokI-R) for the wild-type (WT) CBP allele or paired with 5′-CCAAAATATACAGCTTAGGGCC-3′ (AA-R) for the mutant CBP allele. The PCR conditions were 95 °C for 3 min; 35 cycles of 95 °C for 15 sec, 60 °C for 15 sec and 72 °C for 15 sec; and 72 °C for 1 min.

Assessment of colon length, stool consistency and occult blood in stool under baseline conditions
Colon length was measured from the ileocecal junction to the anus using a plastic ruler. Disease severity was evaluated by an observer blinded to the experimental groups based on stool consistency and fecal occult blood scores. Stool consistency was assessed by pressing a stick onto the feces and scored as follows: 0, well-formed pellets; 1, semi-formed stools; 2, pasty stools; and 3, liquid stools adhering to the anus. Fecal occult blood was determined using a Hemoccult Sensa test (Beckman Coulter, #64151) and scored as follows: 0, negative hemoccult; 1, positive hemoccult; 2, positive hemoccult with visible traces of blood; and 3, gross rectal bleeding. For intermediate severity, half-scores (e.g., 0.5, 1.5) were assigned.

Histological evaluation and scoring
Histological scoring was performed in a blinded manner by a trained veterinary pathologist, Yi-Ting Tsai (Laboratory Animal Center, National Taiwan University College of Medicine, Taipei, Taiwan). For tissue preparation, a 2-cm colon fragment was taken from the distal colon above the anus and submerged in a buffered 10% formalin solution. Hematoxylin and eosin (H&E) staining was performed on 5-μm sections from 10% formalin-fixed, paraffin-embedded samples using the appropriate procedures. Scoring was based on seven parameters: the severity of inflammation, location of the inflammatory lesion, crypt architecture, lesion area in the crypts, mucosal hyperplasia, goblet cell depletion, and crypt dilation and abscesses. Each parameter was scored on a scale from 0 to 3, where 0 indicated normal tissue and 3 indicated severe pathology. For example, the severity of inflammation was scored as 0 (normal), 1 (slight infiltration of mononuclear cells and neutrophils, <30%), 2 (moderate infiltration, 30–60%), or 3 (severe infiltration, >60%). The lesion location was scored as 0 (normal), 1 (mucosa), 2 (mucosa and submucosa), or 3 (all layers of the bowel wall). The remaining parameters were similarly graded based on the severity and extent of tissue involvement. Representative images were captured using a Leica DM2500 upright microscope equipped with MicroVisioneer Manual Whole-Slide Imaging Software.

Assessment of ex vivo intestinal permeability
A 2-cm segment of colon tissue was opened longitudinally along the mesenteric border and mounted in an Ussing chamber (World Precision Instruments, Sarasota, FL). The tissue in each chamber was exposed to 5 mL of circulating oxygenated Krebs buffer (37 °C) containing either glucose (10 mM, serosal buffer) or mannitol (10 mM, mucosal buffer). Fluorescein isothiocyanate (FITC)-dextran (12.5 M; molecular weight, 3,000–5,000 Da; Sigma–Aldrich, FD4) was added to the mucosal buffer at a final concentration of 500 μM. Samples were collected from the serosal buffer at different time points (0, 30, 60, 90 and 120 min) and measured using a multimode plate reader (Paradigm Detection Platform; Beckman Coulter, Indianapolis IN) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

Assessment of intestinal permeability in vivo
The mice were starved for 4–6 h and then orally gavaged with 0.5 mg/g body weight FITC-dextran (molecular weight, 3,000–5,000 Da; Sigma–Aldrich, FD4). After 4 h, 300–400 μL of blood was collected by cardiac puncture and centrifuged at 900 × g for 20 min at room temperature. Serum was diluted with an equal volume of PBS, and the serum FITC-dextran concentrations were measured with a multimode plate reader (Paradigm Detection Platform; Beckman Coulter) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a FITC-dextran standard curve (0, 0.125, 0.25, 0.5, 1, 2, 4, 6, 8, and 12.5 μg/mL). Serum from mice not administered FITC-dextran was used as the background sample.

Immunohistochemical (IHC) assay
Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 4 μm thickness. Immunohistochemistry (IHC) staining was performed using the BioTnA Polymer Detection System (BioTnA, #TAHC04D) according to the manufacturer’s instructions. Heat-induced epitope retrieval (HIER) was performed using Tris-EDTA HIER Solution (pH 9.0; ScyTek, #TES500). The following primary antibodies were used: anti-pCBP (Ser1383/1387 for mouse samples; Ser1382/1386 for human samples; 1:100; GeneTex, customized, GTX90661), anti-CBP (1:50; Cell Signaling Technology, #7389), anti-F4/80 (1:250; Cell Signaling Technology, #70076), anti-Ly6G (1:50; R&D, MAB1037), anti-p53 (1:100; Santa Cruz Biotechnology, sc-126), anti-p21 (1:100; Cell Signaling Technology, #2947), anti-versican (1:100; Abcam, ab19345) and anti-IKKα (1:100; Santa Cruz Biotechnology, sc-7606). Whole-slide images were acquired with a TissueFAXS microscopy system (TissueGnostics GmbH, Vienna, Austria). Quantification of positive staining was performed using ImageJ as described in the corresponding figure legends.

Generation of bone marrow chimeras
Bone marrow transplantation was performed using bone marrow cells isolated from donor mice, which were subsequently transplanted into γ-irradiated recipient mice. B6.SJL-Ptprcα pepcβ/BoyJ mice expressing the CD45.1 alleles were used as WT mice; CBPAA mice (C57BL/6J strain) expressed the CD45.2 allele. Recipient mice were sublethally irradiated with 6 Gy from a cesium source two times at 4 h intervals on the day before transplantation. Bone marrow cells were isolated from the femurs and tibias of the donor mice, and a total of 5 × 106 bone marrow cells were injected into the tail veins of the irradiated recipient mice. The mice were evaluated and sacrificed 6 weeks after reconstitution. Bone marrow reconstitution was evaluated by staining mLNs with anti-CD45.1 (1:200; BUV395; BD Biosciences, #565212) and anti-CD45.2 (1:200; BUV737; BD Biosciences, #612778) antibodies and performing flow cytometry (LSRFortessa, BD).

Frozen sections for immunostaining
The distal colon (approximately 2 cm from the anus) was collected, flushed with cold PBS to remove fecal content, and fixed in 4% paraformaldehyde (PFA) for 30 min at room temperature. The tissues were then dehydrated in 15% (w/v) sucrose in PBS until they sank (6–12 h), followed by 30% (w/v) sucrose in PBS overnight at 4 °C. Subsequently, the colon segments were embedded in FSC 22 Frozen Section Media (Leica Biosystems, #3801480) and frozen at -80 °C.

Frozen sections were cut at 5 μm thickness. Sections were permeabilized with 0.25% Triton X-100 for 20 min and blocked with 5% bovine serum albumin (BSA) in PBS for 1 h at room temperature. After incubation with primary antibodies overnight at 4 °C and secondary antibodies for 1 h at room temperature, slides were mounted using ProLong Gold Antifade Mountant with DAPI (Invitrogen, P36935). Confocal laser scanning microscopy was performed on a Zeiss LSM 780 confocal microscope (Carl Zeiss, Jena, Germany). Quantification of positive staining was performed as described in the corresponding figure legends.

The following primary antibodies were used: anti-pCBP (Ser1383/1387 for mouse, Ser1382/1386 for human; 1:150; GeneTex, customized, GTX90661), anti-CBP (1:200; Cell Signaling Technology, #7389), anti-versican (1:200; Abcam, ab19345), anti-Ki67 (1:200; Cell Signaling Technology, #9129S), anti-E-cadherin (1:200; Invitrogen, #33-4000), and anti-p21 (1:150; Cell Signaling Technology, #2947).

The following secondary antibodies were purchased from Invitrogen and used at a 1:500 dilution: Alexa Fluor® 488-conjugated goat anti-rabbit IgG (A-11034), Alexa Fluor® 488-conjugated goat anti-mouse IgG (A-11029), Alexa Fluor® 594-conjugated goat anti-rabbit IgG (A-11037), and Alexa Fluor 594-conjugated goat anti-mouse IgG (A-11005).

Confocal microscopy and image analysis
Confocal imaging was performed using a Zeiss LSM780 or LSM880 confocal laser scanning microscope (Carl Zeiss, Jena, Germany) equipped with 405 nm, 488 nm, and 561 nm laser lines and detectors, operated with ZEN software. All images were acquired using a 20×/0.8 NA Plan-Apochromat objective in sequential scanning mode to eliminate spectral crosstalk. Within each comparative experimental set, core acquisition parameters were maintained consistently, including pixel resolution (1024 × 1024), pixel dwell time, and pinhole size. To ensure quantitative accuracy, acquisition parameters (e.g., laser power and detector gain) were optimized per staining batch to maintain the fluorescence signal within the linear dynamic range without pixel saturation, and were carefully controlled and kept within a comparable range across all samples within the same comparative experiment. For three-dimensional structures such as organoids, Z-stack images were acquired with optimal step sizes automatically calculated by the ZEN software based on the objective numerical aperture. Image processing and quantification were performed using ImageJ/Fiji. Any adjustments to brightness or contrast were applied uniformly across images within the same experiment and were performed solely for visualization purposes. No nonlinear image manipulation was applied. All original raw image files (.czi), including complete acquisition metadata such as laser power, detector gain, and Z-stack intervals, have been deposited in the EBI BioImage Archive under accession number S-BIAD2810.

Pulse-chase BrdU labeling and IHC detection of BrdU staining
The mice were injected intraperitoneally with 100 mg/kg body weight 5-bromo-2’-deoxyuridine (BrdU; Sigma–Aldrich, St. Louis, MO; B5002) at 2 h, 24 h, 48 h or 96 h before sacrifice. The colon was excised from each mouse, fixed overnight with Formald-Fresh solution (Fisher Scientific, SF-94), and embedded in paraffin. Colon sections were cut at a thickness of 5 μm for IHC staining using a BioTnA Polymer Detection System (BioTnA; Kaohsiung, Taiwan; TAHC04D) and probed with an anti-BrdU antibody (1:100; Abcam, ab6326). A flash label at 2 h was used to designate the baseline position of the proliferating crypt cells. The migration of BrdU-positive (BrdU+) cells from the crypt base to the lumen was measured using ImageJ software.

CUT&RUN assay for CBP and p53 enrichments at the p21 promoter
The CUT&RUN assay was performed using the CUT&RUN Assay Kit (Cell Signaling Technology, #86652) according to the manufacturer’s protocol, with minor modifications. Briefly, 1-3 × 105 freshly isolated colonic crypt cells were fixed with 0.1% formaldehyde, permeabilized with digitonin-containing buffer, and incubated with anti-CBP (1:50; Cell Signaling Technology, #7389) and anti-p53 (1:50; Cell Signaling Technology, #2524) antibodies to capture CBP-bound and p53-bound chromatin, respectively. Nuclei were immobilized with concanavalin A-coated magnetic beads. Targeted chromatin was cleaved by Protein A–micrococcal nuclease (pA-MNase) upon calcium exposure and released at 37 °C. Purified DNA was analyzed by qPCR using primers specific for the p53 binding site within the mouse p21 promoter (forward: 5′-CCACAGCAGAGGAGAAAGAAG-3′; reverse: 5′-GCTGCTCAGAGTCTGGAAATC-3′). Enrichment was calculated as % input, defined as 100 × 2 [Ct (input) – Ct (CUT&RUN)] for each sample. qPCR was performed with SYBR Green (Thermo Fisher Scientific, A46109) on ABI QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific). All reactions were run in at least three biological replicates with corresponding matched input controls.

Colonic crypt isolation and organoid culture
The colons of 8-week-old female CBPWT and CBPAA mice were opened longitudinally. The tissue was minced into 5-mm pieces, washed three times with cold PBS and incubated with 30 mM EDTA in PBS for 5 min at 37 °C in a tube roller at 100 rpm. After the incubation, the tissue fragments were vortexed three times in 10 mL of cold PBS containing 0.1% bovine serum albumin (BSA), and the resulting supernatant, which contained few crypts and debris, was discarded. The tissue pieces were resuspended in 10 mL of cold PBS containing 0.1% BSA and vortexed eight times, and the supernatant enriched in crypts was collected in a clean 50 mL tube. After crypt-enriched supernatants were collected by vortexing three to four times, the samples were centrifuged at 50 × g for 5 min at 4 °C to pellet intact crypt fragments. After the crypts were resuspended in 1–10 mL of cold PBS containing 0.1% BSA, the crypts were counted (400 crypts/well), transferred to 1.5 mL Eppendorf tubes and centrifuged at 9,600 × g for 10 sec. The supernatant was discarded, and 0.5 mL of TrypLE (Gibco, #12563-011) containing 10 μM Y-27632 (LC Laboratories, Y-5301) was then added and incubated at 37 °C for 8 min. After the incubation, the crypts were pipetted fifteen times and centrifuged at 9,600 × g for 10 sec. For culture, the isolated crypts were resuspended in Matrigel (Corning, #356231) (40 μL/well) and seeded in prewarmed 24-well plates. The polymerized Matrigel was overlaid with organoid growth medium (1:1 mixture of basal culture medium and WNR conditioned medium). The basal culture medium was Advanced DMEM/F12 (Gibco, #12634-034) supplemented with penicillin/streptomycin (Gibco, 15140-122), GlutaMAX (Gibco, 35050-061), 10 mM HEPES (Gibco, #15630-080), N2 (Gibco, #17502-048), B27 (Gibco, #17504-044), 1.25 mM n-acetylcysteine (Sigma–Aldrich, A9165-5G), and 10 mM nicotinamide (Sigma–Aldrich, N0636). WNR conditioned medium containing WNT, Noggin, and R-spondin derived from stable cell lines was a kind gift from Dr. Jr-Wen Shui and supplemented with 10 μM Y-27632 (a ROCK inhibitor) and 50 ng/mL mEGF. The overlay medium was changed every 3 days, and the organoids were harvested on Day 6. The organoid-forming efficiency was calculated as the number of organoids formed in the Day 6 culture divided by the total number of isolated crypts seeded in the well and presented as a percentage.

