CDK8 and CDK19 regulate intestinal differentiation and homeostasis via the chromatin remodeling complex SWI/SNF

Initiation and maintenance of transcriptional states are critical for controlling normal tissue homeostasis and differentiation. The cyclin dependent kinases CDK8 and CDK19 (Mediator kinases) are regulatory components of Mediator, a highly conserved complex that orchestrates enhancer-mediated transcriptional output. While Mediator kinases have been implicated in the transcription of genes necessary for development and growth, its function in mammals has not been well defined. Using genetically defined models and pharmacological inhibitors, we showed that CDK8 and CDK19 function in a redundant manner to regulate intestinal lineage specification in humans and mice. The Mediator kinase module bound and phosphorylated key components of the chromatin remodeling complex switch/sucrose non-fermentable (SWI/SNF) in intestinal epithelial cells. Concomitantly, SWI/SNF and MED12-Mediator colocalized at distinct lineage-specifying enhancers in a CDK8/19–dependent manner. Thus, these studies reveal a transcriptional mechanism of intestinal cell specification, coordinated by the interaction between the chromatin remodeling complex SWI/SNF and Mediator kinase.


Introduction
The intestinal epithelium is among the most proliferative tissues, with a complete turnover of intestinal epithelial cells (IECs) every 3 to 5 days. The self-renewal capacity of the intestinal epithelium relies on intestinal stem cells (ISCs), which reside at the bottom of specialized invaginations called crypts of Lieberkühn (1). ISCs continuously divide and give rise to transient amplifying cells while migrating upward through the villus toward the lumen and differentiating into mature lineages such as absorptive enterocytes as well as secretory goblet cells, tuft cells, and enteroendocrine cells (1). One exception to this migration pattern are Paneth cells, which escape the upward migration and instead move down to the bottom of the crypts, where they intersperse with ISCs to support their proliferative capacity (1,2). ISC proliferation, migration of IECs along the crypt-villus axis, and their differentiation are controlled by the coordinated activity of morphogen-driven pathways such as BMP, EGF, Notch, and Wnt/β-catenin signaling. Notch signaling is the main pathway determining secretory cell differentiation.
Cells with active Notch signaling express the transcription factor hairy and enhancer of split-1 (HES1), resulting in atonal BHLH transcription factor 1 (ATOH1) repression and commitment toward absorptive lineages, while cells that escape Notch signaling express ATOH1 and differentiate into the secretory lineages.
Higher-order chromatin structure and organization play an important role in differentiation and cell fate decisions in embryonic stem cells and in adult stem cells during tissue homeostasis (3)(4)(5). Accessible cis-regulatory elements provide binding sites for transcription factors (TFs) (6) to induce lineage-restricted gene expression programs that enable differentiation of tissue-specific cell lineages (7)(8)(9). Intestinal secretory and absorptive progenitors were shown to have a similar degree of accessible and permissive chromatin at the majority of cis-regulatory elements, suggesting that chromatin accessibility at cis elements may set the stage, but TFs such as ATOH1 ultimately control lineage specification (10). In addition, chromatin modifying complexes such as switch/sucrose non-fermentable (SWI/SNF), NuRD, and SRCAP have been linked to intestinal differentiation networks (11)(12)(13)(14)(15), underscoring the importance of chromatin structural dynamics in this process. Nevertheless, the detailed mechanisms determining chromatin access, and TF activity accounting for the high degree of intestinal cell lineage plasticity, remain largely unknown.
The multimeric Mediator complex is implicated in a number of steps of gene transcription, ranging from chromatin organization to RNA polymerase II phosphorylation (16). The Mediator Initiation and maintenance of transcriptional states are critical for controlling normal tissue homeostasis and differentiation. The cyclin dependent kinases CDK8 and CDK19 (Mediator kinases) are regulatory components of Mediator, a highly conserved complex that orchestrates enhancer-mediated transcriptional output. While Mediator kinases have been implicated in the transcription of genes necessary for development and growth, its function in mammals has not been well defined. Using genetically defined models and pharmacological inhibitors, we showed that CDK8 and CDK19 function in a redundant manner to regulate intestinal lineage specification in humans and mice. The Mediator kinase module bound and phosphorylated key components of the chromatin remodeling complex switch/sucrose non-fermentable (SWI/SNF) in intestinal epithelial cells. Concomitantly, SWI/SNF and MED12-Mediator colocalized at distinct lineage-specifying enhancers in a CDK8/19-dependent manner. Thus, these studies reveal a transcriptional mechanism of intestinal cell specification, coordinated by the interaction between the chromatin remodeling complex SWI/SNF and Mediator kinase.
CDK8 and CDK19 regulate intestinal differentiation and homeostasis via the chromatin remodeling complex SWI/SNF To begin to address this possibility, we first performed in silico analyses of publicly available scRNA-Seq data sets delineating the intestinal cell types of normal mouse (32) and human intestinal epithelium (33). Indeed, CDK8 and CDK19 were found to be coexpressed in nearly all cell lineages, albeit CDK8 was generally more highly expressed than CDK19 (Supplemental Figure 1, A-D; supplemental material available online with this article; https://doi. org/10.1172/JCI158593DS1). This raised the intriguing possibility that the Mediator kinases, CDK8 and CDK19, based on their high degree of sequence homology (20) and coexpression, may have functional redundancy in the intestinal epithelium.