Organoid viability assay
Organoid viability was assessed using an MTT assay52. Crypt-derived organoids from 8-week-old female mice were seeded into 96-well plates at a density of 200 crypts/well in 7 μL of Matrigel. After 6 days of organoid culture, 0.5 mg/mL MTT (Sigma–Aldrich, M2128) was added to each well, and the cells were further incubated for 3 h at 37 °C. After the incubation, the medium was discarded, and 20 μL of a 2% sodium dodecyl sulfate (SDS; Invitrogen, #15525017) solution in ddH2O was added to solubilize the Matrigel at 37 °C for 2 h. Then, the purple–blue MTT formazan precipitate was dissolved in 100 μL of DMSO for 2 h for 20 min at 37 °C, and the absorbance was measured at 562 nm in a microplate reader (BioTek Synergy HT, USA). Cell viability was normalized to that of the CBPWT organoids by dividing the OD value of each sample by the mean OD of the CBPWT controls.

Whole-mount staining of organoids
Crypt-derived organoids generated from 8-week-old female mice were seeded in 8-well chamber slides (Merck, PEZGS0816) or 8-well chambered coverglasses (Thermo Scientific, #155411) at a density of 40 crypts/well in 40 μL of Matrigel. After 6 days of organoid culture, the organoids were fixed with 4% paraformaldehyde for 30 min at room temperature on an orbital shaker at 50 rpm, permeabilized with TBS containing 0.1% Triton X-100 and 1% BSA for 30 min at room temperature, immersed in acid–alcohol for 10 min at -20 °C, and blocked with TBS containing 0.1% Triton X-100 and 1% BSA for 1 h at room temperature. Organoids were incubated with primary antibodies overnight at 4 °C. The primary antibodies used included anti-pCBP (Ser1383/1387) (1:150; GeneTex, customized, GTX90661), anti-CBP (1:200; Cell Signaling Technology, #7389 ), anti-Ki67 (1:200; Cell Signaling Technology, #9129S) and anti-E-cadherin (1:200; Invitrogen, #33-4000). After the incubation with the primary antibodies, the organoids were incubated with secondary antibodies at a 1:500 dilution, including Alexa Fluor® 488-conjugated goat anti-mouse IgG (Invitrogen, A-11029) and Alexa Fluor® 594-conjugated goat anti-rabbit IgG (Invitrogen, A-11037), for 30 min at room temperature and then with ProLong Gold Antifade Reagent with DAPI (Invitrogen, P36935) for nuclear staining. Images of most whole-mount sections were obtained via z-stack reconstruction using a Zeiss LSM880 or LSM780 confocal microscope (Carl Zeiss, Jena, Germany). Images were acquired and analyzed as described in the confocal microscopy and image analysis section.

Molecular Cloning and Plasmid Construction
The plasmids utilized in this study, including a control vector expressing EGFP alone (pCMV-puro-IRES2-EGFP) or construct encoding HA-tagged CBP phosphomimetic mutants coexpressing EGFP (DD mutant: pCMV-puro-IRES2-EGFP mCBPS1383D/S1387D-HA; EE mutant: pCMV-puro-IRES2-EGFP mCBPS1383E/S1387E-HA), were all developed at the Biomedical Resource Core (BMRC) of the First Core Labs, National Taiwan University College of Medicine. All plasmids have a total size of 14,236 bp and utilize the Mus musculus CREB binding protein (Crebbp; NCBI Reference Sequence: NM_001025432.1) as the backbone. To ensure proper expression and facilitate cloning, a silent mutation (V292V; c.876 G>T) was introduced into the mCbp sequence. For the phospho-mimetic constructs, site-directed mutagenesis was performed to introduce the following amino acid substitutions:
• S1383D/S1387D (DD): c.4147T>G, c.4148C>A (S1383D) and c.4159T>G, c.4160C>A, c.4161G>C (S1387D).
• S1383E/S1387E (EE): c.4147T>G, c.4148C>A, c.4149T>A (S1383E) and c.4159T>G, c.4160C>A (S1387E).
The oligonucleotide sequences for cloning and sequencing verification are provided in Supplementary Table 2. All constructs were validated by Sanger sequencing through the BMRC facility to confirm the presence of intended mutations and the integrity of the plasmid backbone.

Lipofectamine-mediated plasmid transfection of organoids dissociated into single cells
Lipofectamine transfection of the organoids was performed with modifications to the established protocol53. Briefly, organoids that were dissociated into single cells were transfected with plasmids using Lipofectamine and subsequently re-embedded in Matrigel for organoid reformation. The detailed steps are described below.

The culture medium was removed, and the organoids were briefly washed with prewarmed PBS. The organoids were then collected in cold PBS and centrifuged at 9,600 × g for 10 sec. After the PBS was aspirated, 500 μL of prewarmed TrypLE (Gibco, #12563-011) containing 10 μM Y-27632 (LC Laboratories, Y-5301) was added to the tube. Organoids were mechanically disrupted by pipetting up and down ten times using a P1000 pipette and then incubated at 37 °C for 5 minutes. Following this incubation, further dissociation was performed by pipetting up and down approximately 20 times using a P200 tip. The enzymatic reaction was terminated by the addition of cold Advanced DMEM/F12 supplemented with GlutaMAX, HEPES, and penicillin–streptomycin. The cells were pelleted by centrifugation at 9,600 × g for 10 sec.

Approximately 105 cells were resuspended in 450 μL of organoid growth medium (as described in the organoid culture section) supplemented with 10 μM Y-27632 and kept on ice until transfection. One microgram of plasmid DNA (i.e., pCMV-puro-IRES2-EGFP (vector), pCMV-puro-IRES2-EGFP mCBPS1383D/S1387D-HA (DD), or pCMV-puro-IRES2-EGFP mCBPS1383E/S1387E-HA (EE)) was mixed with 1 μL of Lipofectamine Stem Transfection Reagent (Thermo Fisher Scientific, STEM00001) according to the manufacturer’s protocol. Fifty microliters of reaction mixture were added to 105 single organoid cells suspended in 450 μL of organoid growth medium supplemented with 10 μM Y-27632 (without Matrigel) in a single well of a 24-well plate. The plate was then centrifuged at 600 × g for 1 h at 32 °C, followed by an incubation at 37 °C for 2 h. The cells were then harvested, pelleted, seeded in Matrigel in two wells of a 24-well plate, and grown in organoid growth medium supplemented with 10 μM Y-27632. The overlay medium was changed every 3 days, and the organoids were harvested on Day 5 post transfection. Transfected cells were identified by EGFP fluorescence. Successful delivery and expression of DD and EE mutants were confirmed by Western blotting detection of HA-CBP (Supplementary Figure 5).

Proximity ligation assay (PLA)
PLA was performed according to the manufacturer’s instructions (Sigma–Aldrich, #DUO92101). Primary antibodies were diluted in Duolink antibody diluent as follows: anti-CBP (Cell Signaling Technology, #7389) at 1:200, anti-p65 (Cell Signaling Technology, #6956) at 1:200, and anti-p53 (Santa Cruz Biotechnology, sc-126) at 1:200. Imaging was performed with a Zeiss LSM780 confocal microscope (Carl Zeiss, Jena, Germany).

Western blot analysis
Cells or organoids were lysed on ice for 30 min using RIPA buffer (Thermo Scientific, #89901) supplemented with Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific, #78442) at a 1:100 dilution. Cell lysates were then centrifuged at 16,200 × g for 20 min at 4 °C. The supernatant was collected, and protein concentration was determined using Bio-Rad Protein Assay Dye Reagent (Bio-Rad Laboratories, #500-0006). Equal amounts of protein (30 μg) were resolved on 6–10% SDS-PAGE gels and then transferred to nitrocellulose membranes (Thermo Scientific, #88018). After transfer, membranes were blocked for 1 h at room temperature with 5% (w/v) non-fat milk in TTBS (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween 20). The membranes were then incubated with the appropriate primary antibodies overnight at 4 °C. Following washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 30 min at room temperature. Immunoblots were visualized using the T-Pro LumiDura Chemiluminescence Detection Kit (T-Pro Biotechnology, #JT96-K006M).

The following primary antibodies were used: anti-p53 (1:2000; Santa Cruz Biotechnology, sc-126; Leica, NCL-L-p53-CM5p), anti-p21 (1:1000; Santa Cruz Biotechnology, sc-6246; Cell Signaling Technology, #2947), anti-ALDH1A1 (1:1000; Cell Signaling Technology, #12035), anti-HA-tag (1:1000; Cell Signaling Technology, #3724), anti-Lgr5 (1:1000; Abcam, ab75850), anti-vinculin (1:1000; Santa Cruz Biotechnology, sc-25336), anti-GAPDH (1:2000; Proteintech, #60004-1-Ig), and anti-actin (1:5000; Proteintech, #66009-1-Ig). HRP-conjugated secondary antibodies included anti-mouse IgG (#7076S, 1:10,000) and anti-rabbit IgG (#7074S, 1:10,000) from Cell Signaling Technology.

Spheroid formation
HCT116 cells (4 × 103 cells/mL in 6-well plates) were cultured in serum-free DMEM/F12 supplemented with 1% antibiotic/antimycotic Solution, 1% insulin–transferrin–selenium, 20 ng/mL EGF and 25 ng/mL bFGF. After 5 days, the formation of spheres was evaluated.

Reverse transcription and cDNA preparation
Total RNA was isolated from various samples, including colonic crypts, cultured colonic organoids, and T cells of the indicated mice, using TRIzol reagent (Thermo Scientific, #15596018) according to the manufacturer’s protocol. Briefly, tissues were homogenized in TRIzol, and phase separation was induced by chloroform addition. RNA was then precipitated with isopropanol, washed with 75% ethanol, and finally resuspended in nuclease-free water. For cDNA synthesis, 2 μg of total RNA from each sample was mixed with 1 μL of Oligo(dT)20 primer and nuclease-free water to a total volume of 15 μL. The mixture was incubated at 70 °C for 5 min to denature secondary structures within the RNA template, followed by immediate cooling to 4 °C for at least 1 min to prevent renaturation. Subsequently, 10 μL of Master Mix containing M-MLV RT 5X Reaction Buffer (5 μL, Promega, #M531A), 10 mM dNTP (1 μL, FocusBio, #BTCP-10M), M-MLV Reverse Transcriptase (1 μL, Promega, #M1705), and RNase inhibitor (0.625 μL, Promega, #N2518) was added to each tube. The reverse transcription reaction was performed at 42 °C for 1 h using a Biometra T3 thermocycler (Biometra). The resulting cDNA was stored at -20 °C until further use.

Real-time PCR and primer sequences
Real-time PCR was performed using Applied Biosystems (ABI) PowerTrack SYBR Green Master Mix (Thermo Fisher Scientific, #A46109) on ABI QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific). All samples were analyzed in technical duplicate, and target gene expression levels were normalized to Actin or Gapdh using the 2-ΔΔCT method. The mouse primer sequences used were as follows:
• Fig. 6d, 6e and 7e: Vcan (5′-GGACCAAGTTCCACCCTGACAT-3′ and 5′-CTTCACTGCAAGGTTCCTCTTCT-3′); Actin (5′-CATTGCTGACAGGATGCAGAAGG-3′ and 5′-TGCTGGAAGGTGGACAGTGAGG-3′).
• Supplementary Figure 2 and 3: Tbx21 (5′-CAACAACCCCTTTGCCAAAG-3′ and 5′-TCCCCCAAGCAGTTGACAGT-3′); Ifng (5′-CAAGTGGCATAGATGTGGAAG-3′ and 5′-GAAGAAGGTAGTAATCAGGTGTG-3′); Gata3 (5′-AGAACCGGCCCCTTATCAA-3′ and 5′-AGTTCGCGCAGGATGTCC-3′); Cmaf (5′-GAGCAGCGACAACCCTTC-3′ and 5′-CCCACGGAGCATTTAACAAG-3′); Il4 (5′-TAGTTGTCATCCTGCTCTTCTT-3′ and 5′-GTGTTCTTCGTTGCTGTGAG-3′); Rorc (5′-CGCCTCACCTGACCTACC-3′ and 5′-TTGCCTCGTTCTGGACTATAC-3′); Il17 (5′-AGGCAGCAGCGATCATCC-3′ and 5′-TGGAACGGTTGAGGTAGTCTG-3′); and Il2rb (5′-GTCCATGCCAAGTCGAACCT-3′ and 5′-GGATGCCTGCCTCACAAGAG-3′); and Gapdh (5′-CATCACTGCCACCCAGAAGACTG-3′ and 5′-ATGCCAGTGAGCTTCCCGTTCAG-3′).

RNA sequencing and bioinformatics
Total RNA samples (3–5 μg) were sent to Genomics BioSci & Tech (New Taipei City, Taiwan) for mRNA enrichment using oligo magnetic beads, and indexed dsDNA libraries were constructed using the Illumina TruSeg RNA Sample Preparation Kit (Illumina) for further processing and bioinformatics analysis.

Flow cytometry analysis
CD4+ and CD8+ T cells were isolated from the spleens, peripheral lymph nodes and mesenteric lymph nodes (mLNs) of CBPWT and CBPAA mice and were then stained with anti-CD4 (1:100; PE; eBioscience, #12-0042-82), anti-CD8α (1:200; APC; BioLegend, #100711) and anti-TCR-β (1:100; FITC; BioLegend, #109205) antibodies and gated on CD4+ T cells (TCR-β+CD4+) and CD8+ T cells (TCR-β+CD8+). Naïve CD4+ T cells were isolated from the peripheral lymph nodes of CBPWT and CBPAA mice, stained with anti-CD4 (1:200; FITC; BioLegend, #100510) and anti-CD62L (1:200; PE; BioLegend, #104407) antibodies, and gated on naïve CD4+ T cells (CD4+CD62Lhi). The cells were analyzed by flow cytometry (FACSAriaIII, BD). Representative gating strategies and isotype controls for splenic CD4+ and CD8+ T cell characterization are provided in Supplementary Figure 9 and 10, respectively. Data analysis and gating were performed using FlowJo software.