Intestinal loss of Mediator kinases leads to defective secretory lineage differentiation. Mediator kinases have been intimately implicated in embryonic stem cell maintenance (24,35). To investigate the role of CDK8/19 in intestinal differentiation, we delineate the stem cell, secretory, and absorptive cell lineages in the CDK8/CDK19-deficient intestine. IHC for lysozyme revealed reduced Paneth cell (PC) numbers in Cdk8 IEC-KO /Cdk19 -/mice compared with VillinCreER T2 mice ( Figure 1, D and E). Likewise, CDK8 iIEC-KO /Cdk19 -/mice exhibited significantly reduced numbers of goblet cells (periodic acid-Schiff stain) and tuft cells (doublecortin-like kinase 1 [DCLK1] IHC) (Figure 1, D and E, and Supplemental Figure 3, A and B). Conversely, absorptive cell marker staining for alkaline phosphatase (AP) was significantly increased in CDK8 iIEC-KO /Cdk19 -/compared with VillinCreER T2 mice ( Figure  1, F and G). Olfactomedin 4-positive (OLFM4-positive) cells exhibited a patchy staining pattern in CDK8 iIEC-KO /Cdk19 -/mice compared with VillinCreER T2 mice (Supplemental Figure 3C). No difference in proliferation was observed in VillinCreER T2 and Cdk8 fl/fl / Cdk19 -/mice by Ki-67 staining (Supplemental Figure 3, D and E). These effects were temporally and spatially sustained in the kinase module composed of cyclin-dependent kinase 8 (CDK8), cyclin C (CCNC), Mediator complex subunit 12 (MED12), and MED13, reversibly associates with the core Mediator complex and functions as a catalytic subunit to activate or repress transcription by regulating TF activity via phosphorylation and colocalizing genome-wide at promoters and super-enhancers (17). Precise regulation and different subunit interactions are required for appropriate control of Mediator kinase activity. Consequently, mutation or changes in subunit expression are associated with aberrant activity of the kinase module, resulting in oncogenic signaling fueling tumor growth (18). For example, CDK8 was identified as a colorectal cancer (CRC) oncogene with copy number gains driving tumor growth by positively regulating β-catenin target gene expression (19). Thus, the possibility of inhibiting CDK8/19 to treat CRC has gained traction in the last few years, and several Mediator kinase inhibitors have been developed.
Vertebrates express a paralog of CDK8, called CDK19, which shares 91% overall sequence homology to CDK8 with a nearly identical cyclin binding domain and kinase domain (20). CDK19 was shown to form a separate kinase module in a mutually exclusive manner with respect to the CDK8 kinase module (21). Consequently, CDK8 and CDK19 were shown to have clearly distinct functions as transcriptional regulators in different stimulusspecific pathways by directly interacting with TFs and activating distinct gene sets within the same transcriptional network (22)(23)(24)(25)(26)(27).
Deletion of CDK8 alone, or inhibition of its kinase activity, is compatible with cell viability in cell lines (22,28). Likewise, both intestine-specific and whole-body deletion of CDK8 in adult mice was well tolerated (29). Studies using a CDK8/19 inhibitor revealed sporadic body weight loss in mice and multiorgan toxicity in rats and dogs (30). Follow-up studies suggest that this inhibitor toxicity may be due to off-target effects (31), underscoring the urgent need to better define the function of the Mediator kinases in vivo.
Herein, we generated a series of genetically engineered, inducible mouse models enabling us to test the consequences of CDK8/19 deletion or kinase activity loss in the intestinal epithelium. We find that CDK8/19 deletion in the intestinal epithelium led to severe depletion of the secretory lineage compartment in both animal and ex vivo organoid models. While mice tolerated ablation of Mediator kinases in the intestine, intestinal organoid viability was severely affected owing to loss of Paneth cells. Using temporal RNA-Seq analyses and genome-wide ChIP-Seq studies in CDK8/CDK19-depleted IECs, we identify Mediator kinases as critical regulators of intestinal lineage differentiation. Phospho-proteomic analyses of the Mediator kinase complex targets converged to show a functional association between MED12-Mediator and SWI/SNF complexes that drives transcription from intestinal lineage-defined enhancers. In conclusion, these data highlight a role for Mediator kinases in regulating the intestinal secretory lineage, and demonstrate higher-order mechanisms of lineage differentiation in the intestine.

CDK8 and CDK19 are dispensable for basal intestinal homeostasis.
We have previously reported that individual knockout of CDK8 is well tolerated in the intestinal epithelium (29). We hypothesized that the paralog gene, CDK19, may compensate for CDK8 loss.