Isolation of colonic epithelial cells from crypts for flow cytometry analysis
The colons of Lgr5-eGFP reporter mice were removed, cut longitudinally and washed five times with cold PBS. Following an incubation for 5 min at 37 °C in a 30 mM EDTA solution, the colonic crypts were released from the colon tissue by mechanical vortexing in wash buffer (0.1% BSA in PBS), and this process was repeated three times. The supernatant, which was enriched with colonic crypts, was centrifuged 2 times at 80 × g and 40 × g for 10 min at 4 °C. Isolated colonic crypts were resuspended in 5 mL of TrypLE Express supplemented with Y27632 (1:1000 stock dilution) and transferred into c-tubes. Using the program m-intestine-1, the crypts were dissociated with the GentleMACS dissociator at room temperature, and the c-tube was incubated for 8 min in a water bath at 37 °C. After the incubation, the program m-intestine-1 was run again. Dissociated crypt cells were resuspended in organoid medium and filtered through a 40-μm cell strainer, and colonic single epithelial cells were analyzed by flow cytometry (LSRFortessa, BD) through the service provided by the Flow Cytometric Analysis and Sorting Core (the First Core Laboratory, National Taiwan University College of Medicine). The sequential gating hierarchy for the isolation of colonic Lgr5-GFP+ stem cells, including debris removal and doublet exclusion, is detailed in Supplementary Figure 11. Lgr5-GFP+ cells were gated using GFP-negative crypt cells (from mice without the Lgr5-eGFP reporter) as a negative control to define background autofluorescence. Data analysis and gating were performed using BD FACSDiva software.

Statistical analysis
The sample size (n) refers to the number of independent biological replicates in each group, specifically representing individual mice or individual human donors/patients as indicated in the respective figure legends. For all organoid experiments, n denotes independent biological replicates, with each replicate representing an organoid line established from a separate donor mouse. For organoid diameter and organoid-forming efficiency experiments, each data point represents the average value derived from at least three technical replicates (wells) per mouse. To ensure reproducibility and minimize batch effects, all samples within a comparative set were processed, stained, and imaged in parallel. Data were analyzed using unpaired two-tailed Student’s t-tests with GraphPad Prism 10 software. Data distribution was assumed to be normal, and variances were similar between groups being compared. All quantitative data are presented as mean ± SEM. Statistical significance was defined as p < 0.05, with exact p-values provided directly on the graphs.

Study approval
Human tissue samples were obtained with informed consent from all participants, and the study protocol was approved by the Institutional Review Board (IRB) of National Taiwan University Hospital. All studies involving human paraffin-embedded colon sections were conducted under the approval of the IRB (Nos. 201705031RIND and 202404149RINC). All animal experiments were approved by the National Taiwan University College of Medicine Institutional Animal Care and Use Committee (IACUC; Nos. 20170357, 20170454, 20200054, 20210223, 20220486, 20230281 and 20240106).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This paper explores the role of CBP phosphorylation at Ser1383/1387 in regulating versican expression and maintaining the\nintestinal stem cell (ISC) niche. Using CBP knock-in mice with alanine substitutions (CBP^AA) at these phosphorylation\nsites, the study demonstrates that impaired phosphorylation results in ulcerative colitis (UC)-like symptoms, driven by altered\nCBP binding preferences. Specifically, increased CBP-p53 binding reduces versican levels, a large, extracellular matrix\nproteoglycan known for its role in cell adhesion, proliferation, migration, tissue remodeling. Reduction in versican disrupts\nISC function, promoting colonic inflammation. These findings highlight the critical roles of CBP phosphorylation and\nversican in maintaining colonic health.\nOverall, the study is intriguing and introduces a novel candidate, versican (Vcan), for exploration in ulcerative colitis (UC).\nHowever, it lacks critical rigor required for studies of this nature. Key issues include insufficient sample sizes to substantiate\ntranslational claims, failure to establish causation (relying primarily on associations), and the absence of a clear mechanism\nconnecting Vcan, CBP, and intestinal epithelial cell (IEC) stem cell dysfunction. These gaps significantly temper the\nenthusiasm of this reviewer.\nMAJOR:\n1) The study lacks key rescue and proof-of-causation experiments. Restoring CBP phosphorylation (e.g., by introducing a\nphospho-mimetic CBP mutant at Ser1383/1387) could clarify whether phosphorylation alone rescues organoid formation\nand viability. Similarly, cell-based assays would help establish sufficiency. Introducing interventions such as versican\nsupplementation or targeting ECM components could test whether reduced stemness is mediated by extracellular matrix\nalterations. Additional rescue experiments—such as supplementing CBP^AA mice with versican or phospho-mimetic CBP\nmutants—could confirm whether CBP phosphorylation or its downstream effects, like versican expression, are sufficient to\nreverse colitis and restore IEC stemness, independent of p53 deletion. Testing in vivo effects of versican replenishment\n(e.g., via systemic or localized delivery) in CBP^AA mice would further strengthen the case for versican as a therapeutic\ntarget for intestinal inflammation and stem cell maintenance. reviewer notes that the only reversal was Vcan addition to\norganoids.\n2) the study currently lacks MoA elucidation, i.e., whether impaired CBP phosphorylation alters key inflammatory signaling\npathways (e.g., NF-κB or cytokine profiles) within the colon, contributing to the inflammatory phenotype. Including data on\ninflammatory cytokine levels or signaling pathways, which could connect CBP phosphorylation status with inflammatory\nresponses. RNAseq could provide MOA guidance here.\n3) only looks at a single time point, but it lacks a longitudinal analysis of disease progression or severity. This limits\nunderstanding of whether the UC-like phenotype worsens, stabilizes, or fluctuates over time, which is relevant for\ntranslational applications and understanding IBD’s chronic nature\n4) Th authors are pointing towards IEC driven phenotype but again would be nice to see more MOA...\nIt’s unclear whether the dysfunction arises from changes in cell-cell adhesion, permeability, or specific signaling pathways\nwithin IECs\n5) How does impaired CBP phosphorylation affect epithelial cell signaling and communication with other colonic cells, such\nas fibroblasts or immune cells?\n6) n = 3 seems particularly very low and lacks rigor in a the patient studies (Fig 9)/ stem cell dysfuncton(a key claim) did not\nget carried into this figure.\nMINOR:\n1) Abstract: expand CBP, IEC, define genotype [CBPAAp53 IEC]", "reviewer_2": "This manuscript by Lin and colleagues investigated the significance of the Ser1382/1386 phosphorylation of CREB-binding\nprotein (CBP), a transcriptional coactivator of NF-kB and p53, in the context of “colitis”. The group previously reported that\nCBP phosphorylation at these sites determine its binding preference for NF-kB or p53, thereby playing a role in controlling\ncell proliferation versus apoptosis. In this current study, the team characterized CBP(AA) knockin mice that do not have\nthese serine sites for phosphorylation. They also used an IKK inhibitor that inhibits these phosphorylation events. In Figure\n1-7, they show that, in the mouse intestinal epithelial cells, mouse enteroids, and HCT human cell lines, blocking these\nphosphorylation increased CBP to bind p53, reduced epithelial proliferation, caused a non-immune cell driven “spontaneous\ncolitis” phenotype resembling human ulcerative colitis. Deleting epithelial p53 in CBP(AA) mice reverted the phenotypes.\nIn\nFigure 8-9, they went on to show a reduced versican expressing compartment in CBP(AA) mice and its restoration by\nepithelial p53 deletion, and this loss of versican is also found in human UC patients. The data reporting changes in organoid\nculture and proximity ligation assay showing CBP-p53 interaction are well done. I have some major concerns about the\ninterpretation of their data and the conceptualization of some findings.\nMajor concerns:\n1. The manuscript tried to tie the phenotype to ulcerative colitis. The authors stated that “CBP(AA) mice exhibit a\nspontaneous colitis phenotype resembling clinical patients with ulcerative colitis (UC)”. However, from the provided\nhistology images, for example the very first one shown in Fig. 1f, no immune cell infiltration, glandular distortion, or any sign\nof epithelial death were seen in the tissue of CBP(AA) mice. The majority of colonic images from CBP(AA) mice provided in\nthe following figure panels also do not strongly support a colitis in CBP(AA) mice, except in Fig. 2e, which was used to show\na colon from the WTàAA transplantation experiment. There in Fig. 2e (WTàAA) does contain some chronic inflammation\n(basal lymphoplasmacytosis and mild glandular distortion). This raised the question why WTàAA transplanted mice showed\na stronger phenotype than intact AA mice? Therefore, the mis- or over-interpretation of these histopathology findings by the\nauthors are a major concern. The group may want to include some GI pathology expertise, especially they want to sell the\nwork as an IBD study.\n2. The epithelial intrinsic versus versican-dependent mechanisms are confusing. While the first 7 figures established the\ndefects in epithelial cells, which were supported by the epithelial intrinsic characterization of CBP-p53 interactions in\nenteroid and HCT cell line experiments. The epithelial intrinsic mechanism is also supported by reversion of the phenotype\nby epithelial deletion of p53. However, Fig. 8-9 switched the mechanism to versican expressing compartment, which is in the\nECM or the “mucosal stromal” compartment. It is unclear how deleting p53 in the epithelial cells restored the versican\nexpression in the ECM or the stroma. This versican perspective is especially confusing as the earlier figures indeed showing\nchanged CBP-p53 interactions in the AA epithelial cells or AA enteroids (independent of versican).\n3. In terms of to demonstrate that CBP(AA) binds more to p53 to regulate gene expression, Fig. 4 is considered a bit thin,\nwithout showing any chromatin level data.\n4. Conceptually, Fig. 6 essentially used an inhibitor to show the similar effects observed in the CBP(AA) organoids. These\ndata are cohesive and may be presented together to deliver the concept.\n5. Fig. 3, the BrdU pulse chase, migration assays do not show an impressive difference.", "reviewer_3": "This report builds on previous work demonstrating that phosphorylation of the transcriptional coactivator CBP at\nSer1382/1386 by IKKalpha enhances its interaction with the p65 NF-kB subunit. Moreover, Ser1382/1386 phosphorylation\ninhibits the interaction of CBP with the tumour suppressor p53. Conversely, the unphosphorylated CBP preferentially binds\np53 over p65. Here, to learn more about the in vivo significance of the regulatory mechanism, the authors have created a\nknockin mouse model where Ser1382/1386 of CBP are mutated to alanines. Analysis of these mice revealed the\nspontaneous development of a condition resembling ulcerative colitis (UC). Moreover, the authors convincingly demonstrate\nthat this results from a defect in the renewal of intestinal stem cells rather than immune cells. RNA Seq analysis revealed\nthat this is a consequence, at least in part, from the p53 dependent down regulation of an ECM component called versican in\nthe CBP(AA) mice. In an organoid model, addition of versican rescued the defects seen upon CBP Ser1382/1386 mutation.\nDemonstrating the potential applicability of these finding to humans, intestinal tissues from UC patients were found to have\nreduced level of CBP Ser1382/1386 phosphorylation, a decreased level of p65/CBP interactions, increased levels of\nCBP/p53 interactions and significantly reduced levels of versican.\nOverall, I found the analysis of the UC phenotype of the CBP(AA) mice, together with the identification of versican and its\nregulation by p53, to be convincing. However, there were other aspects of the manuscript where I have major concerns.\nMajor Concerns\n(1) Figures 5 & 6 rely heavily on an IKK inhibitor called CYL-19S as a means of reducing CBP Ser1382/1386\nphosphorylation in cells and determining the effect of this on the interaction between CBP and p53 or p65, together with\nvarious phenotypic assays. There are a number of concerns with use of this inhibitor.\n(a) CYL-19S is also an inhibitor of IKKbeta. Indeed this group has previously reported effects of this compound on p53\n(including induction of p21 seen here) as a result of IKKbeta inhibition (DOI: 10.1111/j.1582-4934.2009.00712.x). Although\nin their previous report looking at CBP Ser1382/1386 phosphorylation they found that IKKbeta cannot phosphorylate these\nresidues in vitro, there are likely to be many other CBP independent mechanisms by which IKKbeta inhibition might\ninfluence the effects seen here. Therefore, additional controls are required to support the data in Figure 5 in HCT116 cells,\nincluding both the effects on the CBP/p53 interaction, p21 induction and spheroid formation. First, the authors need to use\nsiRNAs targeting IKKalpha or beta to confirm which of these kinases are responsible for the effects seen. Second, they\nshould use a commercial IKKbeta inhibitor that does not inhibit IKKalpha and determine whether it has any effect.\n(b) A further control that should be included is to determine whether CYL-19S has any effect on the CBP(AA) organoids. If\nthe effects of CYL-19S are specifically through CBP Ser1382/1386 phosphorylation there should be no effect when these\nare mutated. Testing the effect of CYL-19S on CBP(AA) organoids that have been supplemented with versican (Figure 8)\nwould be a good way of demonstrating that this compound is not affecting organoid growth through other, non-specific,\nmechanisms.\n(c) In Figure 5A, there still seems to be a significant level of CBP Ser1382/1386 phosphorylation after CYL-19S treatment.\nThis data needs to be quantified. Moreover, confirming the specificity of this signal through the use of CBP, IKKalpha and\nIKKbeta siRNAs is also required.\n(d) I could find very little information on CYL-19S as it does not seem to be commercially available. There is a previous\nreport on the synthesis of this compound from these authors (https://doi.org/10.1093/carcin/bgh211) but it is not cited in this\ncurrent manuscript. Indeed I could not find a description of the source of CYL-19S in the methods. This previous report only\nperforms limited analysis of the specificity of CYL-19S. Therefore, unless published elsewhere, the\nspecificity of CYL-19S\nneeds to be demonstrated using a large panel of recombinant kinases. Many companies offer this type of compound\nscreening as a commercial service.\n(2) Is versican also down regulated in the CBP(AA) organoids and in HCT116 cells treated with CYL-19S?\n(3) One conclusion from Figure 8 is the CBP Ser1382/1386 mutation only affects a small subset of p53 regulated genes\n(including versican). One interpretation of this is that these therefore represent genes that are uniquely dependent on CBP\nas a regulator. Assuming that versican is also downregulated in CBP(AA) organoids and HCT116 cells (see (2) above), this\napparent CBP dependence should be demonstrated through the use of a CBP siRNA and a CBP inhibitor.\n(4) Does the RNA Seq data reveal any known NF-kB regulated target genes being affected by the CBP Ser1382/1386\nmutation? The Venn diagram in Figure 8c suggests that there are 35 genes affected by the CBP Ser1382/1386 mutation in\ncells with wild type p53 but only 21 are shown in the heat map in Figure 8a. However, only one gene appears to be affected\nby the CBP (AA) mutation when p53 is mutated, suggesting that effects on NF-kB activity, at least in unstimulated cells, are\nminimal. In the discussion (lines 340-347) the authors discuss the previously described roles of NF-kB in IECs. They\nsuggest that in patients with UC and lowered CBP Ser1382/1386 phosphorylation together with the CBP(AA) mice that NF-\nkB would be inert to environmental stimuli. Using the organoid model, can the authors demonstrate this by stimulating the\ncells with an NF-kB inducer such a TNF and then examining the effects on NF-kB regulated gene expression in cells with\nthe CBP Ser1382/1386 mutation?\n(5) Related to (4), I can't find any information on where the RNA Seq data was deposited. It would also be desirable to\nprovide an Excel file of the RNA Seq data for the reviewers and as Supplementary data.\nOther concerns\n(6) Line 214 states ‘Because increased CBP/p53 binding was demonstrated to dampen stemness in HCT116 IECs’. This is\nnot the case. CYL-19S was shown to do this but as discussed above, it cannot currently be concluded that these effects are\nthrough CBP/p53 binding.", "reviewer_4": "In the present study, the authors followed up on their previous study which showed that phosphorylation of coactivator CBP\nby IKKa promotes NF-κB-dependent transcription while unphosphorylated CBP promotes p53-mediated transcription. Here\nthe authors generated a CBPAA knock-in mouse model with impaired IKKa-dependent phosphorylation of CBP that resulted\nin an Ulcerative Colitis (UC) phenotype, including shortened colon length, fecal occult blood, and stool consistency\nconsistent with clinical UC. Histologically, there were infiltration of neutrophils and macrophages and increased CD4+ and\nC8+ T cells in mesenteric lymph nodes. Bone marrow chimera study suggested that the UC-like phenotype is mediated\nthrough Intestinal Epithelial Cells (IECs) which demonstrate decreased proliferation, migration, and organoid formation in\nCBPAA cells due to increased p53 activity. Predictably, an IKK inhibitor that decreased phosphorylation of CBP in wild-type\nIECs resulted in increased p53 activity, lower expression of stem cell marker ALDH1A1, and decreased organoid formation\nof HCT116 IECs, thereby recapitulating the CBPAA phenotype. The authors then deleted p53 from IECs in the CBPAA\nmouse and the UC phenotype resolved, indicating that these conditions are p53 dependent. RNA-seq showed that versican\nis down-regulated in CBPAA cells from colonic crypts, but deletion of p53 restores versican expression. In addition, CBPAA\norganoids cultured in exogenously added versican showed proliferation similar to wild-type counterparts. Tissue from UC\npatients show increased CPB/p53 binding, decreased CBP/p65 binding, and decreased versican expression.\nOverall, the study provides strong evident to support their major conclusions. However, there are some concerns that need to\nbe addressed to make the study even more compelling.\nPoints to address:\n1. The images presented in Figure 1f are not very clear to an untrained reader. It is unclear what has been scored on the\nright graph. This needs better explanation.\n2. The materials and methods section indicates the use of 8-week old females (line 391) in the study but some of the figure\nlegends indicate the use of 6-8 week old mice. Figure 2 clearly includes the use of male mice as well. The specifics of mice\nused need to be consistent throughout the presentation.\n3. Figure 5: the HCT116 colon tumor cell line is referred to as IEC (intestinal epithelial cells). The data with this cell model\ndoes not seem to add much to the overall study which is otherwise focused on mouse IECs and UC. Moreover, referring\nHCT116 colon tumor cell line as IEC in the text makes the reading confusing because mouse intestinal cells are also\nreferred to as IECs throughout. Consequently, this figure could be moved to supplemental section of the data presentation\nand these cells should be referred to as HCT116 or simply colon tumor cells to avoid confusion.