Mediator kinases are required for IECintrinsic growth. Previous reports have shown that the intestinal secretory lineage is dispensable for ISC maintenance and intestine proliferation in vivo, but not ex vivo, owing to compensatory Wnt signaling from the mesenchymal niche (36)(37)(38). To assess whether CDK8/19-deficient crypts sustain self-renewal and growth in the absence of the mesenchymal niche, we isolated small intestinal organoids from VillinCreER T2 / Cdk8 fl/fl /Cdk19 -/mice. We confirmed loss of CDK8 upon tamoxifen-induction in both VillinCreER T2 /Cdk8 fl/fl /Cdk19 -/and VillinCreER T2 /Cdk8 fl/fl organoids compared with VillinCreER T2 controls ( Figure 2, A-C, and Supplemental Figure 4, A and B). Strikingly, we found that loss of Mediator kinases led to a steady and profound reduction in both organoid number and size from 1 week after deletion with complete loss of organoid viability seen 4 weeks (passage 4) after the deletion of CDK8 ( Figure 2D and Supplemental Figure 4, C-F). The results were confirmed in an additional 2 independently established organoid lines originating from different mice of the same genotype (Supplemental Figure 4, G and H). Importantly, these effects were specifically attributed to complete CDK8/19 loss, as neither single deletion of either CDK8 or CDK19, nor Cre activation alone, affected organoid growth ( Figure 2D and Supplemental Figure 4, D-F). These results demonstrate functional redundancy for CDK8 and CDK19 in IEC-intrinsic growth and maintenance of the intestinal crypt.
CDK8/19 may act through both kinase-dependent and kinase-independent/scaffolding function (17). To test these possibilities in the context of intestinal differentiation, we generated and employed a knockin CDK19 kinase-dead mouse model (Cdk19 D173A ). As homozygous Cdk19 D173A/D173A mice exhibited perinatal lethality (unpublished), heterozygous CDK19 D173A mice were used in which the second allele of CDK19 was constitutively deleted (Cdk19 D173A/-) to generate VillinCreER T2 /Cdk8 fl/fl /Cdk19 D173A/mice. Importantly, the CDK19 kinase-dead allele was expressed at levels similar to those of WT CDK19 (Supplemental Figure 4I). Similarly to CDK8 iIEC-KO /Cdk19 -/mice, CDK8 iIEC-KO /Cdk19 D173A/mice did not exhibit any gross intestinal pathology (Supplemental Figure 4J). Small intestinal organoids were isolated from Villin-CreER T2 /Cdk8 fl/fl /Cdk19 D173A/mice, and tamoxifen treatment resulted in efficient deletion of CDK8 (Supplemental Figure 4K). CDK8 iIEC-KO /Cdk19 D173A/but not VillinCreER T2 /Cdk8 fl/fl /Cdk19 D173A/WT organoids exhibited profoundly reduced organoid growth across intestinal tract of mice lacking CDK8/19, as CDK8 iIEC-KO /Cdk19 -/mice exhibited reduced secretory compartment cell types in both the ileum (Supplemental Figure 3, F-O) and duodenum (Supplemental Figure 3, P and Q) 6 months after CDK8 deletion. Notably, Cdk8 fl/fl /Cdk19 -/mice exhibited a mild (~12%) but statistically significant decrease in Lyz + cells compared with VillinCreER T2 mice at both 3-week and 6-month time points (Supplemental Figure 3, G and H). Together, these results demonstrate that Mediator kinases are necessary for intestinal secretory cell lineage differentiation, but that these effects may be compensated, under basal conditions, to maintain ISCs and basal intestinal viability. several parametric measurements (i.e., number, size, viability) ( Figure  2, E-G, and Supplemental Figure  4L). To confirm these effects, we used an orthogonal pharmacological approach. C57BL/6 organoids were treated with compound 32 (Cp32), a highly potent and specific CDK8/19 kinase inhibitor (39,40). Consistent with our kinasedead knockin genetic approach, WT organoids treated with different concentrations of CDK8/19 chemical inhibitor (Cp32) showed profoundly reduced viability (Figure 2, H and I) even at 0.1 μM. Cp32 on-target activity was confirmed by S727-phospho-STAT1 analysis using the lowest Cp32 concentration that showed viability effects (Supplemental Figure 4M). Importantly, these results were recapitulated in human small intestinal organoids where CDK8/19 was inhibited using orthogonal functional genomic and pharmacological inhibition. Organoids were isolated from duodenal biopsies from human patients and transduced with a lentiviral construct expressing a shRNA targeting CCNC, mimicking the effects of CDK8/19 kinase inhibition. Strikingly, CCNC depletion led to a significant reduction in intestinal organoid growth in comparison with organoids treated with nontargeting control (shNTC) ( Figure 2J). CCNC depletion was validated by quantitative reverse transcriptase PCR (qRT-PCR) (Supplemental Figure 5A). Importantly, this growth defect was recapitulated when human intestinal organoids were treated with the CDK8/19 inhibitor, Cp32 (Supplemental Figure 5, B and C). Taken together, these complementary approaches identify that the kinase activity of CDK8/19 is essential for the maintenance of murine and human small intestinal organoids.