\n4. Figure 5a, 5b and 5g: the differences concluded for these figures are not clear. Quantitative data need to be presented to\nbe convincing.5. Figure 5c: Data for TNFa+/- CYL-19S also need to be included for comparison.\n6. The source of the IKKa inhibitor, CYL-19S, was not described in the manuscript. This needs to be stated in the\nmaterials and methods section. It also needs to be stated how specific this inhibitor is toward IKKa. There is no clear information even\nin the authors’ prior publication (Huang, et al., Mol Cell, 2007).\n7. The source and nature of the recombinant versican used in Figure 8 was not described. Was this generated from\nmammalian expression system or from E. coli? This needs to be stated. How did the authors come to the specific dose of\nversican (100 ng/microL)? This needs to be explained."}, "author_response": "Point-by-point response\nReviewer #1\nMajor:\n1. The study lacks key rescue and proof-of-causation experiments.\n(a) Restoring CBP phosphorylation (e.g., by introducing a phospho-mimetic CBP mutant at\nSer1383/1387) could clarify whether phosphorylation alone rescues organoid formation and\nviability.\n(b) Similarly, cell-based assays would help establish sufficiency. Introducing interventions such as\nversican supplementation or targeting ECM components could test whether reduced stemness\nis mediated by extracellular matrix alterations.\n(c) Additional rescue experiments-such as supplementing CBPAA mice with versican or phospho-\nmimetic CBP mutants-could confirm whether CBP phosphorylation or its downstream effects,\nlike versican expression, are sufficient to reverse colitis and restore IEC stemness, independent\nof p53 deletion. Testing in vivo effects of versican replenishment (e.g., via systemic or localized\ndelivery) in CBPAA mice would further strengthen the case for versican as a therapeutic target\nfor intestinal inflammation and stem cell maintenance. reviewer notes that the only reversal\nwas Vcan addition to organoids.\nReply: The key rescue and proof-of-causation experiments have been conducted.\n(a) Restoring CBP phosphorylation in CBPAA organoids by introducing a phospho-mimetic CBP DD\nor EE mutant at Ser1383/1387 have been conducted and demonstrated, also CBPAA organoid\nformation and viability as well as IECs proliferation and versican are rescued (now Fig.7a-d).\nThese provide key rescue and proof-of-causation that CBP phosphorylation alone maintains\nversican and stem cell niche to preserve IEC stemness. (p. 9, lines 269-273)\n(b) To establish the sufficiency of extracellular matrix on stemness, ex vivo rescue experiments by\nsupplementing recombinant versican into cultured medium of CBPAA organoids were\nperformed. The findings of restoring IECs proliferation and organoid-forming efficiency not only\nstrengthen versican and stem cell niche maintaining IECs stemness but also indicate that\nreduced stemness is mediated by extracellular matrix alteration (now Fig. 6h,i). Furthermore,\ngefitinib, an EGFR inhibitor, abrogated these rescues in CBPAA organoids, suggesting that\nversican may trigger activation of the EGFR pathway (now Fig. 6h,j). (p. 8-9, lines 248-255)\n(c) We thank the reviewer for this insightful comment.\nIn the present study, we explore the role of a component of the extracellular matrix (ECM),\nparticularly the involvement of versican in maintaining IEC stemness, which paves the way for\nnew treatments for IBD. Our findings not only have valuable clinical implications—offering a\nnovel potential therapeutic strategy for IBD—but also demonstrate how molecular-level\ninsights can be translated into clinically significant applications. CBP phosphorylation is\nessential for maintaining versican and stem cell niche, thereby preserving IEC stemness and\nsustain intestinal homeostasis. The in vivo supplementation of versican to restore IEC stemness\nand reverse IBD is another task with great challenge that needs well preparation by consulting\nwith glycan expert. It takes time.\nWe will contact with Dr. Chi-Huey Wong to arrange a face to face discussion. Dr. Wong is a\nformer President of Academia Sinica in Taiwan and also a world-renowned expert in\nglycoscience. He is currently the Scripps Family Chair Professor of Chemistry at the Scripps\nResearch Institute and a joint Distinguished Research Fellow at the Genomics Research Center\nof Academia Sinica.\n1\n2. The study currently lacks MoA elucidation, i.e., whether impaired CBP phosphorylation alters key\ninflammatory signaling pathways (e.g., NF-kB or cytokine profiles) within the colon, contributing\nto the inflammatory phenotype. Including data on inflammatory cytokine levels or signaling\npathways, which could connect CBP phosphorylation status with inflammatory responses.\nRNAseq could provide MOA guidance here.\nReply:\nAs suggested, we have analyzed RNA-seq data to assess whether impaired CBP phosphorylation\nin CBPAA mice alters key inflammatory signaling pathways (e.g., NF-kB or cytokine profiles) within\nthe colon. There are no significant changes in the expression of key inflammatory signaling\ncomponents, such as NF-κB target genes or pro-inflammatory cytokines in CBPAA mice compared\nwith CBPWT, suggesting that the inflammatory phenotype observed in CBPAA mice is not driven by\nconventional inflammatory signaling pathways. Instead, increased CBP binding to p53 leads to\nreduced versican that plays a central role as followings:\nl Genetic deletion of p53 in IECs of CBPAA mice reversed colonic histopathology, restored barrier\nintegrity (now Fig. 5g,f) and expression of versican in vivo and ex vivo (now Fig. 6f, k). (p. 8, lines\n217-219; p. 9, lines 260-263)\nl Introducing phospho-mimetic CBP DD and EE mutants into CBPAA organoids rescued the\ndefective organoid formation and restored versican expression (now Fig. 7b,d). Similarly,\nsupplementation with recombinant versican in CBPAA organoids restored growth and proliferation\n(now Fig. 6h,i), reinforcing that versican is a functional downstream mediator of CBP\nphosphorylation to maintain intestinal homeostasis. (p. 9, Lines 271-273; p8-9, lines 248-251)\n3. only looks at a single time point, but it lacks a longitudinal analysis of disease progression or\nseverity. This limits understanding of whether the UC-like phenotype worsens, stabilizes, or\nfluctuates over time, which is relevant for translational applications and understanding IBD’s\nchronic nature.\nReply:\nIn our pilot study, we did assess colitis progression in CBPAA mice from 4 to 56 weeks of age by\ntracking stool consistency and occult blood (Fig. R1 in this response letter). The colitis severity\npeaked around 8-14 weeks and then partially resolved during adulthood.\nAll experiments in the present study were conducted using 8-week-old mice, both male and\nfemale CBPAA mice showed comparable severity of colitis.\ncolitis severity in male and female CBPAA mice were shown (a), which were assessed by combination of\nindividual weekly stool occult blood and stool consistency scores (b-h).\n4. The authors are pointing towards IEC driven phenotype but again would be nice to see more\nMOA... It’s unclear whether the dysfunction arises from changes in cell-cell adhesion,\npermeability, or specific signaling pathways within IECs.\nReply:\nThe mechanisms underlying IEC dysfunction in CBPAA mice like cell-cell adhesion, barrier\npermeability, specific signaling pathways are all examined.\nCell-cell adhesion: Reduced E-cadherin expression in colonic epithelial cells of CBPAA mice\ncompared with CBPWT is revealed and that is restored in CBPAAp53DIEC mice (now Fig. 6g). Similarly,\nin CBPAA organoids, E-cadherin expression is diminished and rescued by supplementation with\nrecombinant versican as well as overexpressed with phospho-mimetic CBP DD or EE mutants (now\nFig. 6j, 7e). These findings suggest that impaired CBP phosphorylation and subsequent\nsuppression of epithelial versican compromise cell-cell adhesion in IECs. (p. 8, lines 245-246; p. 8-\n9, lines 248-252; p.9, lines 273-274)\nBarrier function: Employing FITC-dextran gavage, increased intestinal permeability in CBPAA mice\nand reversed in CBPAAp53DIEC mice were originally shown (now Fig. 1f, 1g, 5f), supporting an IEC-\nintrinsic defect. (p. 4, lines 96-98; p. 8, lines 217-219)\nSpecific signaling pathways: Rescued functions of CBPAA organoids by supplementing with\n3\nrecombinant versican are shown, the effects of which were abrogated by the treatment with EGFR\ninhibitor, gefitinib (now Fig.6h-j), suggesting the involvement of EGFR pathway in the CBP-versican\nsignaling axis. (p. 9, lines 253-255; p. 11, lines 349-351)\n5. How does impaired CBP phosphorylation affect epithelial cell signaling and communication with\nother colonic cells, such as fibroblasts or immune cells?\nReply:\nIn the present study, we revealed that impaired CBP phosphorylation in CBPAA mice leads to\nincreased CBP/p53 binding, impaired IEC proliferation and reduced IEC stemness through down-\nregulating chondroitin sulfate proteoglycan versican and stem cell niche, resulting in a UC-like\nphenotype. All these findings were restored in CBPAAp53DIEC mice (now Figs.3-6).\nVersican is a matricellular protein playing a key role not only in epithelial integrity but also in\nmodulating the extracellular matrix composition and cytokine milieu. Its crosstalk with adjacent\nstromal and immune cells through paracrine signaling in CBPWT mice is possible, like preventing\nluminal antigen exposure that triggers immune cell infiltration; by contrast, CBPAA mice show\nincreased F4/80+ and Ly6G+ cells (now Fig. 1i). These highlight that epithelial CBP phosphorylation\nstatus can shape the tissue microenvironment not only through stem cell regulation and barrier\nmaintenance, but also by influencing downstream stromal and immune responses. (p. 4, lines\n100-102)\n6. n = 3 seems particularly very low and lacks rigor in the patient studies (Fig 9)/stem cell dysfuncton\n(a key claim) did not get carried into this figure.\nReply:\n(1) We have expanded our immunohistochemical analysis using additional colonic biopsy samples\nto n = 4-7 for normal controls and n = 9-10 for UC patients (now Fig. 8b-d). These expanded data\nstrengthen the statistical power and reinforce our original observations. (p. 31, lines 1036-1038).\n(2) Additionally, we performed Ki-67 staining and observed reduced Ki-67+ staining in epithelium\nof UC patients compared to non-IBD controls (now Fig. 8c), consistent with the stem cell\ndysfunction. (p. 9, lines 281)\n(3) To further support the translational relevance of CBP phosphorylation in human, we\nperformed additional IHC staining for IKKα. Consistent with our previous findings demonstrating\nthat IKKα phosphorylates CBP at Ser1382/1386 to regulate cell fate decisions (Mol. Cell\n2007;26(1):75-87), we observed reduced IKKα expression in the colonic sections of UC patients\n(now Fig. 8d), which may account for the decreased CBP phosphorylation detected in these\nsamples. (p. 9, lines 281-282)\nIn our pilot study, CBP phosphorylation sites in colonic biopsies from UC patients were sequenced\nand there were no point mutations at the corresponding serine residues (Fig.R2). This suggests\nthat reduced CBP phosphorylation in human UC is due to reduced IKKα expression instead of\ngenetic mutations.\n(4) The sample number of UC patients for CBP/p53 proximity ligation assay (PLA) remains at n = 3\n(Fig. 8a), since the results are consistent with those in Fig. 8b-d. Furthermore, the quality of these\nPLA figures were as prominent as those in Figs 3c, 5h and Supplemental Fig. 5b.\nMINOR:\n1) Abstract: expand CBP, IEC, define genotype [CBPAAp53DIEC]\nReply: We have now expanded CBP (CREB-binding protein), extracellular matrix (ECM) and\ndefined genotype of CBPAAp53DIEC mice (CBPAA mice with deletion of p53 in the intestinal\nepithelium) in the Abstract.\nUC p’t#2\nUC p’t#3\nUC p’t#4\nUC p’t#5\nUC p’t#6\nUC p’t#7\nUC p’t#8\nUC p’t#9\nUC p’t#10\nUC p’t#11\nUC p’t#12\nFig. R2 Blood sample sequencing of UC patients. Genomic DNA was extracted from peripheral blood\nsamples of ulcerative colitis (UC) patients. The obtained sequence was aligned to the human reference\ngenome (NCBI RefSeq: NG_009873.1) and showed 100% identity, indicating the absence of point\nmutations.\nMajor concerns:\n1. The manuscript tried to tie the phenotype to ulcerative colitis.\n(a) The authors stated that CBP(AA) mice exhibit a spontaneous colitis phenotype resembling\nclinical patients with ulcerative colitis (UC). However, from the provided histology images, for\nexample the very first one shown in Fig. 1f, no immune cell infiltration, glandular distortion, or\nany sign of epithelial death were seen in the tissue of CBP(AA) mice.\nReply:\nTo address this concern, we have replaced a higher-quality representative image that more clearly\nillustrates the phenotype in Fig. 1 (now Fig. 1h, AA). Additionally, we have included a TUNEL assay\nto provide a quantitative evidence of epithelial cell apoptosis (now Fig. 1j). Increased infiltration\nof F4/80⁺ macrophages and Ly6G⁺ neutrophils in CBPAA colons were shown (now Fig. 1i). The\nobserved increase in immune cell infiltration along with the increased epithelial cell death support\nthe UC-like phenotype in CBPAA mice. (p. 4, lines 100-102)\nThe scoring criteria in histological assessment have been expanded in the revised Methods section\nunder “Histology”. (p.15, lines 473-481)\n(b) The majority of colonic images from CBP(AA) mice provided in the following figure panels also\ndo not strongly support a colitis in CBP(AA) mice, except in Fig. 2e, which was used to show a\ncolon from the WT→AA transplantation experiment. There in Fig. 2e (WT→AA) does contain\nsome chronic inflammation (basal lymphoplasmacytosis and mild glandular distortion). This\nraised the question why WT→AA transplanted mice showed a stronger phenotype than intact\nAA mice?\nReply:\nPrior to chimera experiments, the recipient mice were irradiated (Fig. 2a), which induced g-\nirradiation-mediated intestinal injury. These explain why AA transplanted mice showed a stronger\nphenotype than intact AA mice, because the combination of intestinal injury in intact CBPAA mice\nplus g-irradiation occurs in the WT→AA group (now Fig. 2f). (p. 16-17, lines 528-533)\n2. The epithelial intrinsic versus versican-dependent mechanisms are confusing. While the first 7\nfigures established the defects in epithelial cells, which were supported by the epithelial intrinsic\ncharacterization of CBP-p53 interactions in enteroid and HCT cell line experiments. The epithelial\nintrinsic mechanism is also supported by reversion of the phenotype by epithelial deletion of p53.\nHowever, Fig. 8-9 switched the mechanism to versican expressing compartment, which is in the\nECM or the “mucosal stromal” compartment. It is unclear how deleting p53 in the epithelial cells\nrestored the versican expression in the ECM or the stroma.\nReply: The epithelial intrinsic versus versican-dependent mechanisms are clarified.\nEpithelial origin of versican was examined by employing ex vivo 3D organoids. Consistent with the\nexpression pattern observed in colon tissues (Fig. 6f), versican is also downregulated in CBPAAp53-\nfloxed organoids and restored in CBPAAp53DIEC organoids with IEC-specific p53 deletion (Fig. 6k),\nconfirming its epithelial origin. Furthermore, replenishing recombinant versican protein into the\nculture medium of CBPAAp53-floxed organoids (AA + Vcan) restored the organoid-forming\nefficiency and size, as well as the number of Ki-67+ cells to levels comparable to those in\nCBPWTp53-floxed organoids (Fig. 6h, i). These suggest that epithelial-derived versican is released\ninto the ECM or the “mucosal stromal” to regulate ISCs. (p. 9, lines 259-263; p. 11, lines 333-336)\n3. In terms of to demonstrate that CBP(AA) binds more to p53 to regulate gene expression, Fig. 4 is\nconsidered a bit thin, without showing any chromatin level data.\nat Ser1382/1386 by IKKa enhances NF-kB, while suppressing p53,-mediated gene expression,\nthereby promoting cell growth. However, mutation of serines to alanines, which impairs CBP\nphosphorylation, increases its recruitment to p53- instead of NF-kB-responsive promoters, like\np21. In the present study, we show that our previous mechanistic findings at the molecular level\nconvey translational significance in CBPAA mice and UC patients.\n4. Conceptually, Fig. 6 essentially used an inhibitor to show the similar effects observed in the CBP(AA)\norganoids. These data are cohesive and may be presented together to deliver the concept.\nReply: Original Fig. 3c-e is now Fig. 4.\nSince we have generated ex vivo CBPWT and CBPAA organoids to deliver our concept (now Fig. 4),\nit is not necessary to treat CBPWT organoids with inhibitor (CYL-19s) that mimic CBPAA status like\nHCT116 did. Therefore, the original Fig. 6 is now deleted.\n5. Fig. 3, the BrdU pulse chase, migration assays do not show an impressive difference.\nReply:\nIncreasing the n number up to 8 in CBPWT and 6 in CBPAA mice at 96 h is further conducted to show\nan impressive difference (Fig. 3b). (p. 29, lines 941-944).