Wnt3 is necessary and sufficient for restoring intestinal homeostasis in the absence of Mediator kinases. The intestinal mesenchyme is an important source of Wnt ligands that support ISCs and epithelial renewal (36)(37)(38). We hypothesized that the CDK8/19-depleted organoids showed reduced expression of the key stem cell and early progenitor markers Olfm4, Ascl2, and Msi1 (Supplemental Figure 6D). Notably, Olfm4 gene expression was also reduced in CDK8/19-deficient IECs (Supplemental Figure  6D), highlighting a potential role for CDK8/19 in Olfm4 transcription. We then examined the effect of CDK8/19 loss on Wnt/β-catenin pathway-related gene expression using a signature of 93 Wnt pathway component genes. Strikingly, we found that only Wnt3 was downregulated significantly in organoids (log 2 FC < -1.0, FDR < 0.05) and trended down in IECs as well (log 2 FC < -1.0, FDR < 0.20) (Supplemental Figure 6E). Importantly, the Wnt3 downregulation in IECs was confirmed by qRT-PCR (Supplemental Figure 6F). This suggests that CDK8 and CDK19 do not play a direct role in regulating Wnt/β-catenin mediated transcription in the normal intestine.
Recent RNA-Seq studies mapping the intestine at the singlecell level (scRNA-Seq) have provided a transcriptomic atlas of cell state, helping to define cellular lineage and identity with unparalleled precision. We sought to delineate discrete cell types and clarify differentiation programs affected by loss of Mediator kinase. To do so, we performed gene set enrichment analysis (GSEA) on DEGs from CDK8/19-deficient organoids and IECs using cell type-defined gene sets from scRNA-Seq studies of mouse and human intestine (32,33). GSEA showed a significant negative enrichment in multiple intestinal secretory lineage cell types and positive correlation with enterocyte signatures ( Figure  4, C and D). These data are consistent with the skewed differentiation pattern observed in the intestines of CDK8 iIEC-KO /Cdk19 -/mice and help define distinct cell states regulated by the activity of the Mediator kinases.
Intestinal differentiation is tightly regulated and defined by the acquisition of lineage-specific gene expression programs in a spatial and temporal manner (10,41). To determine the transcriptional changes directly mediated by the Mediator kinases and infer causality, we performed a temporal transcriptomic analysis (RNA-Seq) on intestinal organoids at 3, 5, 7, and 10 days after ablation of Mediator kinases. DEGs were clustered based on 6 temporally defined expression patterns: downregulated early (log 2 FC < -1, days 3-5), downregulated late (log 2 FC < -1, days 5-7, days 7-10), upregulated early (log 2 FC > 1, days 3-5), upregulated late (log 2 FC > 1, days 5-7, days 7-10), stepwise/progressive loss (log 2 FC < -1, days 3-10), and stepwise/progressive gain (log 2 FC > 1, days 3-10) (Supplemental Figure 7A and Supplemental Table 2). GSEA of these 6 clusters with intestinal cell type signatures revealed distinct downregulation of progenitor and Paneth signatures as the earliest changes after loss of CDK8/19. Interestingly, loss of other intestinal secretory cell types (goblet cells, tuft cells) and stem cell signatures correlated with genes whose expression was more gradually lost (progressive loss signature) ( Figure 4E). We confirmed these results using 3 well-established PC markers (Lyz, Ang4, Mmp7), which were highly downregulated in CDK8/19-deficient organoids and IECs (Figure 4, F and G, and Supplemental Figure  7B). Importantly, these results were specific to CDK8/19 depletion, as 4-hydroxytamoxifen (4-OHT) treatment of VillinCreER T2 did not reduce secretory marker expression (Supplemental Figure  7C). Lastly, PC depletion was confirmed at the cellular level by Lyz immunofluorescence staining directly on CDK8 iIEC-KO /Cdk19 -/organoids, confirming the transcriptomic data ( Figure 4H). profound growth phenotype observed in CDK8/19-deficient intestinal crypts ex vivo compared with in vivo may be explained by an IEC-intrinsic function for CDK8/19 that can be compensated by the mesenchymal niche. To test this possibility, primary murine intestinal fibroblasts were cocultured together with Mediator kinase-deficient intestinal organoids to recapitulate extrinsic Wnt sources. Strikingly, we found that intestinal mesenchymal fibroblasts restored growth capacity of CDK8 iIEC-KO /Cdk19 -/organoids ( Figure 3, A and B). While PCs are dispensable for ISCs and intestinal homeostasis in vivo, they are essential for ISC maintenance in organoid cultures (2). Given the loss of PCs in CDK8/19-deficient intestines, we postulated that the growth defect observed in CDK8/19-deficient organoids may be caused by loss of PCderived Wnt3. Consistently, qRT-PCR analysis showed significant downregulation of Wnt3 in CDK8/19-deficient organoids 7 days after CDK8 deletion ( Figure 3C).
Mediator kinases have been implicated in the regulation of Wnt signaling and stemness genes in CRC cells (19,24). Indeed, gene expression analysis of 11 stem-related genes revealed that To determine whether the CDK8/19 requirement for secretory cell types is Wnt3 dependent, we subjected RNA from EtOH-and 4-OHT-treated VillinCreER T2 /Cdk8 fl/fl /Cdk19 -/organoids cultured in Wnt3-CM for 10 days to whole-transcriptome sequencing (RNA-Seq). Gene expression signatures from CDK8/19-deficient organoids cultured in Wnt3-CM maintained a negative enrichment for PCs and other secretory cell lineages, but no longer showed negative enrichment for ISCs (Supplemental Figure 8A). The reduction in PC cells (Lyz, Ang4, Mmp7) and tuft cells (Pou2f3) was validated by qRT-PCR in CDK8/19-deficient organoids cocultured with fibroblasts and Wnt3-CM (Supplemental Figure 8, B-D, and Supplemental Table 1). These results suggest that CDK8/19 act independently of Wnt3 to produce the secretory cell types.