\nMajor Concerns\n1. In Figures 5 & 6, there are a number of concerns with use of an IKK inhibitor, CYL-19S as a means\nof reducing CBP Ser1382/1386 phosphorylation in cells and determining the effect of this on the\ninteraction between CBP and p53 or p65, together with various phenotypic assays.\n(a) CYL-19S is also an inhibitor of IKKbeta reported by authors (DOI: 10.1111/j.1582-\n4934.2009.00712.x). Although this group previously found that IKKbeta cannot phosphorylate\nCBP Ser1382/1386 in vitro, there are likely to be many other CBP independent mechanisms by\nwhich IKKbeta inhibition might influence the effects seen here. Therefore, additional controls are\nrequired to support the data in Figure 5 in HCT116 cells, including both the effects on the CBP/p53\ninteraction, p21 induction and spheroid formation.\nFirst, the authors need to use siRNAs targeting IKKalpha or beta to confirm which of these kinases\nare responsible for the effects seen. Second, they should use a commercial IKKbeta inhibitor that\ndoes not inhibit IKKalpha and determine whether it has any effect.\n(b) A further control that should be included is to determine whether CYL-19S has any effect on the\nCBP(AA) organoids. If the effects of CYL-19S are specifically through CBP Ser1382/1386\nphosphorylation there should be no effect when these are mutated. Testing the effect of CYL-19S\non CBP(AA) organoids that have been supplemented with versican (Figure 6) would be a good way\nof demonstrating that this compound is not affecting organoid growth through other, non-specific,\nmechanisms.\n(c) In Figure 5A, there still seems to be a significant level of CBP Ser1382/1386 phosphorylation after\nCYL-19S treatment. This data needs to be quantified. Moreover, confirming the specificity of this\nsignal through the use of CBP, IKKalpha and IKKbeta siRNAs is also required.\n(d) I could find very little information on CYL-19S as it does not seem to be commercially available.\nThere is a previous report on the synthesis of this compound from these authors\n(https://doi.org/10.1093/carcin/bgh211) but it is not cited in this current manuscript. Indeed I\ncould not find a description of the source of CYL-19S in the methods. This previous report only\nperforms limited analysis of the specificity of CYL-19S. Therefore, unless published elsewhere, the\nspecificity of CYL-19S\nneeds to be demonstrated using a large panel of recombinant kinases. Many companies offer this type of compound\nscreening as a commercial service.\nReply (a) to (d): CYL-19s synthesized by our collaborator, Prof. Cherng-Chyi Tzeng, a medicinal\nchemist is now cited as reference 29 and its non-specific mechanisms have been excluded.\n(1) We have now included the proper citation of our previous work describing synthesis and initial\ncharacterization of CYL-19s as Ref. 29 (Carcinogenesis. 2004;25(10):1925-34), and this information\nhas also been added to the Methods section. (p.13, 418-419)\n(2) As suggested, we have tested the effect of CYL-19s on CBPAA organoids that have been\nsupplemented with versican and demonstrated that this compound is not affecting organoid growth\nand Ki-67+ cells (Fig. R3), confirming that CYL-19S specifically acts through reducing CBP\nSer1383/1387 phosphorylation without non-specific mechanisms.\nSince we have generated ex vivo primary CBPWT and CBPAA organoids (now Fig. 4), in line with the\nsuggestion of Reviewer #4, HCT116 cells treated with CYL-19s that mimic CBPAA status (originally\nFigure 5) is now moved to Supplementary Figure 5.\nby CYL-19s treatment. Organoids were\ncollected on day 6. The experimental\ngroups included: CBPWTp53f/f (WT),\nCBPAAp53f/f (AA), CBPAAp53f/f organoids\ntreated with 100 ng/µL versican from day\n0 (AA + Vcan), and CBPAAp53f/f organoids\ntreated with 100 ng/µL versican from day\n0 and 5 µM CYL-19s added on day 3 (AA\n+ Vcan + CYL-19s). a Microscopy images\nof day 6 cultured organoids (upper panel,\n10X; lower panel, 4X). Scale bar, 100 µm.\nb Representative immunofluorescence\nstaining of Ki-67 in colon sections. Nuclei\nwere counterstained with DAPI. Scale bar,\n100 µm. Image shown is a reused panel\nfrom Fig. 6h and 6i of the manuscript,\nprovided here to facilitate direct\ncomparison with the additional data.\n2. Is versican also down regulated in the CBP(AA) organoids and in HCT116 cells treated with CYL-\n19S?\nReply:\n(1) Versican is also downregulated in CBPAA Fig. R4 CYL-19s\ndownregulated Ki-\norganoids compared with CBPWT organoids 67, versican, and E-\n(now Fig. 6k). cadherin expression\nin HCT116 cells.\n(2) HCT116 cells treated with CYL-19S also show HCT116 cells were\ndownregulation of versican as well as Ki-67+ treated with 10 μM\ncells and E-cadherin expression (right\nCYL-19s for 24 h.\nFig.R4), consistent with the results of CBPAA\nRepresentative\norganoids (Fig.6i-k).\nimmunofluorescence\nimages of Ki-67,\nversican and E-\ncadherin expression.\nNuclei were\ncounterstained with\nDAPI (blue). Scale\nbars, 20 μm.\n3. One conclusion from Figure 8 is the CBP Ser1382/1386 mutation only affects a small subset of p53\nregulated genes (including versican). One interpretation of this is that these therefore represent\ngenes that are uniquely dependent on CBP as a regulator. Assuming that versican is also\ndownregulated in CBP(AA) organoids and HCT116 cells (see (2) above), this apparent CBP\ndependence should be demonstrated through the use of a CBP siRNA and a CBP inhibitor.\nReply:\nWe employed CBP AA organoids overexpressing phospho-mimetic CBP DD or EE mutant at Ser1383/\n1387 to restore CBP phosphorylation in CBP AA organoid and organoids formation and versican\nexpression are rescued, clarifying the unique dependence of CBP phosphorylation in regulating\ndownstream versican expression (Fig. 7), as reply to Reviewer #1 (comment 1a). (p. 9, lines 269-273)\n4. Does the RNA Seq data reveal any known NF-kB regulated target genes being affected by the CBP\nSer1382/1386 mutation? The Venn diagram in Figure 8c suggests that there are 35 genes affected\n9\nby the CBP Ser1382/1386 mutation in cells with wild type p53 but only 21 are shown in the heat\nmap in Figure 8a. However, only one gene appears to be affected by the CBP (AA) mutation when\np53 is mutated, suggesting that effects on NF-kB activity, at least in unstimulated cells, are\nminimal.\nIn the discussion (lines 340-347) the authors discuss the previously described roles of NF-kB in IECs.\nThey suggest that in patients with UC and lowered CBP Ser1382/1386 phosphorylation together\nwith the CBP(AA) mice that NF-kB would be inert to environmental stimuli. Using the organoid\nmodel, can the authors demonstrate this by stimulating the cells with an NF-kB inducer such a TNF\nand then examining the effects on NF-kB regulated gene expression in cells with the CBP\nSer1382/1386 mutation?\nReply:\nTo address whether CBP Ser1382/1386 phosphorylation influences NF-κB signaling, CBPWT and\nCBPAA organoids were respectively stimulated with TNF-α and abundant CBP/p65 binding was only\nobserved in CBPWT organoids (Supplementary Fig. 8c, lower left panel), indicating the activation\nof NF-κB pathway. In contrast, CBP/p65 binding was not seen in CBPAA organoids (Supplementary\nFig. 8c, lower right panel), indicating that CBPAA organoids do not respond to pro-inflammatory\ncues. These findings suggest that impaired phosphorylation of CBP renders IECs inert to NF-κB-\nmediated transcriptional activation upon environmental stimulation. (p. 11-12, lines 356-362)\nFurthermore, RNA-seq analysis did not reveal significant differences between CBPWT and CBPAA\nmice in canonical NF-κB target gene expression under basal conditions, consistent with the idea\nthat impaired phosphorylation of CBP affects inducibility rather than baseline NF-κB activity.\nTherefore, reduced CBP phosphorylation dampens stimulus-induced NF-κB activation, providing\na potential explanation for inert response in CBPAA mice to environmental stimuli and possibly in\nUC patients harboring low CBP phosphorylation.\nThe discrepancy in gene numbers between the Venn diagram (original Fig. 8c) and the heat map\n(original Fig. 8a) was due to employing inconsistent threshold criteria during the initial analysis.\nTo address this, we have reanalyzed the RNA-seq data with consistent threshold parameters:\n|log FC| > 0.5 and padj < 0.05. The revised data show the correct number of 13 genes in both\nthreshold. (p. 30, lines 988, 991).\n5. Related to (4), I can't find any information on where the RNA Seq data was deposited. It would\nalso be desirable to provide an Excel file of the RNA Seq data for the reviewers and as\nSupplementary data.\nReply: The RNA sequencing data reported in this study have been deposited in the Gene\nExpression Omnibus (GEO) database under accession codes: GSE295556\n(https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE295556). The following secure token\nhas been created to allow review while the record remains private: qjuveqwkhzyjbyd.\n6. Line 214 states “Because increased CBP/p53 binding was demonstrated to dampen stemness in\nHCT116 IECs”. This is not the case. CYL-19S was shown to do this but as discussed above, it cannot\ncurrently be concluded that these effects are through CBP/p53 binding.\nReply:\n“Because increased CBP/p53 binding was demonstrated to dampen stemness in HCT116 IECs” is\nnow revised into “Since increased CBP/p53 binding suppressed sphere formation and reduced\nstemness markers in HCT116 cells”. (p. 7, lines 195-196)\nPoints to address:\n1. The images presented in Figure 1f are not very clear to an untrained reader. It is unclear what has\nbeen scored on the right graph. This needs better explanation.\nReply:\nWe have replaced a higher-quality representative image that more clearly illustrates the\nphenotype in Fig. 1 (now Fig. 1h, AA). Additionally, we have included a TUNEL assay to provide a\nquantitative evidence of epithelial cell apoptosis (Fig. 1j). (p. 4, lines 100-102)\nThe scoring criteria in histological assessment have been expanded in the revised Methods section\nunder “Histology”. (p.15, lines 473-481)\n2. The materials and methods section indicates the use of 8-week-old females (line 391) in the study\nbut some of the figure legends indicate the use of 6–8-week-old mice. Figure 2 clearly includes the\nuse of male mice as well. The specifics of mice used need to be consistent throughout the\npresentation.\nReply:\nAll experiments in the present study employed female mice except the bone marrow chimera\nexperiments shown in Figure 2. Dr. Chien-Kuo Lee (Graduate Institute of Immunology, College of\nMedicine, National Taiwan University) kindly provided us congenic CD45.1 (B6.SJL-Ptprcα\npepcβ/BoyJ) mice in both sexes. Due to the limited availability of CD45.1 female mice, male mice\nwere also included. We have revised the legend of Figure 2 to explicitly state the use of both sexes.\nThe results shown in the new Figure 2 combine both male and female mice. To further address\nthe concern about sex-related issues, we present separate data in Supplementary Figure 2. All\nreadouts, including stool scores, colon length, histological damage, and immune cell infiltration\nare consistent between male and female mice, supporting that the colitis phenotype is not sex-\ndependent. (p.28, lines 923-927)\n3. Figure 5: the HCT116 colon tumor cell line is referred to as IEC (intestinal epithelial cells). The data\nwith this cell model does not seem to add much to the overall study which is otherwise focused on\nmouse IECs and UC. Moreover, referring HCT116 colon tumor cell line as IEC in the text makes the\nreading confusing because mouse intestinal cells are also referred to as IECs throughout.\nConsequently, this figure could be moved to supplemental section of the data presentation and\nthese cells should be referred to as HCT116 or simply colon tumor cells to avoid confusion.\nReply:\nAs suggested, Figure 5 regarding HCT116 colon tumor cell line is now moved to supplemental Fig.\n5. Also, we now refer this cell line as HCT116 instead of IEC to avoid confusing.\n4. Figure 5a, 5b and 5g: the differences concluded for these figures are not clear. Quantitative data\nneed to be presented to be convincing.\n5. Figure 5c: Data for TNFa+/- CYL-19S also need to be included for comparison.\nReply (4-5):\nAs suggested, we have moved CYL-19s-related HCT116 cell data (original Fig. 5) to Supplementary\nFig. 5. Since we have already generated CBPAAp53DIEC mice and performed ex vivo organoid assays\nto get core findings in this manuscript, we believe it is not essential to further quantify HCT116\ndata.\n6. The source of the IKKa inhibitor, CYL-19S, was not described in the manuscript. This needs to be\nstated in the materials and methods section. It also needs to be stated how specific this inhibitor\n11\nis toward IKKa. There is no clear information even in the authors’ prior publication (Huang, et al.,\nMol Cell, 2007).\nReply:\nAs stated in the reply to Reviewer #3, point 1, we have now included the proper citation of our\nprevious work describing synthesis and initial characterization of CYL-19s as Ref. 29\n(Carcinogenesis. 2004 Oct;25(10):1925-34), and this information has also been added to the\nMethods section. (p.13, 418-419)\nThe size and number of organiods formation and Ki-67+ IECs in CBPAA organoids supplemented\nwith versican are not affected by CYL-19s (Fig. R3), confirming that CYL-19s specifically acts\nthrough reducing CBP Ser1383/1387 phosphorylation without non-specific mechanisms.\nOur prior work (Mol Cell. 2007 Apr 13;26(1):75-87) demonstrated that CYL-19s effectively\nabolished TNF-α-induced CBP phosphorylation at Ser1382/1386 in HeLa cells (mentioned in p.6,\nlines 175-176).\n7. The source and nature of the recombinant versican used in Figure 8 was not described. Was this\ngenerated from mammalian expression system or from E. coli? This needs to be stated. How did\nthe authors come to the specific dose of versican (100 ng/microL)? This needs to be explained.\nReply:\nThe recombinant mouse versican protein (Abbexa, abx069663) used in original Fig. 8 (now Fig. 6)\nwas produced using a prokaryotic (E. coli) expression system and now clarified in the Methods\nsection. (p.13, lines 422-423)\nTo determine the optimal concentration, we first follow a recent study that employed 100 ng/mL\nfor in vitro treatment (Circulation. 2024;149:1004–1015; now reference 52). Based on this, we\nperformed a preliminary dose-response test from 50, 100, to 200 ng/mL. The concentration of 50\nng/mL already elicited a detectable biological response, and 100 ng/mL induced a more robust\neffect, and no further enhancement was observed at 200 ng/mL. Therefore, we selected 100\nng/mL as the optimal working concentration for the experiments presented. (p.13-14. Lines 423-\n426)\nResponse to Reviewer #1:\nMAJOR CONCERNS (1-11)\n1. Figure Legends and Clarity: Figure legends throughout the manuscript lack sufficient detail to\ninterpret the data presented. Critical information needed to understand the panels is missing,\nforcing the reader to repeatedly consult the Methods section. Authors must significantly improve\nlegend clarity and completeness to make their key findings more accessible and self-contained.\nReply 1: We thank Reviewer’s important suggestions and apologize for not providing sufficient details\nin the prior figure legends.\nIn the current revised form, sufficient details highlighted in red have been added into all the figure\nlegends to interpret the data presented throughout the manuscript. We have significantly improved\nthe legend clarity and completeness to make our key findings more accessible and self-contained.\nThank you.\n2. Figure 1I & 1K: Quantification and Inconsistencies in CBP Staining: Panel K quantifies total CBP+\ncells, yet images in panel I reveal absence of CBP at the top of the crypts. This may be due to CBP\ninstability in cells lacking phosphorylation; however, such interpretations are speculative without\ncorroborating images or dual-staining for pCBP and total CBP on serial or the same tissue sections.\nMoreover, it's unclear how quantification was derived—specifically, the method of thresholding\nused for defining “positive” cells based on integrated density is not described. The chosen\nUlcerative Colitis (UC) field does not reflect the same features as in the mouse model, making\ntranslational claims tenuous.\nReply 2: We sincerely thank the Reviewer’s valuable comments to point out the issues regarding CBP\nstaining, quantification, and translational relevance.\n(1) To address the concern about absence of CBP at the top of the crypts in CBPAA mouse colons\n(original panel 1k not panel 1l), we have now employed frozen sections of CBPWT and CBPAA\ncolons to perform immunofluorescence (IF) staining\ninstead of IHC by formalin-fixed paraffin-embedded (FFPE)\n(now Fig. 1i). CBP stainings are prominent in both\ngenotypes (now Fig. 1i, lower panels), and pCBP staining is\nstill detected in CBPWT colons but not CBPAA colons (now\nFig. 1i, upper panels), similar to our original IHC results\n(original Fig. 1k, upper panels). These results indicate that\nCBP is not destabilized in cells lacking phosphorylation.\nIF staining can better preserve antigenicity compared to\nIHC, where suboptimal antigen retrieval may result in\nuneven staining. As our custom-made anti-pCBP antibody\nand the commercial anti-CBP antibody are both rabbit-\nderived, dual staining is not feasible due to potential cross-\nreactivity.