To assess whether these effects are kinase dependent, we characterized the secretory lineage in the Cdk19 kinase-dead-expressing mouse (Cdk19 D173A ). CDK8 iIEC-KO /Cdk19 D173A/intestines showed a significantly lower number of PCs by IHC analysis (Supplemental Figure 8E) and reduced expression of the PC markers Lyz and Ang4 by qRT-PCR (Supplemental Figure 8F), phenocopying CDK8/19 protein depletion. To extend these findings, we performed whole-transcriptome (RNA-Seq) analysis on both CDK8 iIEC-KO / Cdk19 D173A/mouse IECs and organoids. Strikingly, both IECs and organoids showed a significant negative enrichment of Paneth and tuft cell signatures (Supplemental Figure 8G). Lastly, we inhibited CDK8/19 activity using CCNC shRNA in human intestinal organ-oids and with Cp32 in both mouse and human intestinal cells. Consistent with our genetic model above, we observed loss of PC markers by qRT-PCR after both pharmacological (Cp32) and genetic (CCNC shRNA) kinase inhibition of CDK8/19 in mouse and human intestinal organoids (Supplemental Figure 8, H-J). Collectively, these data demonstrate an indispensable role for CDK8/19 kinase activity, specifically for PC production and more broadly for intestinal secretory lineage differentiation.
Phospho-proteomic analysis reveals a functional interaction between the CDK8/19 kinase module and the SWI/SNF complex in the intestinal epithelium. CDK8 and CDK19 have been shown to regulate transcription in both a Mediator-dependent and a Mediator-independent manner. To determine whether loss of Mediator kinases in the intestine leads to dysregulated core Mediator composition and activity, we performed quantitative (tandem mass tagging) immunoprecipitation-coupled mass spectrometry using Mediator components in WT and CDK8/19-deficient cells. Using an antibody directed at MED1, we coimmunoprecipitated the majority (22 of 26) of the core Mediator subunits at equivalent levels in both WT and CDK8/19-deficient intestinal organoids. Importantly, total levels of Mediator complex subunits were largely unaltered (Supplemental Figure 9A). Strikingly, however, CDK8/19 ablation led to dissolution of MED1/core Mediator from the kinase module protein MED12 ( Figure 5A and Supplemental Table 3). These data suggest that loss of Mediator kinases perturbs MED12 is a critical regulatory subunit of the Mediator kinase complex that is required for priming CDK8/19 kinase activity and linking CDK8/19 to additional kinase substrates (42,43). Our work, herein, shows that the ability of Mediator kinases to control intestinal secretory cell differentiation is kinase dependent. To identify potential direct substrates of CDK8/19 activity required for intestinal differentiation, we took a 2-pronged approach. Firstly, we subjected WT and CDK8/19-depleted intestinal organoids to phospho-proteomic analysis. In total, 103 high-confidence phosphorylation events (in 68 unique nuclear proteins) were found to be significantly reduced upon loss of CDK8/19 (log 2 FC > 1, FDR < 0.05) (Supplemental Table 4). Of these 103 sites, 66 harbored canonical CDK active sites (SP/TP sites) (Supplemental Figure 9B), suggesting that they may be directly phosphorylated by CDK8/19. STRING network analysis of the 68 putative CDK8/19 kinase substrates revealed strong enrichment in chromatin remodeling and epigenetic regulatory complexes (Supplemental Figure 9, C and D).
To identify which of these proteins may be direct targets of Mediator kinases, we performed quantitative immunoprecipitation-coupled mass spectrometry in IECs using a MED12-directed antibody. After filtering for nuclear proteins, we found 72 significant MED12 binding partners (log 2 FC > 1, FDR < 0.05) compared with isotype IgG pull-down controls ( Figure 5B and Supplemental Table 5). As expected, these included the majority of core Mediator and CDK8 kinase module components (23 of 28; 82%). Strikingly, we found that MED12 associated with several other enhancer regulatory proteins, most notably more than half of the SWI/SNF subunit proteins ( Figure 5, B and C). Overlay of CDK8/19-regulated phospho-proteome and MED12 interactome analyses revealed 9 proteins, including 3 SWI/SNF subunits (ARID1A, SMARCC2, and PBRM1), that both bound to MED12 and were phosphorylated in the presence of Mediator kinases ( Figure 5, B and D, and Supplemental Figure 9E). Importantly, 4 of the other 6 non-SWI/ SNF subunits were also found as CDK8/19 targets in HCT116 CRC cells (44) ( Figure 5D). Interestingly, HCT116 harbors mutations in SMARCA4 (BRG1), an important component of SWI/SNF com- kinase and functional implications are not known. To determine whether Mediator kinases directly phosphorylate ARID1A, we expressed and purified ARID1A protein fragments that were WT or harbored mutations at the putative CDK8/19 phosphosites (Ser365, Ser696, Ser699, Ser703, Ser716). ARID1A protein was then subjected to an in vitro kinase assay using a reconstituted human CDK8 kinase module (CDK8, MED12, and CCNC). We found site-specific CDK8-dependent phosphorylation of ARID1A at Ser365, but not Ser696-Ser716. Importantly, protein levels were equivalent for the plex function (45), providing a potential explanation of why these phosphorylation events were not identified in this cell line.
We confirmed MED12 and SWI/SNF complex interaction by performing reciprocal immunoprecipitation and Western blotting for MED12 and a key SWI/SNF regulatory subunit, ARID1A. Indeed, we found that endogenous ARID1A and MED12 bound to each other in human and murine IECs, confirming our proteomic analysis ( Figure 5, E and F, and Supplemental Figure 9F). While ARID1A phosphorylation has been previously reported (46), the responsible tested WT and mutant forms of ARID1A ( Figure 5G). Cumulatively, these data show that the CDK8/19 kinase module (MED12 and CDK8) associates with and phosphorylates components of the SWI/ SNF complex in the context of the intestinal epithelium.
Deletion of Mediator kinases leads to perturbed enhancer activity in a distinct subset of ARID1A-regulated genes. ARI-D1A has been intimately linked to intestinal differentiation programs and colon carcinogenesis (12,47). As both Mediator and SWI/SNF have been implicated in enhancer-mediated transcriptional control, we performed ChIP-Seq to investigate their genomic occupancy in intestinal organoids in the presence and absence of CDK8/19. Firstly, ChIP-Seq for RNA polymerase II revealed that overall RNA polymerase II binding and distribution were unchanged after CDK8/19 deletion (Supplemental Figure 10A), indicating that, in contrast to cancer cells (40), loss of CDK8/19 does not affect global gene regulation in normal intestine. Similarly, ChIP-Seq analysis showed no change in the amount or distribution of H3K27ac sites, MED1, and MED12 upon loss of CDK8/19 (Supplemental Figure 10, B-D). In contrast, ARID1A genomic occupancy was both reduced (log 2 FC = 1.15; P < 0.001) and showed changes in binding distribution to enhancer and promoter regions after CDK8/19 ablation ( Figure  6A). Given the critical role of SWI/SNF in enhancer-mediated transcription, we then categorized MED1/MED12/ H3K72ac-defined enhancers based on whether they were lost (n = 1,350) or retained (n = 263) upon CDK8/19 ablation. Strikingly, we observed a near-complete loss of ARID1A binding to enhancers that were dependent on the presence of CDK8/19 ( Figure  6B and Supplemental Table 6). Importantly, overall ARID1A levels were not changed in CDK8/19-deficient intestinal organoids. Overall, these data show that Mediator kinases are required for ARID1A genomic binding, and are consistent with our proteomic studies suggesting a regulatory role for CDK8/19 in SWI/SNF activity.
been intimately linked with cell fate specification by their enabling of master transcription factors (TFs) to bind and control cell type-specific transcriptional programs (48). Given the critical role of CDK8/19 in secretory intestinal cell specification, we sought to map the super-enhancer gene regulatory landscape of the intestinal epithelium in the presence and absence of CDK8/19. We found 633 MED12-defined SE regions, of which 222 were lost upon CDK8/19 deletion. We then used the Genomic Regions Enrichment of Annotations Tool (GREAT) (49) to identify cis-regulated genes associated with MED12-defined SE elements. From a total of 92 SE cis-associated genes, we found 8 TFs and developmental regulator genes associated with these SE elements, including genes previously implicated in ISC maintenance and differentiation: ATOH1 (50), MYB (51), and HOPX (52) ( Figure 6C). To determine whether the expression of these SE-associated genes was dependent on CDK8/19, we investigated their expression in intestinal organoids and IECs lacking CDK8/19. Gene expression across all 92 SE-associated genes was globally downregulated, with the majority of genes below the 0 point of log 2 FC (Figure 6D). In particular, ATOH1, a critical lineage specification TF of the intestinal secretory pathway, was one of the most significantly downregulated SE-associated genes upon CDK8/19 loss ( Figure 6D). These data pinpoint specific SE-associated genes that may require CDK8/19 for their appropriate expression and, as such, offer potential effector proteins that are necessary for intestinal differentiation.
Our previous data indicate that the SWI/SNF complex may act as an important mediator of CDK8/19 function in the intestine. To test whether the ARID1A-bound site enhancer region is critical for ATOH1 transcription, we used CRISPR activation (CRISPRa) to direct a strong transcriptional activator (dCas9-VP64) to the ARID1A-defined Atoh1-associated enhancer (peak 3). The system was confirmed using a guide RNA targeting the Atoh1 promoter, which showed robust activation of Atoh1 transcription ( Figure 7D). Strikingly, CRISPRa targeted activation of the ARID1A-defined enhancer (peak 3) showed similar induced Atoh1 expression in CDK8 iIEC-KO /Cdk19 -/organoids compared with negative controls ( Figure 7D). Interestingly, CRISPRa-induced ATOH1 expression from peak 3 was lower in CDK8/19-deficient organoids compared with controls. Lastly, to test whether this regulatory mechanism applies in vivo, we assessed ARID1A and MED12 binding at the Atoh1 super-enhancer peak 3 in CDK8/19-deficient IECs by ChIP-qPCR. Using 2 primer pairs targeting peak 3 of this enhancer, we found that ARID1A and MED12 binding at the Atoh1 superenhancer peak 3 was significantly reduced in CDK8 iIEC-KO /Cdk19 -/mice compared with VillinCreER T2 IEC controls (Figure 7, E and F). These results show that the Mediator kinase module is a direct regulator of ATOH1 expression and secretory cell differentiation in vivo as well. Taken together, these data define a CDK8/19dependent, enhancer-mediated mechanism of Atoh1 transcription that involves the interplay of both MED12-Mediator and SWI/SNF complex components.