\n(2) Regarding quantification, our original analysis was performed using StrataQuest software with\nfixed ROIs. In this revised version, we have re-quantified the data using ImageJ with fixed ROIs\nand integrated density thresholding. Detailed descriptions are now added to the legends of Fig.\n1i and 1j (p.31, lines 966-973).\n(3) We acknowledge the Reviewer’s concern regarding the UC field selection. We have carefully\nreviewed our data and noted that the same feature of reduced pCBP expression is detected in\nboth human UC colon tissues and CBPAA colons without changes in total CBP level. This finding,\n1\ntogether with the similar results of increased CBP–p53 binding, reduced versican expression, and\nfewer Ki-67+ proliferative IECs in both colons (Fig. 8a–c; Fig.3c, 5h, 5i, 6f), collectively support the\ntranslational relevance of the current study. We are grateful to the Reviewer for prompting us to\nclarify and better substantiate this point.\n3. Internal Inconsistency Regarding Inflammation: Panel 1i shows prominent infiltration by\nneutrophils and macrophages, consistent with inflammation. However, the rebuttal document\ncontradicts this, claiming no upregulation of inflammatory pathways in colonic gene expression.\nThis discrepancy is not resolved and undermines the internal consistency of the manuscript.\nReply 3:\nWe sincerely thank the Reviewer for highlighting this important point regarding potential internal\ninconsistency between histological observations and gene expression data.\n(1) In response to this concern along with the related comments\nfrom the Reviewer #2, we have consulted two gastrointestinal\npathologists—one veterinary pathologist at our Laboratory\nAnimal Center and one attending physician specializing in\ngastrointestinal pathology at the Department of Pathology, NTU\nHospital. Consistent with the Reviewer #2’s assessment, both\nexperts confirmed that CBPAA mice under basal conditions has no\ncolonic inflammation. To avoid overinterpretation, we have\ntherefore described the lesions in CBPAA mice as colonic\nabnormalities throughout the manuscript (p.4, lines 101-106),\nand concomitantly removed two original Fig. 1i and 1j from prior\nrevised form shown in the right which presented F4/80+ and\nLy6G+, and TUNEL assay.\n(2) In bone marrow chimera experiments (Fig. 2), chimeric CBPAA mice exhibit substantial immune\n\ncell infiltration in all layers of the bowel wall, severe crypt distortion with the loss of entire crypts,\nand moderate mucosal hyperplasia, accompanied by increased histological scores and elevated\nmesenteric lymph node (mLN) cellularity (Fig. 2f, g, h). These mice also showed greater\ninfiltration of macrophages (F4/80+) and neutrophils (Ly6G+) than chimeric WT mice did\n(AA→WT) (Fig. 2i, j), in which minimal levels of infiltration markers were observed, likely because\nof irradiation prior to the chimera experiments (p. 5, lines 124-131). These results indicate that\nchimeric CBPAA mice exhibit colonic inflammation driven by nonhematopoietic cells, such as\nepithelial cells.\n(3) Regarding RNA-seq analysis, we would like to clarify that these data were generated from\nisolated colonic crypts enriching epithelial cells without inclusion of the lamina propria, which\ncontains the primary source of inflammatory cells. Therefore, the lack of upregulation in\ninflammatory pathways in this dataset is expected. We have revised the text to make this\nmethodological detail more explicit (p.8, lines 243-244).\nWe are grateful to the Reviewer for prompting us to carefully reassess the presentation of our data\nand ensure the internal consistency between histology and colonic gene expression.\n4. Figure 1i & 1j: Overcropping of Panels: The panels are cropped too tightly, raising concerns about\nselective presentation. Larger fields with insets should be included to demonstrate reproducibility\nand allow assessment of generalizability across the tissue.\nReply 4: We sincerely thank the Reviewer’s helpful suggestion regarding image cropping and the\nimportance of showing larger fields with insets to assess reproducibility and generalizability.\nAs stated in Reply 3, we have removed the original Fig. 1i and 1j (F4/80+, Ly6G+, and TUNEL panels) to\n2\navoid overinterpretation and retained F4/80+ and Ly6G+ stainings in bone marrow chimera\nexperiments by larger fields with insets (now Fig. 2i and 2j). The lower panels in these two figures\nclearly show greater infiltration of macrophages (F4/80+) and neutrophils (Ly6G+) in chimeric CBPAA\nmice compared to those in chimeric WT mice, thereby demonstrating the reproducibility and\ngeneralizability of our findings across the examined tissues.\n5. Figure 3c: PLA Localization of p53-CBP Interaction: PLA signals indicating interaction between\np53 and CBP are predominantly extranuclear, which contradicts the model proposed in the\nmanuscript. This could reflect poor antibody specificity, background noise, or suboptimal staining\nprotocols. Validation using negative controls (e.g., CBP–/– mice) is strongly advised.\nReply 5: We sincerely thank the Reviewer for raising this important point regarding the localization\nand specificity of PLA signals for p53–CBP interactions.\nAs suggested, we have validated the assay using multiple negative controls, including CBPAA mouse\ntissues, UC patient samples, and HCT116 cells. As shown in Fig. R1, no PLA signals were detected in\nany of these negative controls, supporting the specificity of the PLA signals shown in Fig. 3c and Fig.\n8a. In these figures, the majority of PLA signals are observed within the nuclei, with some located\nimmediately adjacent to these signals. In UC patient tissues (Fig. 8a, right), some PLA signals are also\nobserved at more distant extranuclear locations.\nWe note that the mouse colonic sections in Fig. 3c were cryopreserved, whereas those in UC patients\nin Fig. 8a were formalin-fixed paraffin-embedded (FFPE) because of the lack of frozen human tissues.\nTherefore, more distant extranuclear background in UC patient tissues may be attributed to technical\nlimitations of FFPE processing, because a similar background signal is also present in the adjacent\nnormal tissues (Fig. 8a, left).\nImportantly, enhanced CBP–p53 PLA signals were specifically in CBPAA mice and UC patient tissues,\nbut not the corresponding negative controls, supporting the conclusion that CBP–p53 interactions are\nincreased in these pathological contexts. We have taken care to present these data clearly,\naccompanied by appropriate validation, as they represent an important part of our mechanistic\nfindings.\nFig. R1 Negative controls for PLA.\nRepresentative images of negative\ncontrol staining from PLA using anti-\np53 antibody only. Tissues and cells\ninclude mouse colon (a), human\ncolon biopsy samples (b), and\nHCT116 cells (c). No PLA signal is\ndetected in the absence of the\nsecond primary antibody, confirming\nthe specificity of the PLA signal in\nexperimental conditions. Nuclei are\ncounterstained with DAPI (blue).\n6. Organoid Immunofluorescence Imaging: All IF images lack visible lumens, whereas the brightfield\nimages show clear lumen structures. This discrepancy should be explained—are lumens\ncollapsing in fixed samples, or is the imaging method insufficient to capture them?\nReply 6: We thank the Reviewer for pointing out this important observation regarding the appearance\nof lumens in our organoid immunofluorescence images.\nAs described in Current Protocols in Mouse Biology (2013 Dec 19; 3(4):217–240), we followed a whole-\n3\nmount staining procedure, and images were acquired by confocal Z-stacking to generate maximum\nintensity projections. This approach optimizes the visualization of fluorescence signals but sometimes\nmay obscure three-dimensional architecture including lumens.\nTo address this, we have now included the representative single optical sections along with full Z-\nstack reconstructions adapted from the AA+Vcan panel in Fig.6q, in which the lumen is clearly visible.\nThese additions clarify that lumen structures are preserved in fixed samples.\nFig. R2 Representative optical sections and\na Z-stack projection of an organoid stained\nby immunofluorescence for versican\nadapted from Fig.6q AA+Vcan. Left panels:\nTwelve individual optical sections (1–12)\nacquired by confocal microscopy from a\nwhole-mount-stained organoid, showing\nlocalization of versican (green) and nuclei\n(blue). Lumen structures are indicated by\narrows. Right panel: Corresponding Z-stack\nmaximum intensity projection clearly reveals\nthe central lumen.\n7. Figure 4c: MTT Assay Interpretation Is Unclear: The MTT readout is not intuitive. The Y-axis label\nlacks context, and the legend does not clarify the nature of the perturbation or the comparison\nbeing made. MTT reduction indicates loss of viability, yet no baseline or control is specified. For\nassessing organoid biogenesis, a growth assay over time, rather than endpoint viability, would\nbe more appropriate.\nReply 7: We sincerely thank the Reviewer for the helpful comments regarding the interpretation and\npresentation of the MTT readout. We apologize for the lack of clarity in the original Fig. 4c and have\nrevised it according to the Reviewer’s guidance to improve the presentation.\nThe MTT assay was performed following the established method described in Cell Death & Disease\n(2014, vol. 5, e1228). In the current revised manuscript, we have clarified the Y-axis label to indicate\ncell viability normalized to CBPWT organoids (Methods, p. 20, lines 641-642; Fig. 4c legend, p.32-33,\nlines 1026-1027). Consistent with Ki-67 staining (Fig. 4b), the MTT assay also revealed reduced\nproliferative capacity in CBPAA organoids compared to those in CBPWT organoids. These two\nindependent assays corroboratively indicate impaired proliferation of CBPAA organoids. We also\nmeasured organoid formation efficiency, which reflects the initiation of new organoids, and organoid\ndiameter, which reflects subsequent growth. Both measurements indicate the evaluation of organoid\nbiogenesis (Nat Commun. 2023 Sep 27;14(1):6016.) (Fig. 4d; p.7, lines 215–216).\n8. Mechanistic Gaps in Vcan Role: While the model points toward stem cell senescence (e.g.,\nincreased Gadd45 expression, p53 activation), and bone marrow transplant data suggest\nimmune involvement, it remains unclear which cells produce Vcan and how it impacts the stem\ncell niche. These mechanistic gaps weaken the central hypothesis.\nReply 8: We sincerely thank the Reviewer for the thoughtful comments on the mechanistic aspects\nof versican (Vcan) production and its impact on the stem cell niche.\n(1) We appreciate the Reviewer’s insights regarding stem cell senescence markers such as increased\nGadd45 expression and p53 activation. Gadd45 was beyond the scope of the current study. The\ncentral hypothesis of our study is that impaired CBP phosphorylation enhances CBP–p53\ninteraction, thereby repressing versican (Vcan) expression.\n(2) Our bone marrow chimera experiments, together with evaluation of the differentiation potential\nof naïve T cells and T subsets, support the conclusion that the colonic abnormalities (originally\nH\n4\ndescribed as colitis) in CBPAA mice are attributed to impaired CBP phosphorylation in IECs per se,\nrather than to the regulation of baseline immune homeostasis (p.5, lines 129-131 and 133-134;\np. 5-6, lines 145-147).\n(3) We provide multiple complementary lines of evidence to demonstrate that versican (Vcan) is\nproduced by intestinal epithelial cells (IECs) and regulated by epithelial CBP-p53 binding:\nIn addition to the RNA-seq and qPCR results (Fig. 6a, d, e), the epithelial-intrinsic defect and\nreduced versican expression observed in CBPAA mice were reversed by epithelial-specific p53\ndeletion (Fig. 6f), suggesting that versican is derived primarily from epithelial cells. Consistent\nwith these tissue-level changes (Fig. 6f), versican expression was also reduced in organoids\nderived from the crypts of CBPAAp53-floxed mice and restored in those derived from\nCBPAAp53IEC mice (Fig. 6q, r), further confirming its epithelial origin (p.9, lines 272-277).\n(4) The number of Lgr5+ stem cells in the IECs did not differ between Lgr5-CBPAA mice and Lgr5-\nCBPWT mice (Supplementary Fig. 6). However, intestinal stem cell function was compromised in\nCBPAA mice, as evidenced by the reduced organoid formation, decreased number of Ki-67+ IECs,\nand reduced versican expression. The unchanged number of Lgr5+ stem cells suggests that the\nobserved impairment in stemness may be attributed to a disruption in the stem cell niche, likely\ncaused by alteration in the extracellular matrix (ECM), rather than a direct loss of the stem cell\npopulation. Currently, epithelial-derived versican, an ECM, may contribute to the local regulation\nof the stem cell niche, as replenishing versican into the culture medium of CBPAA organoids\nrestored organoid formation, the organoid diameter and the number of Ki-67+ cells to levels\ncomparable to those of CBPWT organoids (Fig. 6j–p; p. 9, lines 263-267 and Discussion, p. 11,\nlines 351-360), supporting the role of versican in modulating stem cell niche environment to\npromote stem cell function and regulating intestinal epithelial cell behavior.\nThe loss of CBP–p53 binding in CBPAAp53IEC mice restored versican expression and IEC stemness to\nprevent colonic abnormalities. In contrast, impaired CBP phosphorylation in CBPAA mice increases\nCBP–p53 binding, leading to the downregulation of versican expression and contributing to colonic\nabnormalities (Figs. 5–6). An analysis of the versican gene promoter and enhancer regions using the\nPROMO and EPD databases did not reveal classical p53-binding sites. The precise mechanism by\nwhich CBP–p53 binding negatively regulates versican expression in IECs seems to be indirect and\nremains to be investigated.\n9. Figure 7: Rescue Experiments Lack Credibility: While rescue experiments are commendable in\nprinciple, the methodology is problematic. The authors report dramatic phenotype reversal using\nDD/EE CBP mutants transfected via Lipofectamine into 1–5 cells/organoid. However, such low-\nefficiency transfection would not plausibly result in 100% rescue of all phenotypes. The\ndiscrepancy between representative images and quantification also raises concerns about data\nintegrity.\nReply 9: We thank the Reviewer for raising these important points regarding the rescue experiments\nand data interpretation.\n(1) We performed gain-of-function experiments using phosphomimetic CBP mutants to further\ndetermine whether CBP phosphorylation affects versican levels. Single cells dissociated from\ncrypt-derived CBPAAp53f/f (AA) organoids were transfected with either a control vector\nexpressing EGFP alone (pCMV-puro-IRES2-EGFP), or constructs encoding HA-tagged CBP\nphospho-mimetic mutants (DD or EE; HA-tagged and co-expressing EGFP) using Lipofectamine™\nStem Transfection Reagent. Transfected cells were identified by EGFP fluorescence. Successful\ndelivery of HA-tagged CBP mutants into AA organoids was confirmed by HA-CBP expression\ndetected by Western blotting (Supplementary Fig. 5). Compared with the vector control, AA\n5\norganoids overexpressing DD or EE showed restored versican expression and a reversal of AA\nphenotype, as evidenced by the rescued organoid-forming efficiency and organoid diameter (Fig.\n7a–b). Additionally, these organoids exhibited increased IEC proliferation and E-cadherin\nexpression (Fig. 7c–d). These findings indicate that CBP phosphorylation plays important roles\nin maintaining versican expression and supporting the intestinal stem cell niche, thereby\ncontributing to the preservation of epithelial homeostasis in the colon. Despite as few as 1-5\nEGFP⁺ cells per AA organoid, overexpression of DD or EE upregulated versican expression in AA\norganoids (Fig. 7a, e). This finding parallels the concept that localized alterations within the stem\ncell niche can have broad effects on epithelial structure and function. We have now added\ndetailed descriptions of the experimental design into the main text and the legend of Fig. 7 to\nensure clarity for readers (p.9-10, lines 282-299 and p.34-35, lines 1098-1104).\n(2) Regarding the discrepancy between representative images and quantification, we apologize for\nany misunderstanding caused by this important issue.\nIn addition to the updated representative images (Fig.7b, left, upper panels), low-magnification\n(4×) images were included to provide a broader view of organoids (Fig.7b, left, lower panels),\nwhich clearly show the rescue of organoid formation after DD/EE transfection. Quantification\nwas performed by analyzing all organoids in the entire field of view, including organoids of\nvarying sizes to ensure unbiased quantification. The quantitative data reveal a significant\ndifference between the vector and DD or EE group regarding relative organoid diameter and\nformation efficiency (Fig.7b, right).\n10. Figure 8a: Questionable PLA Signal Localization: As in Figure 3c, PLA signals are largely\nextranuclear, despite the proposed interaction (p53-CBP) occurring in the nucleus. This\ninconsistency must be addressed with proper controls.