Discussion
Using genetic mouse models combined with transcriptomic, epigenetic, and phospho-proteomic profiling, we identify a kinase-dependent function for the Mediator kinases CDK8 and tion factor ATOH1 is the master regulator of intestinal secretory cell differentiation (1). ATOH1 overexpression results in expansion of secretory cells (53), and conversely, deletion of ATOH1 results in the loss of intestinal secretory cells (50,54), similar to the phenotype we observe in the intestine upon loss of the Mediator kinases. To confirm whether ATOH1 is a target of CDK8/19, we performed qRT-PCR for Atoh1 expression on both organoids and IECs after CDK8/19 ablation. Indeed, Atoh1 expression was significantly reduced in both IECs and organoids after CDK8/19 ablation ( Figure 6E). Consistently, there was a significant reduction in the small intestinal ATOH1 target gene expression (ATOH1 targetome) (55) in both CDK8/19-ablated small intestinal IECs (normalized enrichment score [NES] = -3.15, P < 0.001) and organoids (NES = -2.26, P = 0.002) ( Figure 6F). Consistent with a kinase-dependent effect, the expression of both ATOH1 itself and its target genes was significantly reduced in CDK8 iIEC-KO /Cdk19 D173A/-IECs and organoids (Supplemental Figure 10, E-H). We then tested whether restoration of ATOH1 could rescue the growth defect in CDK8/19depleted intestinal organoids. To do so, we stably transduced Vil-linCreER T2 /Cdk8 fl/fl /Cdk19 -/organoids with a lentivirus encoding a doxycycline-regulated ATOH1 construct and confirmed its inducible expression (Supplemental Figure 10, I and J). Strikingly, exogenous expression of ATOH1 in CDK8/19-deficient organoids restored expression of PC markers and Wnt3 and partially rescued the growth defect in Mediator kinase-depleted organoids ( Figure  6, G and H, and Supplemental Figure 10K). Since overexpression of ATOH1 at least partially restores Wnt3 expression, this suggests that the Mediator kinase/ATOH1 axis is upstream of PC differentiation and Wnt3 expression. ATOH1 overexpression also restored expression of other secretory cell types in CDK8/19-deficient organoids, including goblet cells (Muc2) and tuft cells (Dclk1) (Supplemental Figure 10, L and M).
Notch signaling and its downstream transcription factor HES1 are known to repress Atoh1 transcription and limit secretory pathway differentiation in a process known as lateral inhibition (1). We then asked whether CDK8/19-mediated Atoh1 regulation is Notch pathway dependent. To assess whether Mediator kinases control secretory cell differentiation as part of the Notch/HES1/ATOH1 axis, CDK8/19-deficient organoids (3 days after 4-OHT) were treated with the γ-secretase inhibitor DAPT for 3 days to inhibit Notch signaling. We found that CDK8/19-depleted organoids subjected to Notch inhibition still retained the ability to upregulate a number of secretory lineage cell markers to an extent comparable to that seen in DMSO-treated controls (e.g., 30-fold [CDK8/19 knockout] vs. 37-fold [control] for Lyz expression) (Supplemental Figure 11A). Consistent with this, Hes1 mRNA expression was unchanged in CDK8/19-deficient IECs, slightly elevated in CDK8/19-depleted organoids, and unchanged at the protein level (Supplemental Figure 11, B and C). Likewise, a HES1-associated super-enhancer was unaffected by loss of Mediator kinases (Supplemental Figure 11D). Collectively, these data provide evidence that the Notch pathway and its downstream effector HES1 are intact in CDK8/19-depleted organoids and, moreover, imply that Mediator kinases may regulate ATOH1 and secretory lineage differentiation independently of Notch/HES1 signaling.
CDK8 and CDK19 control Atoh1 expression by regulating a distinct ARID1A-defined enhancer. Given the Notch-independent Strikingly, we show that the MED12-Mediator complex interacts with most components of the SWI/SNF complex, suggestive of a broader association between Mediator and SWI/SNF complexes. Importantly, SWI/SNF enhancer occupancy was CDK8/19 dependent, as we observed a widespread reduction of ARID1A binding across the genome and particularly at MED1/12-defined enhancers after CDK8/19 loss. While the exact nature of these interactions remains to be determined, it is exciting to speculate that Mediator complex function may extend beyond its canonical role in transcription to control of higher-order chromatin accessibility via its ability to regulate SWI/SNF activity.
The mechanism of lineage specification in the intestinal tract requires the hierarchical activation of discrete transcriptional programs. Previous work has demonstrated that Notch inhibition, HES1 deletion, and overexpression of ATOH1 result in overabundance of secretory cells, while ATOH1 deletion leads to a loss of secretory cells (50,54,(61)(62)(63). While the Notch pathway and its role in regulating ATOH1 via HES1 have been extensively studied, the cell-intrinsic mechanisms through which ATOH1 is expressed have remained elusive. Our work directly implicates Mediator kinases as cell-intrinsic regulators of enhancer-mediated ATOH1 transcription.
Importantly, pharmacological inhibition of Notch overcame CDK8/19-mediated ATOH1 suppression, highlighting that the Notch/HES1 pathway is still intact in the absence of CDK8/19. Consistent with this, previous reports show that deletion of either ATOH1 or ARID1A has a more drastic impact on the intestinal secretory cell compartment compared with CDK8/19 ablation (12,50,54). This highlights the diverse mechanisms that contribute to fine-tuning of the expression of ATOH1 and subsequent production of secretory cell types.
In our study, CDK8/19-depleted intestinal cells did not show significant changes in Wnt/β-catenin-mediated transcriptional output. Consistent with this, Wnt stimulation did not rescue the effects of CDK8/19 loss on secretory type differentiation. Hence, we propose that the requirement of CDK8/19 for secretory cell type differentiation is independent of the Wnt/β-catenin pathway. This contrasts with several previous studies, using genetic and pharmacological inhibitors, which have reported a direct role for CDK8/19 in mediating Wnt/β-catenin transcription in the context of CRC. Further studies comparing CDK8/19 activity in the normal and neoplastic intestine will be helpful to delineate the contextual differences of CDK8/19 activity in the gut. Taken together, these findings highlight the ability of signal-driven transcriptional effectors and chromatin-mediated regulators to act as molecular rheostats of intestinal secretory lineage specification.
Limitations of the study. Forced expression of ATOH1 from a constitutive promoter did not completely rescue the phenotype of CDK8/19-deficient organoids. While the incompleteness of the rescue effect may be related to technical challenges of expressing the appropriate amount of ATOH1 in the correct cell type, we also cannot rule out that CDK8/19-mediated secretory cell type differentiation may also involve ATOH1-independent mechanisms. Our study was conducted in intestinal epithelial cells, which enables us to place the function of Mediator kinases within the context of one of the most well-studied tissue homeostatic mechanisms. While our conclusions, with regard to SWI/SNF regula-CDK19 in intestinal secretory cell type differentiation. Importantly, we find that CDK8 and CDK19 act in a functionally redundant manner to control transcriptional output and differentiation. We show that the Mediator complex interacts with the SWI/SNF complex members and is required for proper ARID1A binding across MED1/12-defined intestinal enhancer regions, suggesting that the Mediator kinase complex may act beyond transcriptional regulation to regulate chromatin accessibility.
Previous studies have suggested that CDK8 and its paralog CDK19 have nonredundant roles as transcriptional regulators (23,27). Moreover, other studies have identified both kinasedependent and -independent functions for CDK8/19 (25,26,56). Herein, we show that CDK8 and CDK19 have redundant and kinase-dependent functions in regulating transcription in normal intestinal homeostasis. This is consistent with our own previous work in CRC, where we found that single gene ablation of CDK8 or CDK19 has minor effects on gene transcription and functional output. Intriguingly, CDK8/19 loss produced broad effects in the cancer state compared with normal tissue. While CDK8/19 ablation led to global loss of transcription and enhancer deposition (40), depletion of CDK8/19 in the normal intestine was marked by enhancer perturbation and transcriptional loss of only a distinct subset of lineage-specific genes. These results suggest divergent, yet kinase-dependent, functions for Mediator kinases in regulating transcription in the normal versus oncogenic states. We speculate that these differences may be attributed to cell type-specific functions of Mediator kinases or the fact that other studies have been performed using transformed cell lines. Intestinal epithelial cells are generally characterized by broadly permissive chromatin (10) combined with a set of unique active enhancers allowing the activity of cell lineage-specific TFs as essential determinants of intestinal cell lineage identities (57). We provide evidence that CDK8 and CDK19 act within this framework and are required to maintain the activity of super enhancers important for TFs regulating intestinal epithelial cell identity.
Our study provides a comprehensive phospho-proteomic analysis of Mediator kinase module interactors and potential phosphorylation substrates. Remarkably, 3 of the top 9 phosphorylated targets were SWI/SNF components, including ARID1A, a critical tumor suppressor gene (47,(58)(59)(60). Intriguingly, CDK8/19 ablation in the intestine phenocopies defects in secretory progenitor differentiation seen upon ARID1A loss in the intestine, although the phenotype of ARID1A-deficient mice is more severe with additional loss of intestinal stem cells (12). Curiously, while we identify a number of CDK8/19 kinase substrates that are consistent with a previous phospho-proteomic analysis in a CRC cell line (e.g., CHD4, MED12, MED13; ref. 44), the SWI/SNF components that we identified were only found in the context of the normal intestine. It is interesting to note that the cell line used in the previous study, HCT116 cells, harbors a SMARCA4 mutation (L1149P) predicted to compromise the SWI/SNF complex (45). This may explain the context-specific regulation of SWI/SNF by Mediator kinases in the normal intestine compared with the cancer state. Given the ubiquitous nature of both Mediator and SWI/ SNF complexes, it will be of great interest to broaden our understanding of how Mediator kinases regulate SWI/SNF activity in other tissues as well.