\nReply 10: We appreciate the Reviewer’s continued attention to the localization of PLA signals. As\nReply 5, we have thoroughly addressed this concern by including appropriate negative controls to\ndemonstrate the specificity of the PLA signals. These negative controls support our conclusion that\nthe enhanced CBP–p53 interaction occurs predominantly in the nucleus, with some extranuclear\nsignals in UC patients (Fig.8a) potentially due to technical limitations as previously discussed.\n11. Response to Reviewers Is Inadequate and Lacking Transparency: The authors have paraphrased\nthe reviewers’ original comments rather than providing the full unedited critiques. This prevents\nan objective evaluation of whether concerns have been adequately addressed. Additionally, the\nmanuscript contains numerous grammatical errors and inconsistencies between results and\nrebuttal statements, further undermining confidence in the revision.\nReply 11: We thank the Reviewer for the feedback regarding our prior response to Reviewer’s\ncomments raised on Dec. 31, 2024. In those responses, only Major Comment 1 was subdivided into\n1a, 1b, and 1c to improve clarity, as those remarks were interrelated. All other comments were\naddressed in original form in our point-by-point response on April 30, 2025 revision.\nIn the current revision, we have included the full, unedited text of the Reviewer’s comments to ensure\ntransparency and to demonstrate that all concerns have been adequately addressed. Additionally, we\nhave carefully re-checked the manuscript for grammatical accuracy and ensured consistency between\nall results and rebuttal statements, and have sent it to Springer Nature Editing Service for professional\nEnglish language editing.\n12. Language and Grammar: The manuscript continues to suffer from grammatical errors and\nawkward phrasing, e.g., in the abstract (page 2, lines 24–27). A thorough language edit by a\nnative English speaker or professional service is necessary.\nReply 12: We thank the Reviewer for highlighting the importance of clear language and grammar. The\nmanuscript has been already undergone professional English language editing by Springer Nature\nEditing Service, and the certificate was issued on August 20, 2025 and may be verified on the SNAS\nwebsite using the verification code E4F9-7033-AEC4-0C87-D582 as below.\n13. Figure 2f (left panel, WT→AA): This image is blurry and low-resolution. A higher-quality version\nshould be provided to ensure proper interpretation.\nReply 13: The images in Fig. 2f now have been replaced with a higher-resolution version, re-acquired\nby a board-certified veterinary pathologist at our Animal Center, College of Medicine, National Taiwan\nUniversity, to ensure optimal image clarity and correct the histopathological interpretation.\nResponse to Reviewer #1’s prior major comment 6 dated Dec. 31, 2024 following our very\nfirst submission on October 1st, 2024\n6. n = 3 seems particularly very low and lacks rigor in the patient studies (Fig 9).\nReply: We thank the Reviewer for the important comment regarding sample size in the patient studies.\nWe have now increased the number of UC patient samples to n = 7 for CBP–p53 proximity ligation\nassay (PLA) (now shown in Fig. 8a; original Fig.9a in the first submission). Because of a temporary\nshortage of reagents from the distributor, Sigma Aldrich, earlier this year, we were unable to expand\nthe sample number in the initial revision on April 30, 2025. We appreciate the Reviewer’s\nunderstanding and believe this expanded dataset strengthens the robustness of our findings.\n1. In my original point #1, I pointed out that there was no colitis in any of the AA tissue images. The\nauthors responded that a new Fig. 1h was included along with macrophage and neutrophil\nstaining panels in Fig. 1i, as well as TUNEL staining in Fig. 1j. Unfortunately, I am still not\nconvinced there is inflammation in AA mouse colons. First, there is no inflammation in Fig. 1h.\nSecond, the WT and AA colons shown in Fig. 1i looked the same. The Ly6G stained cells do not\nlook like \"neutrophils\", as neutrophils are smaller than these stained cells. Third, the TUNEL\nstained cells in AA tissue do not look like apoptotic cells, instead they look quite healthy. Overall,\nI do not believe there is inflammation or colitis in these AA mouse colons. This was one of the\nmajor issues I raised in prior review. The authors may want to consult some GI pathologists.\nReply 1: We sincerely thank the Reviewer for the detailed and professional feedback.\n(1) As suggested, we consulted Yi-Ting Tsai, a veterinary pathologist at Laboratory Animal Center,\nCollege of Medicine, NTU, and Dr. Jia-Huei Tsai, a gastrointestinal pathologist at the Department\nof Pathology, National Taiwan University Hospital. Both experts concur with the Reviewer #2’s\nassessment that the histological evaluation of CBPAA mice revealed mild pathological changes in\nthe distal colon, including localized low-grade immune cell infiltration in the mucosa and\nsubmucosa as well as mild crypt architectural irregularities (Fig. 1h, right panels and\nSupplementary Fig. 1e). Since these changes do not meet the criteria for colitis, we therefore\ndescribed these observed lesions as colonic abnormalities (p. 4, lines 101-106),\nand\nconcomitantly removed two original Fig. 1i and 1j presenting F4/80+ and Ly6G+, and TUNEL assay\nfrom prior revised form.\n(2) In bone marrow chimera experiments (Fig. 2), chimeric CBPAA mice exhibit substantial immune\ncell infiltration in all layers of the bowel wall, severe crypt distortion with the loss of entire crypts,\nand moderate mucosal hyperplasia, accompanied by increased histological scores and elevated\nmesenteric lymph node (mLN) cellularity (Fig. 2f, g, h). These mice also showed greater\ninfiltration of macrophages (F4/80+) and neutrophils (Ly6G+) than chimeric WT mice did\n(AA→WT) (Fig. 2i, j), in which minimal levels of infiltration markers were observed, likely because\nof irradiation prior to the chimera experiments (p. 5, lines 124-131). These results indicate that\nchimeric CBPAA mice exhibit colonic inflammation driven by nonhematopoietic cells, such as\nepithelial cells.\nWe appreciate the Reviewer’s comments, which have helped us improve the clarity and rigor of\nour study.\n2. My #3 point is to suggest more data, hopefully chromatin level results, to support CBP(AA) binds\nmore to p53 to regulate gene expression. Instead of addressing the point using their mouse\nmodel, the authors responded by pointing to a study published in 2007.\nReply 2: We thank the Reviewer for the valuable suggestion regarding additional chromatin-level\nevidence to support enhanced CBP(AA) binding to p53. We have now completed the chromatin level\nexperiments and further confirmed that CBPAA preferentially binds to p53 to regulate gene expression,\nand the results showed increased binding of p53 to the p21 promoter (now Fig. 3f; p. 6, lines 171-\n173). During the prior revision, the Cut&Run assays were still being optimized, therefore chromatin-\nlevel data were not available yet. We appreciate the Reviewer’s understanding and believe the new\ndata further strengthen the mechanistic insights of our study.\n3. Thanks for addressing #4.\nReply 3: We sincerely thank the Reviewer for the appreciation of our ex vivo CBPWT and CBPAA\norganoid models, which support and deliver the key concepts of our study, thus the figures of CBPWT\norganoids treated with inhibitor CYL-19s as employed in HCT116 cells that effectively mimic CBPAA\nstatus were deleted. We appreciate the Reviewer’s constructive comments.\n8\n4. The last point was about the BrdU pulse chase assay to show migration. The authors responded\nby adding more animal numbers. However, from the provided images, this reviewer failed to see\nany difference in terms of migration.\nReply 4: We thank the Reviewer for the helpful comment. To better illustrate the migration trend,\nwe\nhave updated Fig. 3b with higher-resolution BrdU images, including enlarged insets and yellow dashed\nlines to indicate the direction of cell migration. Hope these improvements clarify the migration\ndifferences.\nResponse to reviewer #2:\n1. For the original point #1: The authors dampened the claim of inflammatory\nphenotype in these AA mice, which improved the rigor of their experimental\ninterpretations.\nReply 1:\nWe sincerely appreciate the Reviewer’s professional comments, which guided us to\ntemper the claim of an inflammatory phenotype in CBPAA mice and improved the\nclarity and rigor of our study. We also gained valuable insights from these\nsuggestions.\n2. For the point regarding chromatin level evidence of CBP binding and regulation of\np53, they showed a new result in panel Fig. 3f to demonstrate more p53 binding\nto p21 promoter. Although this indirectly showed an increased p53 activity in\nthese AA tissues, the direct regulation of CBP on p53 was not demonstrated in this\nexperiment. The n-number in this new experiment was 3 for each group. The\npanels of 3a, 3d, and 3e were also showing n=3 in all comparison, which did not\nseem to support a high confidence of these experimental results.\nOverall, the paper has been improved but figure 3 remains to be a weak\ncomponent of the work.\nReply 2: We sincerely thank the Reviewer for the constructive comments.\n(a) In addition to p53, we also use anti-CBP antibody to perform CUT&RUN assay. The\nresults reveal that both CBP- and p53-precipitated chromatin are enriched at the\np21 promoter (Fig. 3f, g), supporting that enhanced recruitment of CBP by p53\nregulates p21 expression in CBPAA mice (p.6, lines 171–173). However, CBP\nenrichment at the p21 promoter is absent in CBPAAp53IEC mice, accompanying a\nreduction in p21 expression (Supplementary Fig. 5), in which CBP–p53 binding is\nlost (Fig. 5h, right two panels). These results confirm that direct regulation of CBP\non p53 is enhanced at the chromatin level by impaired CBP phosphorylation in\nCBPAA mice, providing further evidence that differential phosphorylation of CBP\nregulates binding to the p53 locus and subsequent p21 expression (p.8, lines 240–\n246). We believe these new data strengthen the mechanistic insights of our study.\nWe have now increased the n-number of CBPAA mice to 5 for each group to\nsupport a high confidence of the results of chromatin level experiments (Fig.3f, g\nand Supplementary Fig. 5a).\n(b) Also, we have now increased the sample size in Fig. 3a to 8 for all comparisons. In\nFig. 3d and Fig. 3e, sample sizes were 5 for CBPWT and 6 for CBPAA, thereby\nstrengthening the confidence in these results.\nIn Fig. 3a, we replaced IHC with Ki-67 immunofluorescence (IF) staining on frozen\ncolon sections from CBPWT and CBPAA mice, which provides much clearer and\nhigher-resolution signals.\nPoint-by-point response\nResponse to reviewer #2:\n1. The newly revised Fig. 3a showed Ki67 and DAPI staining. The AA image on the right is overall\ndimmer than the WT image. The DAPI signal is brighter in the WT image than the DAPI signal in\nthe AA image. The figure legend stated \"The nuclei were counterstained with DAPI for\nnormalization. Ki-67 expression was quantified as the number integrated density (IntDen)\nnormalized to the DAPI IntDen within a fixed region\". If the quantification was based on\nfluorescent intensity and the two images were clearly taken with different settings, I am concerned\nwhether the authors carefully examined these images before including them in the manuscript.\nReply 1:\nWe sincerely thank the reviewer for the careful and insightful assessment, and we apologize for the\nconfusion caused by the original selection of representative images. We have addressed this\nconcern as follows:\n1. Updated Representative Images: We appreciate the reviewer’s careful attention to the image\npresentation in Fig.3a. To address this concern, we re-examined our complete dataset from the\nentire cohort (n = 8 mice per group; Fig. R1). We retained the original WT representative image\n(WT-03) and replaced the AA image (AA-08) with AA-07. We believe that this updated\nrepresentative pair (WT-03 and AA-07; highlighted in the red box) offers a more comparable DAPI\nintensity profile, thereby ensuring a fair and balanced visual comparison between the WT and AA\ngroups (Fig. R2).\n2. Clarification of Imaging Settings and Data Consistency: To address the concerns regarding\nacquisition settings for Fig. 3a, we have now provided a comprehensive summary of imaging\nparameters (Laser Power and Detector Gain) for the entire cohort in Table R1. To maintain\nconsistent visualization of Ki-67 staining across the manuscript, the Ki-67 signal in Fig. 3a has been\ndisplayed using a green pseudo-color to match Fig. 5i. Specifically, Fig. 3a was detected using Alexa\nFluor 594 (red fluorescence, 561 nm channel), but the signal was pseudo-colored green to ensure\nconsistency with Fig. 5i, which was acquired using Alexa Fluor 488 (green fluorescence).\nAfter consultation with imaging specialists at the First Core Labs, National Taiwan University\nCollege of Medicine, variations in DAPI intensity are commonly attributable to unavoidable\ntechnical factors such as tissue section thickness.\nAs shown in Table R1, the Detector Gain for each channel was individually optimized to ensure\nappropriate dynamic range and to prevent signal saturation. Importantly, even in cases where AA\nsamples required higher Detector Gain (up to 700 V), the Ki-67 signal remained consistently\nweaker than that in WT controls. This strongly supports the conclusion that the observed\nreduction reflects a genuine biological phenomenon rather than an imaging artifact.\nFurthermore, the reduction of Ki-67 in the AA group was consistently observed in our earlier IHC\nstaining (Fig. 3a in NCOMMS-24-62813B-Z). We expanded the cohort size (n = 8) when\ntransitioning to immunofluorescence staining to ensure consistency with the experimental\nconditions in Fig. 5i (NCOMMS-24-62813C).\n1\n3. Data Accessibility and Transparency: To ensure full transparency, all raw confocal image files (.czi)\ncorresponding to Fig. 3a, along with all other imaging data contributing to quantification in this\nstudy, have been deposited in the EBI BioImage Archive. Additionally, all processed figures and\nthe detailed parameter summary table are available via Figshare.\n EBI BioImage Archive (Accession: S-BIAD2810):\nhttps://www.ebi.ac.uk/biostudies/bioimages/studies/S-BIAD2810?key=6d2c7865-f2a6-\n42c3-9667-c29da769d403\n Figshare (Table R1 & Source data): https://figshare.com/s/94654a07dcdcda6cb64e\nFig. R1 Full dataset of Ki-67/DAPI staining for Figure 3a. Top panels: Wild-type (WT) group (n = 8 mice). Bottom panels: AA\ngroup (n = 8 mice). The blue box indicates the original AA representative image (AA-08). The red boxes highlight the\nupdated representative pair selected for the revised manuscript: WT-03 (which remains unchanged from the original\nversion due to its high structural integrity) and AA-07 (the newly selected image to provide a more balanced DAPI intensity\nfor visual comparison). Scale bar = 100 µm. All raw confocal image files are available in the EBI BioImage Archive (S-\nBIAD2810).\nFig. R2 Original and revised representative images for Figure 3a.\nKi-67 DAPI\nSample ID Group Attenuation: Attenuation:\nDetector Gain Detector Gain\nLaser 561nm Laser 405nm\nWT-01 WT 0.6 650 0.75 540\nWT-02 WT 0.6 650 0.75 550\nWT-03 WT 0.6 650 0.75 550\nWT-04 WT 0.55 660 0.7 550\nWT-05 WT 0.55 660 0.7 550\nWT-06 WT 0.5 650 0.75 520\nWT-07 WT 0.5 650 0.75 520\nWT-08 WT 0.5 650 0.75 520\nAA-01 AA 0.6 650 0.7 550\nAA-02 AA 0.65 630 0.7 550\nAA-03 AA 0.65 600 0.7 550\nAA-04 AA 0.65 600 0.75 540\nAA-05 AA 0.65 700 0.75 530\nAA-06 AA 0.65 700 0.75 530\nAA-07 AA 0.65 700 0.75 505\nAA-08 AA 0.6 700 0.75 530\n3"}, {"round": 2, "reviewer_comments": {"reviewer_2": "For the original point #1: The authors dampened the claim of inflammatory phenotype in these AA mice, which improved the rigor of their experimental interpretations.\nFor the point regarding chromatin level evidence of CBP binding and regulation of p53, they showed a new result in panel Fig. 3f to demonstrate more p53 binding to p21 promoter. Although this indirectly showed an increased p53 activity in these AA tissues, the direct regulation of CBP on p53 was not demonstrated in this experiment. The n-number in this new experiment was 3 for each group. The panels of 3a, 3d, and 3e were also showing n=3 in all comparison, which did not seem to support a high confidence of these experimental results.\nOverall, the paper has been improved but figure 3 remains to be a weak component of the work."}, "author_response": ""}, {"round": 3, "reviewer_comments": {"reviewer_2": "The newly revised Fig. 3a showed Ki67 and DAPI staining. The AA image on the right is overall dimmer than the WT image. The DAPI signal is brighter in the WT image than the DAPI signal in the AA image. The figure legend stated \"The nuclei were counterstained with DAPI for normalization. Ki-67 expression was quantified as the number integrated density (IntDen) normalized to the DAPI IntDen within a fixed region\". If the quantification was based on fluorescent intensity and the two images were clearly taken with different settings, I am concerned whether the authors carefully examined these images before including them in the manuscript."}, "author_response": ""}]

Decision letter